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A splice site mutation in the TSEN2 causes a new syndrome with craniofacial and central nervous system malformations, and atypical hemolytic uremic syndrome

Authors:
  • Istanbul University-Cerrahpaşa Cerrahpaşa Faculty of Medicine
  • Istanbul University-Cerrahpasa

Abstract

Recessive mutations in the genes encoding the four subunits of the tRNA splicing endonuclease complex (TSEN54, TSEN34, TSEN15, and TSEN2) cause various forms of pontocerebellar hypoplasia, a disorder characterized by hypoplasia of the cerebellum and the pons, microcephaly, dysmorphisms, and other variable clinical features. Here, we report an intronic recessive founder variant in the gene TSEN2 that results in abnormal splicing of the mRNA of this gene, in six individuals from four consanguineous families affected with microcephaly, multiple craniofacial malformations, radiological abnormalities of the central nervous system, and cognitive retardation of variable severity. Remarkably, unlike patients with previously described mutations in the components of the TSEN complex, all the individuals that we report developed atypical hemolytic uremic syndrome (aHUS) with thrombotic microangiopathy, microangiopathic hemolytic anemia, thrombocytopenia, proteinuria, severe hypertension, and end-stage kidney disease (ESKD) early in life. Bulk RNA sequencing of peripheral blood cells of four affected individuals revealed abnormal tRNA transcripts, indicating an alteration of the tRNA biogenesis. Morpholino-mediated skipping of exon 10 of tsen2 in zebrafish produced phenotypes similar to human patients. Thus, we have identified a novel syndrome accompanied by aHUS suggesting the existence of a link between tRNA biology and vascular endothelium homeostasis, which we propose to name with the acronym TRACK syndrome (TSEN2 Related Atypical hemolytic uremic syndrome, Craniofacial malformations, Kidney failure). This article is protected by copyright. All rights reserved.
ORIGINAL ARTICLE
A splice site mutation in the TSEN2 causes a new syndrome
with craniofacial and central nervous system malformations,
and atypical hemolytic uremic syndrome
Nur Canpolat
1
| Dingxiao Liu
2,3
| Emine Atayar
4
| Seha Saygili
1
|
Nazli Sila Kara
5
| Trudi A. Westfall
6
| Qiong Ding
2
| Bartley J. Brown
7
|
Terry A. Braun
7
| Diane Slusarski
7
| Kader Karli Oguz
8
| Yasemin Ozluk
9
|
Beyhan Tuysuz
10
| Tugba Tastemel Ozturk
11
| Lale Sever
1
|
Osman Ugur Sezerman
5
| Rezan Topaloglu
11
| Salim Caliskan
1
|
Massimo Attanasio
2
| Fatih Ozaltin
4,11
1
Department of Pediatric Nephrology, Istanbul University-Cerrahpasa, Cerrahpasa Faculty of Medicine, Istanbul, Turkey
2
Department of Internal Medicine, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA
3
Department of Vascular Surgery, Second Xiangya Hospital, Central South University, Changsha, Hunan, China
4
Nephrogenetics Laboratory, Department of Pediatric Nephrology, Hacettepe University, Faculty of Medicine, Ankara, Turkey
5
Biostatistics and Medical Informatics Program, Faculty of Medicine, Graduate School of Health Sciences, Acibadem Mehmet Ali Aydinlar University, Istanbul, Turkey
6
Department of Biology, University of Iowa, Iowa City, Iowa, USA
7
Center for Bioinformatics and Computational Biology, University of Iowa, Iowa City, Iowa, USA
8
Department of Radiology, Hacettepe University Faculty of Medicine, Ankara, Turkey
9
Department of Pathology, Istanbul University Faculty of Medicine, Istanbul, Turkey
10
Department of Pediatric Genetics, Istanbul University-Cerrahpasa, Cerrahpasa Faculty of Medicine, Istanbul, Turkey
11
Department of Pediatric Nephrology, Hacettepe University Faculty of Medicine, Ankara, Turkey
Correspondence
Massimo Attanasio, Department of Internal
Medicine, Carver College of Medicine
University of Iowa, 285 Newton Road, Iowa
City, IA 52242, USA.
Email: massimo-attanasio@uiowa.edu
Fatih Ozaltin, Department of Pediatric
Nephrology, Hacettepe University Faculty of
Medicine, 06100, Sihiye, Ankara, Turkey.
Email: fozaltin@hacettepe.edu.tr
Funding information
National Institute of Diabetes and Digestive
and Kidney Diseases, Grant/Award Number:
R01DK126759; Scientific Research Projects
Coordination Unit of Istanbul University-
Cerrahpasa, Grant/Award Number:
3736-55436; Turkish Pediatric Association,
Grant/Award Number: 05-2019
Abstract
Recessive mutations in the genes encoding the four subunits of the tRNA splicing
endonuclease complex (TSEN54,TSEN34,TSEN15, and TSEN2) cause various forms
of pontocerebellar hypoplasia, a disorder characterized by hypoplasia of the cerebel-
lum and the pons, microcephaly, dysmorphisms, and other variable clinical features.
Here, we report an intronic recessive founder variant in the gene TSEN2 that results
in abnormal splicing of the mRNA of this gene, in six individuals from four consan-
guineous families affected with microcephaly, multiple craniofacial malformations,
radiological abnormalities of the central nervous system, and cognitive retardation of
variable severity. Remarkably, unlike patients with previously described mutations in
the components of the TSEN complex, all the individuals that we report developed
atypical hemolytic uremic syndrome (aHUS) with thrombotic microangiopathy, micro-
angiopathic hemolytic anemia, thrombocytopenia, proteinuria, severe hypertension,
and end-stage kidney disease (ESKD) early in life. Bulk RNA sequencing of peripheral
blood cells of four affected individuals revealed abnormal tRNA transcripts, indicating
Nur Canpolat and Dingxiao Liu contributed equally to this work.
Received: 19 November 2021 Revised: 19 December 2021 Accepted: 26 December 2021
DOI: 10.1111/cge.14105
Clinical Genetics. 2022;113. wileyonlinelibrary.com/journal/cge © 2021 John Wiley & Sons A/S . Published by John Wiley & Sons Ltd 1
an alteration of the tRNA biogenesis. Morpholino-mediated skipping of exon 10 of
tsen2 in zebrafish produced phenotypes similar to human patients. Thus, we have
identified a novel syndrome accompanied by aHUS suggesting the existence of a link
between tRNA biology and vascular endothelium homeostasis, which we propose to
name with the acronym TRACK syndrome (TSEN2 Related Atypical hemolytic uremic
syndrome, Craniofacial malformations, Kidney failure).
KEYWORDS
atypical hemolytic uremic syndrome, cranio-facial malformation, novel syndrome, tRNA splicing
endonuclease, TSEN2
1|INTRODUCTION
Nuclear encoded transfer RNAs (tRNA) must be processed and modi-
fied to generate functional tRNAs in humans. It has been reported that
28 out of 429 predicted high confidence tRNA genes contain introns
that need to be removed from pre-tRNAs through splicing
mechanism.
13
Excision of the introns and ligation of the 50and 30
exons of tRNA are accomplished by two multiprotein assemblies with
enzymatic activity, namely tRNA splicing endonuclease (TSEN) and
tRNA ligase complex, respectively.
4,5
The TSEN complex consists of
two catalytic subunits (i.e., TSEN2 and TSEN34) and two structural sub-
units (i.e., TSEN15 and TSEN54),
4,6
which work in association with
RNA kinase cleavage factor polynucleotide kinase subunit 1, CLP1.
7
Mutations in all four subunits of the TSEN complex or in CLP1 have
been shown to impair tRNA splicing in vitro and to cause central and
peripheral nervous system pathologies mainly pontocerebellar hypopla-
sia (PCH), a heterogeneous group of neurodegenerative disorders char-
acterized by cerebellar hypoplasia and microcephaly (MIM #277470,
#225753, #610204, #612389, #612390, #615803), however their con-
tribution to the disease pathogenesis remains unclear.
815
Very
recently, it was shown that PCH mutations cause thermal destabiliza-
tion of the TSEN complex resulting in assembly defects and reduced
pre-tRNA cleavage activity that leads to an imbalanced pre-tRNA
pool.
15
It was suggested that critical reduction of neuron-specific
isodecoders (tRNAs with the same anticodon but sequence differences
in the tRNA body) and/or aberrant accumulation of pre-tRNAs would
contribute to the pathogenesis of PCH by impairing vital cellular pro-
cesses and molecular signaling pathways as well as synaptic plasticity.
15
aHUS is a rare and severe form of thrombotic microangiopathy
(TMA). The kidneys are invariably involved, with consequent loss of
renal function that frequently leads to end-stage kidney disease
(ESKD) when left untreated. All forms of hemolytic uremic syndromes
arise from vascular endothelial cell injury of the microvasculature of
the kidney and other organs.
16
In aHUS, the trigger of endothelial cell
damage is endogenous and is caused by genetic mutations or autoan-
tibodies against some of the complement components, or mutations
that cause loss of function of the lipid kinase diacyl-glycerol kinase
epsilon (DGKε) independent of complement dsyregulation.
17,18
The
underlying genetic etiology has still not been identified in 30%40%
of all cases of aHUS.
19,20
We described six children from four consanguineous pedigrees,
affected with microcephaly and craniofacial malformations, severe
growth failure, intellectual retardation, and aHUS that progressed to
ESKD requiring kidney replacement therapy within the first decade of
life. Our research identified of a homozygous intronic variant in the
gene TSEN2 (tRNA splicing endonuclease subunit 2) associated with
this undescribed new syndrome.
2|CONCISE METHODS (COMPLETE
METHODS AVAILABLE IN THE
SUPPLEMENTARY DATA)
2.1 |Exome capture and sequencing
They were performed by standard techniques according to the
manufacturing instructions.
2.2 |Sequencing data and relatedness analyses
Reads were mapped against the human reference genome assembly
(hg19 genome, GRCH37Hg19), and variants were annotated using the
Qiagen
®
Clinical Insight Translationalsoftware (Qiagen, Redwood
City, CA, USA), and the Integrative Genomics Viewer (IGV).
21
The pathogenicity of variants was estimated using the in silico pre-
diction software Mutation Taster (http://www.mutationtaster.org), Poly-
morphism Phenotyping v2(PolyPhen2, http://genetics.bwh.harvard.
edu/pph2), Sorting Intolerant From Tolerant (SIFT, https://sift.bii.a-star.
edu.sg/sift4g/), MaxEntScan (http://hollywood.mit.edu/burgelab/
maxent/Xmaxentscan_scoreseq.html),
22
and NetGene2
23
version 2.42
(DTU Technical University, Lyngby, Denmark). Relatedness analysis was
performed using PLINK (v1.9)
24
and Peddy.
25
2.3 |Sanger sequencing
Sanger sequencing was performed by standard technique. The
GeneBank (National Center for Biotechnology Information, NCBI)
2CANPOLAT ET AL.
sequence TSEN2 NM_001145394 (corresponding to Ensembl refer-
ence ENST00000454502.6) was used as reference sequence.
2.4 |Alignments
Protein alignments were performed using Clustal Omega version 1.2.2
(https://www.ebi.ac.uk/Tools/msa/clustalo/) and visualized using
Jalview 2.11.1.0.
2.5 |Bulk RNA sequencing and analysis
Four affected individuals (PN556-II3, PN878-II2, PN1093-II1,
PN1939-II3 in Figure S1) and one healthy mother of an affected indi-
vidual (PN878-I2) were RNA sequenced obtained from peripheral
blood cells using Novaseq 6000 (Illumina, Inc., San Diego, CA, USA)
sequencer according to the manufacturer's recommendations. Details
of the analyses are given in the Supplemental Data.
2.6 |RNA Sanger sequencing
RNA sequencing was performed by standard techniques.
2.7 |In vitro splicing assay
TheexontrapvectorpET01(MoBiTecGmbH,Goettingen,
Germany) was used for in vitro characterization of the splicing
(Figure S2A). Details of the assay are given in the Supplemen-
tal Data.
2.8 |Zebrafish experiments
Zebrafish experiments were approved by the University of Iowa's
IACUC, PHS Assurance No. A3021-01, under animal protocol
No. 5091513. The detailed experiments are described in the Supple-
mental Data.
FIGURE 1 Facial features of patients with homozygous TSEN2 variant. Representative images of the craniofacial malformations in five
homozygous carriers of the c.914-5T>A variant. Note the occurrence of triangular faces, high and broad forehead, sparse and broad eyebrows,
deeply set eyes, high nasal bridge, long narrow nose, retro-micrognathia, and sparse cone-shaped teeth. The full description of signs is given in
Table 1 [Colour figure can be viewed at wileyonlinelibrary.com]
CANPOLAT ET AL.3
3|RESULTS
3.1 |An intronic variant in the gene TSEN2 causes
a syndrome characterized by microcephaly,
craniofacial malformations, growth and intellectual
retardation, and aHUS
In a large cohort of patients affected with aHUS, we ascertained four
individuals from two consanguineous families (pedigrees PN556 and
PN878 in Figure S1) who showed additional similar extrarenal symp-
toms (Figure 1). Kidney pathology showed lesions of glomerular TMA
(Figure 2). Detailed clinical data are available in the Supplemental Data.
In addition to these symptoms, all four individuals presented micro-
cephaly, multiple craniofacial malformations, and cognitive and devel-
opmental delay of variable severity (Figure 1 and Table 1). We excluded
mutations in the genes CFH, CFI, CFB, C3, CD46 (MCP), CFHR1-5,DGKE,
PLG,andTHBD, known to be relevant for aHUS in all four affected indi-
viduals, by Sanger sequencing as well as the presence of anti-
complement factor H antibodies by ELISA. We then performed WES of
three affected and one unaffected individual as well as the healthy con-
sanguineous parents of pedigree PN556, and of one affected and one
unaffected sibling, and the consanguineous parents of family PN878
(Figure S1A,B). We applied a stepwise filtering strategy to prioritize
clinically relevant variants among all the ones annotated by WES
(Figure S3 and Supplemental Data). After stringent filtering, the only
variant that was homozygous in all four affected individuals and s-
egregated in the pedigrees compatibly with autosomal recessive inheri-
tance was the intronic change c.914-5T>A in the gene TSEN2
(NM_001145394, Figure S4A,B). Both families came from the South-
eastern Anatolia with no clear record of known relatedness. However,
relatedness analysis showed relatedness values >0.5 indicating kinship
in these two families (Table S1 and Supplemental Data). Moreover,
haplotype analysis using WES data showed shared homozygosity
of approximately 4.2 Mb between single nucleotide polymorphisms
rs17222536 and rs2279017 on chromosome 3 in all affected individ-
uals of families PN556 and PN878. This region contains seven genes
(i.e., SLC6A1, TAMM41, TSEN2, RAF1, CAND2, NUP210,andXPC;
Table S1). We excluded variations in all genes but TSEN2 based on their
relatively high MAFs and high ratio of homozygosities in the Genome
Aggregation Database (gnomAD; https://gnomad.broadinstitute.org) as
well as in slico analyses with Mutation Taster, which predicted them to
be polymorphisms (Table S1). Therefore, we considered the variation
FIGURE 2 Pathological findings of kidney biopsy. Representative images of kidney biopsies of individual PN556-II2. Glomeruli show
mesangiolysis (A, B), glomerular basement membrane wrinkling and duplication (B, arrows), and segmental sclerosis (C, red arrow). Hyperplastic
and hyalin changes in arterioles (C, bottom left, black arrows) and myxoid intimal thickening in the arterial wall are also visible (D, arrow) (AC
PAS 200; D HE 200) [Colour figure can be viewed at wileyonlinelibrary.com]
4CANPOLAT ET AL.
TABLE 1 Summary of the clinical features of the six affected individuals from four pedigrees included in the study
Case #1 Case #2 Case #3 Case #4 Case #5 Case #6
Identifier PN556-II1 PN556-II2 PN556-II3 PN878-II2 PN1093-II1 PN1939-II3
Sex Male Female Female Female Male Male
Chromosome
analysis
46, XY 46, XX NA NA NA 46, XY
Birth
characteristics
Term, SGA
(BW 2000 g)
Term, AGA
(BW 3100 g)
Term, SGA
(BW 2200 g)
Term, SGA
(BW 2600 g)
Term, AGA
(BW 3400 g)
Term, SGA
(BW 1700 g)
Consanguinity ++++++
Clinical characteristics at the presentation
Age (year) 5 4 3/12 2.5 6 4 7/12 10 9/12
Height (cm)
/ SDS
97/2.96 90/3.21 85/1.97 102/2.78 110/0.39 133/1.99
Weight (kg) /
SDS for
height age
9.0/5.53 11/1.61 9.5/1.93 11.4/3.81 16/0.89 20/3.79
BMI (kg/m
2
)/
SDS for
height age
9.6/6.50 13.6/2.02 13.1/3.16 10.9/5.11 13.2/2.02 11.3/4.14
Head
circumference
(cm) SDS
for age
45/4.41 45/3.62 44.5/2.7 44.5/4.61 50.3/2.39 NA
Presentation
symptoms
and signs
TMA Growth failure and
proteinuria
Growth failure
and positive
family history
TMA TMA TMA
Dysmorphic features
Microcephaly +++++
Triangular
facies
+++
Upslanting
palpebral
fissures
+
Retro-
micrognathia
+Severe +Mild ++Severe +
Cone-shaped
and sparse
teeth
++++++
Deeply
set eyes
++++++
Sparse and
broad
eyebrows
++++++
Gray sclera ++++++
High nasal
bridge
++++++
Long-
narrow nose
++++
Bulbous nose ++
Deep and
shrill voice
++++++
Right frontal
upsweep
of hair
++++++
Long philtrum ++++++
(Continues)
CANPOLAT ET AL.5
TABLE 1 (Continued)
Case #1 Case #2 Case #3 Case #4 Case #5 Case #6
Thin upper lip ++++++
High and
broad
forehead
++++++
Prominent
supraorbital
ridge
++++++
Skin N N N Hyperkeratosis, thin
prominent
superficial vessel
N Hyperkeratosis, thin
prominent
superficial vessel
Renal involvement
Severe
hypertension
++++++
Proteinuria ++++++
Biopsy
proven TMA
++NA NA NA NA
Chronic
kidney
disease
++++++
End stage
kidney
disease
(ESKD)
++++++
Age at
development
of
ESKD (year)
9 9.5 8 8/12 8 10/12 4 10/12 11 5/12
Renal
replacement
therapy
PD PD PD, KTx HD, KTx HD
Extrarenal involvements
Hematological MAHA
thrombocytopenia
MAHA
thrombocytopenia
MAHA MAHA
thrombocytopenia
MAHA
thrombocytopenia
MAHA
thrombocytopenia
Neurological
Intellectual
disability
(IQ score)
78 NA 59 (WISC-R) 69 (WISC-R) NA 82 (WISC-R)
Language and
motor skills
Mild delay Normal Mild delay Normal Normal Normal
Seizures +
Cardiac LV hypertrophy,
reduced LV
systolic function,
mild MR, LVNC
LV hypertrophy,
mild MR
LV hypertrophy,
reduced LV
systolic
function, mild
to moderate
MR
LV hypertrophy
reduced LV
systolic function,
LVNC
Reduced LV systolic
function,
moderate MR
Reduced LV systolic
function, dilated
cardiomyopathy
Pulmonary Recurrent
pneumonia and
acute respiratory
distress
Acute respiratory
distress
Pulmonary
edema
Acute respiratory
distress
Endocrine Partial GH
deficiency
Normal Hypothyroidism Low levels of ACTH,
somatomedin-C,
LH and FSH
NA NA
6CANPOLAT ET AL.
detected in TSEN2 as responsible for the phenotype. This variation was
not present in our in-house database composed of 187 WES and
39 whole genome sequencing data from Turkish individuals without
renal phenotype. Later, we identified from our large aHUS cohort two
more individuals with aHUS, and similar craniofacial abnormalities from
two consanguineous families (i.e., PN1093 and PN1939 in Figure S1
and Figure 1) in which disease-causing variations in complement and
other relevant genes had been previously excluded (Table 1). Sanger
sequencing of the 10 coding exons of TSEN2 identified the same
intronic variant in both individuals (Figure S4C,D), indicating a founder
effect and strongly supporting the causal role of the variant in deter-
mining the observed phenotype.
Cranial magnetic resonance imaging (MRI) of the six patients
showed distinctive radiological abnormalities of the central nervous
system, including hypoplasia of the anterior pituitary gland with pre-
served neurohypophysis and thin corpus callosum (Figure S5). Each
patient's MRI findings are presented in the Supplemental Data.
3.2 |The intronic variant c.914-5T>A affects
TSEN2 mRNA splicing
The c.914-5T>A change is localized within the pyrimidine-rich region
of intron 9 that is normally not evolutionary conserved. However, in
the TSEN2 intron 9 the Tand the surrounding bases are highly con-
served in vertebrates (Figure S6). The c.914-5T>A change was also
predicted to affect the splicing efficiency by MaxEntScan
22
(score
decrease of 31% from 11.34 to 7.71), to be deleterious by Combined
TABLE 1 (Continued)
Case #1 Case #2 Case #3 Case #4 Case #5 Case #6
Eye Hypertensive
retinopathy
Posterior
embryotoxon and
hypertensive
retinopathy
Hypertensive
retinopathy
NNA
Radiological findings
Skeletal
abnormalities
Decrease in bone
density
Cranial MRI Thin
adenohypophysis,
thin corpus
callosum with a
short
anteroposterior
diameter
hyperintensity of
the bilateral basal
ganglia suggesting
calcification,
possible cortical
malformation
Large sella hosting
very thin pituitary
gland and thin
corpus callosum
coarse anterior
temporal cortices
Relatively small
pons, Very thin
pituitary gland,
Thin corpus
callosum,
Possible
cortical
malformation
Very thin band of
anterior pituitary
gland and slight
hyperintensity of
the basal ganglion,
Possible cortical
malformation
Small anterior
pituitary
hyperintensity of
the basal ganglion
periventricular
hyperintense
lesions prominent
posteriorly
Thin
adenohypophysis,
diffuse subtle
hyperintensity in
the lentiform
nuclei on FLAIR
image
Outcome Died (at the age of
11.5 years)
Died (at the age of
10 years)
Alive on PD (at
the age of
9 years)
Alive with a
functioning
transplanted
kidney (at the age
12 years)
Alive with a
functioning
transplanted
kidney (at the age
10 years)
Died (at the age of
12 9/12 years)
Clinical characteristics at the last visit
Age (year) 11.5 10 9 12 10 3/12 12 4/12
Height (cm)/SDS 110/5.45 108/4.59 107/4.32 114/6.08 126/2.22 133/2.72
Weight (kg)/SDS
for height age
12.4/3.30 12.5/3.26 12.5/3.84 17.7/1.43 24/2.03 20/4.04
BMI (kg/m
2
)/
SDS for height
age
10.3/6.17 10.7/5.28 10.9/4.97 13.6/1.4 15.1/1.12 11.3/4.17
Head
circumference
(cm)/SDS
47/4.8 49/2.54 45.5/4.64 47/4.86 50.5/2.18 52.5/1.61
Abbreviations: ACTH, adrenocorticotrophic hormone; AGA, appropriate for gestational age; BMI, body mass index; BW, body weight; FLAIR, Fluid-
Attenuated-Inversion-Recovery; FSH, follicle stimulating hormone; GH, growth hormone; HD, hemodialysis; KTx, kidney transplantation; LH, luteinizing
hormone; LV, left ventricle; LVNC, left ventricle non-compaction; MRI, magnetic resonance imaging; NA, not available; PD, peritoneal dialysis; SDS,
standard deviation score; SGA, small for gestational age; TMA, thrombotic microangiopathy.
CANPOLAT ET AL.7
Annotation Dependent Depletion (CADD),
26
and to be disease-
causing by Mutation Taster. We reasoned that, since the c.914-5T is
followed by a c.914-4G, the transition to A may give rise to an alter-
native AG acceptor splice site three base pairs before the canonical
one, which could interfere with the normal splicing process. In silico
splice-site analysis using NetGene2
23
supported this hypothesis
(Figure S7). We, therefore, performed Sanger sequencing of cDNA
from the patients. Detailed scrutiny of the chromatogram showed the
presence of two transcripts: one in which exon 9 is normally spliced
to exon 10, and the other in which exon 10 is skipped, and exon 9 is
spliced to exon 11 (Figure 3). Furthermore, we also noticed the pres-
ence of a putative third transcript with a very low-intensity sequenc-
ing signal. For this reason, to further characterize the splicing defect,
we performed a minigene-based splicing assay (Figure S2AC). The
results of the assay showed the coexistence of transcripts that are
normally spliced from exon 9 to exon 10, but also a splicing variant in
which exon 9 is spliced to the newly created alternative splice site
(Figure S2D), and a transcript in which exon 9 is spliced to exon 11 (Sup-
plementary Figure S2E). The splicing to the alternative splicing site
givesrisetoaTSEN2 transcript with an additional in-frame codon
(CAG) between exon 9 and exon 10 that results in the inclusion of two
extra amino acids, a proline and a valine, in the protein (Figure S8),
whereas the splicing to exon 11 results in the skipping of exon
10, which is highly conserved in the evolution (Figure S9). Collectively,
our results indicate that the intronic change c.914-5T>A results in the
creation of alternative splice site that stochastically competes with the
canonical one giving rise to a protein in which exon 10 is skipped or
another protein with two additional amino acids. These results also sug-
gest that the variant results in only partial loss-of-function of TSEN2 as
a complete loss of function of any of the components of the TSEN
complex is not expected to be compatible with life.
13
3.3 |TSEN2 exon 10 skipping in zebrafish causes
vascular and cranial anomalies
Morphological analysis of the splice-block knockdown larva
(tsen2sbMO) presented blood cells pooling,a phenotype compatible
with vascular anomalies (Figure 4). The tsen2sbMO knockdown was
performed in a transgenic zebrafish line that expresses enhanced GFP
under the control of endothelial cell-specific fli1 promoter Tg(fli1:
EGFP)y
127
and that allows the visualization of the vasculature in live
embryos. We observed constriction of the Common Cardinal Vein,
enlarged pericardium and extra numbers of vessel branching, and bra-
nches with no connections. We also observed smaller anterior cra-
nium, forebrain and mid-brain, and kidney cysts and edema at 7dpf
(Table S2).
3.4 |tRNA composition is altered in the presence
of the TSEN2 c.914-5T>A intronic variant
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.
28,29
While no published in vivo data are
available for individuals with mutations in one of the four TSEN
components of the spliceosome, depletion of mature RNAs and accu-
mulation of unspliced RNAs have been documented in patient-derived
induced neurons.
10
To establish if the c.914-5T>A variant affects the
tRNAs biogenesis, we performed bulk sequencing of the RNA
extracted from peripheral blood cells of four affected individuals and
one of their heterozygous mothers (PN878-I2) and compared them to
healthy children of similar ages. Four affected children shared five
abnormal tRNA transcripts that were absent in the heterozygous
mother and the healthy children (Table S3). These tRNAs were
searched in the annotation files from GENCODE
30
and FASTA files
for High confidence tRNA sequencesfrom GtRNAdb.
31
The tRNAs
with gene ID 26988, 25052, 22262 and 13036 were found both in
the FASTA file from GtRNAdb and GFF3 file from GENCODE,
whereas tRNA with gene ID 8789 was found in GtRNAdb only. Inter-
estingly, some of these tRNAs, such as tRNA
TyrGTA
represent
isodecoders as intron-containing precursors for which splicing has
been reported to be essential for their production.
3
Pathway enrichment analysis of downregulated genes shared by
all four children identified 20 pathways in total; Synaptic vesicle
cycle(pathway ID: hsa04721), Glutamergic synapse(pathway ID:
hsa04724) and Hippo signaling pathway(pathway ID: hsa04392) as
the top three most significantly enriched terms with high-fold value
and lowest p-values (Figure 5). On the other hand, pathway enrich-
ment analysis of upregulated genes from differential expression analy-
sis from the mother resulted in 226 pathways, whereas 71 pathways
were observed in her child. Ten pathways with the highest signifi-
cance from mother and her child were compared. We showed that
the mother and her child share the Spliceosome(pathway ID:
FIGURE 3 Abnormal splicing of TSEN2 exon 9 to exon 10.
Chromatogram of the cDNA of the homozygous affected individual
PN1093 II-1 corresponding to the exon 9exon 10 junction showing
three different splicing events: normal splicing to exon 10; splicing
that occurs to exon 11; and splicing that seems to occur to the newly
created alternative splice site (asterisks) [Colour figure can be viewed
at wileyonlinelibrary.com]
8CANPOLAT ET AL.
hsa03040) and the Ribosome(pathway ID: hsa03010) terms in their
top 10 pathways with the highest significance (Figure 6).
4|DISCUSSION
Here we report a homozygous intronic sequence variant in TSEN2
that results in an undescribed syndrome characterized by multiple
dysmorphic features, intellectual retardation, radiological abnor-
malities of the central nervous system, and aHUS that leads to
ESKD.Thismutationsegregatedwiththephenotypeinsixaffected
individuals from four consanguineous families of Turkish descent,
which identifies this variant as a founder mutation as confirmed by
relatedness and haplotype analyses. The mutation results in a
complex combination of transcripts of the TSEN2 mRNA, with the
retention of a normally spliced transcript, transcripts in which exon
10 is skipped, and transcripts that contain two extra amino acids,
all of which may contribute to the determination of the observed
phenotype.WedemonstratedfiveabnormaltRNAsinallaffected
individuals that are absent in the healthy individuals clearly indicat-
ing that the mutation impairs the function of the TSEN complex
and modifies the tRNA inventory.
TSEN2 constitutes the catalytic subunit of the tRNA splicing com-
plex and is conserved from Archea.
32
Complete loss of function in any
of the components of the tRNA splicing complex is thought to be
incompatible with life. On the other hand, bi-allelic hypomorphic
mutations in any of the four components of the TSEN complex,
including TSEN2, have been associated with a spectrum of clinical
FIGURE 4 (AC0) Splice block morpholino in fli1:EGFP at 30 h postfertilization. A. Lateral view of uninjected control embryo. (A') Lateral view
of MO-injected embryo. White arrows indicate blood pooling (in A'). (B) Lateral view and (C) Dorsal view of fli1:EGFP uninjected control embryo.
(B0) Lateral view and (C0) dorsal view of fli1:EGFP MO-injected embryo focused on the vasculature including the Duct of Cuvier (arrows), which
will become the future common cardinal vein. The vascular defects are not due to developmental delay as the defect persists through 7 days. (D
I) Splice block morpholino in fli1:EGFP at 7 days postfertilization. Lateral brightfield microscopy images of (D) uninjected control and (E) MO-
injected embryo. Gray arrow notes the enlarged pericardium and white arrows the smaller anterior cranial/forebrain and mid-brain. Boxes in
brightfield images indicate the area shown in immunofluorescence microscopy images in (F) and (G). (FG) Lateral immunofluorescence
microscopy images of larvae in (D) and (E). (HI) Lateral immunofluorescence microscopy images of two different fli1:EGFP MO-injected embryos,
showing extra numbers of vessel branching and even branches with no connections [Colour figure can be viewed at wileyonlinelibrary.com]
CANPOLAT ET AL.9
manifestations, ranging from cerebral malformations to pontocerebellar
hypoplasia, that partially overlap with the phenotype presented by our
patients: while hypoplasia of the pons was observed in only one of the
affected individuals reported here, all the individuals presented hypo-
plasia of the anterior pituitary gland and corpus callosum, in addition to
aHUS, all features that have not been described in other forms of
pontocerebellar hypoplasia.
The tRNA splicing machinery is involved in the processing of
other RNA species, including the processing of mRNAs and rRNAs,
thereby directly affecting vital pathways in the cell.
4,33,34
The ques-
tions that remain unanswered are why mutations in the genes
encoding four components of the TSEN complex, which is ubiqui-
tously expressed, lead to isolated PCH, how these mutations affect
only specific neurons in the central nervous system and what are
the mechanisms behind the brain malformations and neuronal
damage associated with disruption of the TSEN complex. From the
present study one more also arises: why does this particular variant
result in a very distinct phenotype? As the TSEN complex partici-
pates to the biogenesis of tRNAs,
28
the defective complex is
expected not only to modify the composition of mature tRNAs that
are critical for protein synthesis but also to cause the aberrant
accumulation of pre-tRNAs at levels that could be toxic in select
cells.
15
Indeed, an adequate supply of tRNAs is essential for protein
biosynthesis in neurons that require rapid and localized protein
synthesis for synaptic plasticity, which needs to be coordinated
with the transport of the translational machinery, mRNAs, and
tRNAs.
35,36
Moreover, transfer RNA-derived fragments (tRFs) have
been identified as small noncoding RNAs contributing to gene regu-
lation, silencing and translational control, and that can be involved
in progressive motor neuron loss.
37
Thus, while a reduction in
TSEN activity can be well tolerated in most cell types, it could have
drastic effects in neurons as these cells may be particularly suscep-
tible to subtle translation and consequent proteostasis defects.
38
This mechanism has been suggested to explain neuronal diseases
that result from defects in many mRNA and tRNA processing path-
ways.
35
Studies also showed that changes in tRNA levels could
affect the local speed of mRNA translation in a tissue-specific man-
ner based on the availability of cognate tRNAs, at the same time
affecting protein levels.
35,39
tRNAs also function as signaling molecules in the regulation of
numerous metabolic and cellular processes, as stress sensors, and in
tRNA-dependent biosynthetic pathways, and thus are expected to
impair many cellular, molecular, and signaling events.
40
Our pathway
enrichment analyses indeed support all these possibilities, identifying
spliceosome,”“ribosome,”“synaptic vesicle cycleand glutamergic
synapsesas the most highly relevant terms.
In addition, we also identified the Hippo signaling pathwayas a
relevant ontogenetic term that is downregulated in the homozygous
individuals compared to the healthy individuals. The Hippo-YAP (Yes-
associated protein) signaling plays a significant role in the craniofacial
and dental development of humans
41
and may be responsible for the
craniofacial and significant dental abnormalities observed in our
patients.
Finally, the presence of a vascular phenotype (i.e., TMA) in indi-
viduals carrying the c.914-5T>A mutation is arguably the most intrigu-
ing finding of our study. The link between tRNAs and angiogenesis is
not entirely new: tRFs have not only been associated with neuronal
damage but also with angiogenesis.
42,43
The ribonucleases angiogenin
was demonstrated to be essential in blood vessel development,
growth, and angiogenesis
44,45
; full-length tRNAs were shown to be
angiogenin substrates in vitro,
46
and angiogenin-mediated accumula-
tion of stress-induced tRFs correlated with pro-survival signaling of
endothelial cells under stress.
42
Reports also exist of anti-angiogenic
effects of tRFs underlining the complexity between the tRNAs and
endothelial cells biology.
43
Other than corroborating the participation
of the tRNAs in the endothelial pathophysiology, our data also sug-
gest the involvement of the Hippo-YAP signaling in this syndrome.
47
YAP is highly expressed in aged-vascular tissue and inhibition of YAP
FIGURE 5 Illustration of top 10 most significantly enriched
pathways identified by functional enrichment analysis of
downregulated genes shared by four children. The X-axis shows the
fold enrichment; y-axis shows the name of pathways. The darkness of
the color of the nodes represents the significance of the p-value. The
size of the node represents the number of differentially expressed
genes involved in the specified pathway. The figure was obtained
using PathFindR from the R package [Colour figure can be viewed at
wileyonlinelibrary.com]
10 CANPOLAT ET AL.
activity reduces senescence while its overexpression enhances senes-
cence in both vascular endothelial cells and vascular tissues.
48
More-
over, modulation of YAP localization (i.e., cytoplasmic or nuclear) was
shown to delay senescence of vascular endothelial cells through sub-
cellular YAP localization.
49
Further studies will be needed to clarify
the mechanisms by which the Hippo signaling pathway affects endo-
thelial homeostasis.
In conclusion, our data link a pre-tRNA splicing defect to a novel
syndrome characterized by microcephaly, craniofacial malformations,
cognitive retardation, and aHUS, which we propose to name with the
acronym of TRACKsyndrome (TSEN2 Related Atypical hemolytic
uremic syndrome, Craniofacial malformations, Kidney failure). Our
data also clearly indicate the existence of a link between tRNA biology
and vascular homeostasis. Further studies, possibly aimed to analyze
neuron- and endothelium-specific mRNAs will be required to uncover
uncharacterized functions of TSEN2 and the mechanisms that lead to
CNS and vascular endothelial defects.
ACKNOWLEDGMENTS
The authors thank all the families for partaking in this study and the
referring clinicians for their generous help; Berk Gürdamar from
Acıbadem University for his help with the bulk RNA-sequencing ana-
lyses; Professor Nurten Akarsu from the Department of Medical
Genetics, Faculty of Medicine, Hacettepe University for her critical
contribution to the haplotype analyses and interpretation; Tugce
Bozkurt from Acibadem University for her contribution to the related-
ness analyses.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
AUTHOR CONTRIBUTIONS
Patient recruitment and evaluation: Nur Canpolat, Seha Saygili,
Tugba Tastemel Ozturk, Lale Sever, Rezan Topaloglu, Salim Cal-
iskan, Fatih Ozaltin. Research formulation and study design:NurCan-
polat, Salim Caliskan, Massimo Attanasio, Fatih Ozaltin. Data
acquisition: Nur Canpolat, Dingxiao Liu, Trudi A. Westfall, Qiong
Ding, Bartley J. Brown, Terry A. Braun, Beyhan Tuysuz, Seha
Saygili, Yasemin Ozluk, Lale Sever, Rezan Topaloglu, Tugba
Tastemel Ozturk, Kader K. Oguz, Fatih Ozaltin. Genetic analyses:
Emine Atayar, Fatih Ozaltin. Data analysis/interpretation:NazliSila
Kara, Diane Slusarski, Kader K. Oguz, Osman Ugur Sezerman, Mas-
simo Attanasio, Fatih Ozaltin.
ETHICS APPROVAL
All individuals participating in this study were enrolled after obtaining
signed informed consent, per human subject research protocols
approved by the Hacettepe University in Ankara (HEK12/112-113) and
by The Ethical Committee of Istanbul University-Cerrahpasa, Cerrahpasa
Medical Faculty (No: 02-2 911 316). Explicit permission was obtained to
publish the photographs of the individuals shown in the figures.
PEER REVIEW
The peer review history for this article is available at https://publons.
com/publon/10.1111/cge.14105.
FIGURE 6 (A) Illustration of top 10 most significantly enriched pathways identified by functional enrichment analysis of genes differentially
expressed and upregulated in the heterozygous mother (i.e., PN878-I2). The X-axis shows the fold enrichment; the y-axis shows the name of
pathways. The darkness of the color of the nodes represents the significance of the p-value. The size of the node represents the number of
differentially expressed genes involved in the specified pathway. The figure was obtained by PathFindR from the R package. (B) Illustration of top
10 most significantly enriched pathways identified by functional enrichment analysis of genes differentially expressed and upregulated in the
homozygous child (i.e., PN878-II2). The X-axis shows the fold enrichment, the y-axis shows the name of pathways. The darkness of the color of
the nodes represents the significance of the p-value. The size of the node represents the number of differentially expressed genes involved in the
specified pathway. The figure was obtained by PathFindR from the R package [Colour figure can be viewed at wileyonlinelibrary.com]
CANPOLAT ET AL.11
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available within
the paper or from the corresponding authors upon reasonable
request.
ORCID
Nur Canpolat https://orcid.org/0000-0002-3420-9756
Dingxiao Liu https://orcid.org/0000-0002-7581-2527
Emine Atayar https://orcid.org/0000-0001-8423-3431
Seha Saygili https://orcid.org/0000-0002-2424-6959
Nazli Sila Kara https://orcid.org/0000-0002-6765-2543
Diane Slusarski https://orcid.org/0000-0003-3793-8636
Kader Karli Oguz https://orcid.org/0000-0002-3385-4665
Yasemin Ozluk https://orcid.org/0000-0002-7191-0488
Beyhan Tuysuz https://orcid.org/0000-0002-9620-5021
Tugba Tastemel Ozturk https://orcid.org/0000-0003-3419-1661
Lale Sever https://orcid.org/0000-0002-5918-6204
Osman Ugur Sezerman https://orcid.org/0000-0003-0905-6783
Rezan Topaloglu https://orcid.org/0000-0002-6423-0927
Salim Caliskan https://orcid.org/0000-0002-3316-8032
Massimo Attanasio https://orcid.org/0000-0002-1278-3650
Fatih Ozaltin https://orcid.org/0000-0003-1194-0164
REFERENCES
1. Parisien M, Wang X, Pan T. Diversity of human tRNA genes from the
1000-genomes project. RNA Biol. 2013;10:1853-1867.
2. Chan PP, Lowe TM. GtRNAdb 2.0: an expanded database of transfer
RNA genes identified in complete and draft genomes. Nucleic Acids
Res. 2016;44:D184-D189.
3. Gogakos T, Brown M, Garzia A, Meyer C, Hafner M, Tuschl T. Charac-
terizing expression and processing of precursor and mature human
tRNAs by hydro-tRNAseq and PAR-CLIP. Cell Rep. 2017;20:1463-
1475.
4. Paushkin SV, Patel M, Furia BS, Peltz SW, Trotta CR. Identification of
a Human Endonuclease Complex Reveals a Link between tRNA Splic-
ing and Pre-mRNA 30End Formation. Cell. 2004;117(3):311-321.
https://doi.org/10.1016/s0092-8674(04)00342-3
5. Popow J, Englert M, Weitzer S, et al. HSPC117 is the essential sub-
unit of a human tRNA splicing ligase complex. Science. 2011;331:
760-764.
6. Rauhut R, Green PR, Abelson J. Yeast tRNA-splicing endonuclease is
a heterotrimeric enzyme. J Biol Chem. 1990;265:18180-18184.
7. Weitzer S, Martinez J. The human RNA kinase hClp1 is active on 30
transfer RNA exons and short interfering RNAs. Nature. 2007;447:
222-226.
8. Hanada T, Weitzer S, Mair B, et al. CLP1 links tRNA metabolism to
progressive motor-neuron loss. Nature. 2013;495:474-480.
9. Karaca E, Weitzer S, Pehlivan D, et al. Human CLP1 mutations alter
tRNA biogenesis, affecting both peripheral and central nervous sys-
tem function. Cell. 2014;157:636-650.
10. Schaffer AE, Eggens VR, Caglayan AO, et al. CLP1 founder mutation
links tRNA splicing and maturation to cerebellar development and
neurodegeneration. Cell. 2014;157:651-663.
11. Bierhals T, Korenke GC, Uyanik G, Kutsche K. Pontocerebellar hypo-
plasia type 2 and TSEN2: review of the literature and two novel
mutations. Eur J Med Genet. 2013;56:325-330.
12. Breuss MW, Sultan T, James KN, et al. Autosomal-recessive muta-
tions in the tRNA splicing endonuclease subunit TSEN15 cause
pontocerebellar hypoplasia and progressive microcephaly. Am J Hum
Genet. 2016;99:228-235.
13. BuddeBS,NamavarY,BarthPG,etal.tRNAsplicingendonucleasemuta-
tions cause pontocerebellar hypoplasia. Nat Genet. 2008;40:1113-1118.
14. Namavar Y, Barth PG, Kasher PR, et al. Clinical, neuroradiological and
genetic findings in pontocerebellar hypoplasia. Brain. 2011;134:143-156.
15. Sekulovski S, Devant P, Panizza S, et al. Assembly defects of human
tRNA splicing endonuclease contribute to impaired pre-tRNA
processing in pontocerebellar hypoplasia. Nat Commun. 2021;12:5610.
16. Fakhouri F, Zuber J, Frémeaux-Bacchi V, Loirat C. Haemolytic
uraemic syndrome. Lancet. 2017;390:681-696.
17. Ozaltin F, Li B, Rauhauser A, et al. DGKE variants cause a glomerular
microangiopathy that mimics membranoproliferative GN. J Am Soc
Nephrol. 2013;24:377-384.
18. Lemaire M, Frémeaux-Bacchi V, Schaefer F, et al. Recessive muta-
tions in DGKE cause atypical hemolytic-uremic syndrome. Nat Genet.
2013;45:531-536.
19. Noris M, Caprioli J, Bresin E, et al. Relative role of genetic comple-
ment abnormalities in sporadic and familial aHUS and their impact on
clinical phenotype. Clin J Am Soc Nephrol. 2010;5:1844-1859.
20. Fremeaux-Bacchi V, Fakhouri F, Garnier A, et al. Genetics and outcome
of atypical hemolytic uremic syndrome: a nationwide French series
comparing children and adults. Clin J Am Soc Nephrol. 2013;8:554-562.
21. Robinson JT, Thorvaldsd
ottir H, Winckler W, et al. Integrative geno-
mics viewer. Nat Biotechnol. 2011;29:24-26.
22. Yeo G, Burge CB. Maximum entropy modeling of short sequence
motifs with applications to RNA splicing signals. J Comput Biol. 2004;
11:377-394.
23. Hebsgaard SM, Korning PG, Tolstrup N, Engelbrecht J, Rouzé P,
Brunak S. Splice site prediction in Arabidopsis thaliana pre-mRNA by
combining local and global sequence information. Nucleic Acids Res.
1996;24:3439-3452.
24. Purcell S, Neale B, Todd-Brown K, et al. PLINK: a tool set for whole-
genome association and population-based linkage analyses.
Am J Hum Genet. 2007;81:559-575.
25. Pedersen BS, Quinlan AR. Who's who? Detecting and resolving sam-
ple anomalies in human DNA sequencing studies with Peddy.
Am J Hum Genet. 2017;100:406-413.
26. Rentzsch P, Witten D, Cooper GM, Shendure J, Kircher M. CADD:
predicting the deleteriousness of variants throughout the human
genome. Nucleic Acids Res. 2019;47:D886-d894.
27. Lawson ND, Weinstein BM. In vivo imaging of embryonic vascular
development using transgenic zebrafish. Dev Biol. 2002;248:307-318.
28. Trotta CR, Miao F, Arn EA, et al. The yeast tRNA splicing endonucle-
ase: a tetrameric enzyme with two active site subunits homologous
to the archaeal tRNA endonucleases. Cell. 1997;89:849-858.
29. Lowe TM, Eddy SR. tRNAscan-SE: a program for improved detection
of transfer RNA genes in genomic sequence. Nucleic Acids Res. 1997;
25:955-964.
30. Harrow J, Frankish A, Gonzalez JM, et al. GENCODE: the reference
human genome annotation for The ENCODE project. Genome Res.
2012;22:1760-1774.
31. Liao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose
program for assigning sequence reads to genomic features. Bioinfor-
matics. 2014;30:923-930.
32. Tocchini-Valentini GD, Fruscoloni P, Tocchini-Valentini GP. Structure, func-
tion, and evolution of the tRNA endonucleases of archaea: an example of
subfunctionalization. Proc Natl Acad Sci U S A. 2005;102:8933-8938.
33. Volta V, Ceci M, Emery B, et al. Sen34p depletion blocks tRNA splic-
ing in vivo and delays rRNA processing. Biochem Biophys Res Commun.
2005;337:89-94.
34. Dhungel N, Hopper AK. Beyond tRNA cleavage: novel essential func-
tion for yeast tRNA splicing endonuclease unrelated to tRNA
processing. Genes Dev. 2012;26:503-514.
12 CANPOLAT ET AL.
35. Kirchner S, Ignatova Z. Emerging roles of tRNA in adaptive translation,
signalling dynamics and disease. Nat Rev Genet. 2015;16:98-112.
36. Kirchner S, Cai Z, Rauscher R, et al. Alteration of protein function by
a silent polymorphism linked to tRNA abundance. PLoS Biol. 2017;15:
e2000779.
37. Anderson P, Ivanov P. tRNA fragments in human health and disease.
FEBS Lett. 2014;588:4297-4304.
38. Wilusz JE. Controlling translation via modulation of tRNA levels.
Wiley Interdiscip Rev RNA. 2015;6:453-470.
39. Qian W, Yang JR, Pearson NM, Maclean C, Zhang J. Balanced codon
usage optimizes eukaryotic translational efficiency. PLoS Genet. 2012;
8:e1002603.
40. Raina M, Ibba M. tRNAs as regulators of biological processes. Front
Genet. 2014;5:171.
41. Wong JS, Meliambro K, Ray J, Campbell KN. Hippo signaling in the
kidney: the good and the bad. Am J Physiol Renal Physiol. 2016;311:
F241-F248.
42. Ivanov P, Emara MM, Villen J, Gygi SP, Anderson P. Angiogenin-induced
tRNA fragments inhibit translation initiation. Mol Cell. 2011;43:613-623.
43. Li Q, Hu B, Hu GW, et al. tRNA-derived small non-coding RNAs in
response to ischemia inhibit angiogenesis. Sci Rep. 2016;6:20850.
44. Shapiro R, Vallee BL. Human placental ribonuclease inhibitor abol-
ishes both angiogenic and ribonucleolytic activities of angiogenin.
Proc Natl Acad Sci U S A. 1987;84:2238-2241.
45. Shapiro R, Vallee BL. Site-directed mutagenesis of histidine-13 and
histidine-114 of human angiogenin. Alanine derivatives inhibit -
angiogenin-induced angiogenesis. Biochemistry. 1989;28:7401-7408.
46. Saxena SK, Rybak SM, Davey RT Jr, Youle RJ, Ackerman EJ.
Angiogenin is a cytotoxic, tRNA-specific ribonuclease in the RNase a
superfamily. J Biol Chem. 1992;267:21982-21986.
47. Panciera T, Azzolin L, Cordenonsi M, Piccolo S. Mechanobiology of
YAP and TAZ in physiology and disease. Nat Rev Mol Cell Biol. 2017;
18:758-770.
48. Pan X, Wu B, Fan X, Xu G, Ou C, Chen M. YAP accelerates vascular
senescence via blocking autophagic flux and activating mTOR. J Cell
Mol Med. 2021;25:170-183.
49. Arisaka Y, Masuda H, Yoda T, Yui N. Delayed senescence of human
vascular endothelial cells by molecular mobility of supramolecular bio-
interfaces. Macromol Biosci. 2021;21:e2100216.
SUPPORTING INFORMATION
Additional supporting information may be found in the online version
of the article at the publisher's website.
How to cite this article: Canpolat N, Liu D, Atayar E, et al. A
splice site mutation in the TSEN2 causes a new syndrome with
craniofacial and central nervous system malformations, and
atypical hemolytic uremic syndrome. Clinical Genetics. 2022;
1-13. doi:10.1111/cge.14105
CANPOLAT ET AL.13
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