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Expanding the Clinical and Molecular Spectrum of HARS2-Perrault Syndrome:

Identification of a Novel Homozygous Missense Variant in the HARS2 gene

Article  in  Genetic Testing and Molecular Biomarkers · August 2021

DOI: 10.1089/gtmb.2021.0092

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 GENETIC TESTING AND MOLECULAR BIOMARKERS
Volume 25, Number 8, 2021
ª Mary Ann Liebert, Inc.
Pp. 528–539
DOI: 10.1089/gtmb.2021.0092

Expanding the Clinical and Molecular

Spectrum of HARS2-Perrault Syndrome:
Identification of a Novel Homozygous Missense
Variant in the HARS2 gene

Amal Souissi,1 Mariem Ben Said,1,i Fakher Frikha,2 Ines Elloumi,1

Saber Masmoudi,1 and Andre Megarbane3
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Background: Variants in the HARS2 gene have been reported to be associated with nonsyndromic hearing loss
(HL) and Perrault syndrome (PS), a rare recessive disorder marked by bilateral sensorineural HL and ovar-
ian dysgenesis. Given the low number of pathogenic variants described in the HARS2 gene, no geno-
type/phenotype correlations have been established between variants in this gene and the clinical data.
Materials and Methods: Whole blood was collected from four members of a Lebanese family with PS. An
affected woman was evaluated for HL by clinical examination and audiological tests. Primary ovarian failure
was analyzed according to age of primary or secondary amenorrhea, follicle stimulating hormone levels, and
pelvic ultrasound. The existence of neurological symptoms and other associated conditions was checked. To
identify the causative variant, we used a custom HaloPlexHS panel for next-generation sequencing of the coding
sequences of six genes implicated in this syndrome.
Results: We identified a novel homozygous HARS2 missense variant (c.260G>A; p.Arg87His), which is only
the second homozygous variant in the HARS2 gene identified to date worldwide. This variant is predicted to be
deleterious by multiple in silico analysis tools, moreover the Arg87 amino acid nearly is invariant among eight
species. Based on molecular modeling analysis, this variation is predicted to disturb the proper folding of
HARS2, which may reduce its aminoacylation efficiency. Clinical data are compared with the other cases
recorded in the literature to help gain further knowledge with regard to the phenotype.
Conclusion: Our results provide strong evidence corroborating the etiological association of this mutation with
the HARS2-PS phenotype. HARS2 variants need to be searched for in patients with early-onset bilateral
sensorineural HL and ovarian dysfunction in women so as to guarantee accurate endocrinological surveillance
and management to minimize secondary complications.

Keywords: Perrault syndrome, HARS2, variant, next-generation sequencing

Introduction involves sensory neuronal hearing loss (HL) in both sexes

and premature ovarian insufficiency (POI) or infertility in

H ARS2 corresponds to an enzyme belonging to the

class II family of aminoacyl-tRNA synthetases.
Aminoacyl-tRNA synthetases are a class of enzymes that
charge tRNAs with their cognate amino acids. Functioning
in the synthesis of histidyl-transfer RNA, the enzyme plays
an accessory role in the regulation of protein biosynthesis.
Pathogenic variants in the HARS2 gene are associated with
the pathogenesis of Perrault syndrome (PS) type 2, which
females (Pierce et al., 2011). Basically, fertility in affected
males is reported as normal (Newman et al., 1993; Pierce
et al., 2011). However, a few cases of male infertility such
as azoospermia have been reported in males (Demain et al.,
2017).
Biallelic pathogenic HARS2 variations prove to result in
a phenotype confined to sensorineural HL and POI with-
out neurological manifestations (Karstensen et al., 2020).

1
Laboratory of Molecular and Cellular Screening Processes, Center of Biotechnology of Sfax, University of Sfax, Sfax, Tunisia.
2
Faculty of Sciences of Sfax, University of Sfax, Sfax, Tunisia.
3
Department of Human Genetics, Gilbert and Rose-Marie Chagoury School of Medicine, Lebanese American University, Byblos, Lebanon.
i
ORCID ID (https://orcid.org/0000-0003-4098-4537).

528
 NOVEL HARS2 VARIANT IN PERRAULT SYNDROME
Neurologic features described in some individuals with
PS include learning difficulties and developmental delay,
cerebellar ataxia, and motor as well as sensory peripheral
neuropathy.
Basically, biallelic pathogenic variants in one of the six
genes CLPP, ERAL1, HARS2, HSD17B4, LARS2, and TWNK
are known to cause PS. Variants in HSD17B4 gene are as-
sociated with PS type 1. CLPP and ERAL1 have active roles
in the formation of the mitochondrial ribosome. Variants in
the CLPP gene are associated with PS type 3, while variants
in the ERAL1 gene are associated with PS type 6. HARS2
and LARS2 are significant in terms of the translation of
mitochondrial proteins. Variants in the HARS2 gene are as-
sociated with PS type 2, whereas variants in the LARS2 gene
are associated with PS type 4. TWNK maintains mitochon-
drial DNA and variants in the TWNK gene are associated
with PS type 5 (Newman et al., 1993).
Currently, these genes account for *40% of the causes
of PS, but the genetic bases of more than half the cases of
Downloaded by 41.228.57.218 from www.liebertpub.com at 08/23/21. For personal use only.
PS remain unclear (Lerat et al., 2016). Nineteen cases of
HARS2 variants spanning 10 families have been reported
(Pierce et al., 2011; Lerat et al., 2016; Demain et al., 2020;
Karstensen et al., 2020; Yu et al., 2020; Zou et al., 2020).
As far as our work is concerned, we report a novel HARS2
missense pathogenic variant with detailed clinical and mo-
lecular investigations of a patient with PS from a Lebanese
family. Depending on our molecular modeling analysis,
this variation may reduce the aminoacylation efficiency of
HARS2.
Materials and Methods
Subjects and sample preparation
Whole blood was collected from four members of a
Lebanese family with PS. DNA from all samples was extracted
using the phenol/chloroform standard method. The quantity of
the DNA was assessed with fluorometry-based Qubit dsDNA
HS and Qubit BR DNA Assay Kits according to the manu-
facturer’s instructions. Besides, all samples underwent a 1%
agarose-Tris-borate-EDTA blend gel so as to detect any deg-
radation. One affected woman had a clinical diagnosis of PS.
Pure-tone audiometry was performed for frequencies of 250,
500, 1000, 2000, 4000, and 8000 Hz. Primary ovarian failure
was analyzed according to age of primary or secondary
amenorrhea, follicle stimulating hormone (FSH) levels, and
pelvic ultrasound. The existence of neurological symptoms and
other associated conditions was checked. Informed consent was
obtained from all individual participants included in the study.
The ethics committee of Gilbert and Rose-Marie Chagoury
School of Medicine (Lebanon) approved this study.
Capture design
A custom gene panel was designed using a web application
from Agilent Technologies, the SureDesign (Agilent Tech-
nologies, Inc., Santa Clara, CA). The design was based on
GRCh37/hg19 reference sequences, with target sources ob-
tained from the RefSeq database. In this custom target en-
richment library design, all coding exons were targeted
involving 25 bp of the flanking intronic sequence of 36 HL
genes, including the 6 Perrault genes (HARS2, TWNK,
HSD17B4, LARS2, CLPP, and ERAL1).
529
Target capture sequencing
For target capture sequencing, libraries were prepared
according to the HaloPlexHS Target Enrichment System
(Version C1; Agilent Technologies, Inc.) as recently high-
lighted in Souissi et al. (2021). High-throughput sequencing
was carried out using Illumina MiSeq instrument (Illumina,
Inc., San Diego, CA). The instrument was set to generate
FASTQ files only, without adapter trimming. HaloPlexHS
index sequences were manually added through editing the
sample sheet.
Bioinformatic pipeline
Primary data analysis was conducted using MiSeq Repor-
ter to generate a pair of FASTQ files for each sample and for
adaptor trimming from all reads. Generated FASTQ files
were analyzed using SureCall NGS data analysis software
version 3.0.3.1 (Agilent Technologies, Inc.).
Variants suspected to be pathogenic were assessed through
Sanger sequencing on a 3100 ABI instrument (Applied Bio-
systems, Foster City, CA).
The allele frequency in the genome Aggregation Database
(gnomAD) was invested to evaluate variant’s frequency.
Furthermore, several other databases were used to predict the
severity of missense variants (Varsome, ClinVar, SIFT,
Provean, MutationTaster, BayesDel addAF, BayesDel noAF,
DEOGEN2, EIGEN, EIGEN PC, FATHMM-MKL,
FATHMM-XF, MVP).
3D structural analysis
Homology modeling. The Molecular Operating Environ-
ment MOE 2019 (MOE) software was used for homology
modeling, molecular dynamics (MD), and structure visuali-
zation. The models were stereochemically evaluated. Visu-
alization was performed with PyMOL version 0.99beta06.
The figures were generated by PyMOL program.
The structure model of the HARS2 was built, using as
template the crystal structure of human histidine-tRNA ligase
(HisRS) (PDB code: 4G85), to provide an insight into the
effect of the R87H mutation. The two proteins share an amino
acid identity of about 78%. The model of the HARS2 was
then subjected to molecular mechanics optimization using
OPLS-AA force field. Energy minimization (geometry opti-
mization) was performed until the gradient of 0.01 kcal/
(Å.mol) was reached. The root mean square deviations
(RMSDs) involving a-carbons between the template and the
optimized models were 1.299Å involving the 309 amino
acids (G62-Q396) (N-terminal domain) and 1.238Å involv-
ing the 108 amino acids (R397-S504) (C-terminal domain).
The Ramachandran plot statistics of the final HARS2 model,
determined using the PROCHECK program, showed that
96.23% of the residues were either in the most favored or in
the additional allowed regions. The structure of the HARS2wt
was also subjected to molecular mechanics optimization
using OPLS-AA force field to compare the two structures.
The RMSD involving a-carbons between the two optimized
models was 0.254Å implying the 453 amino acids. The dimer
structure model of mHARS2 and wtHARS2 was built.
MD simulations. The 3D structure models of mHARS2
and wtHARS2 dimer were used for the MD simulation study
with OPLS-AA/L all-atom force field. MD simulations were
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530
FIG. 1. (A) Pedigree and sequence analysis of a Perrault family. Affected individuals are marked in black. Sanger sequencing electropherograms of the variant in HARS2
gene are shown; (B) tonal audiogram on both sides in the patient; (C) Sequence comparison of the HARS2 orthologs. The ARG87 is marked in gray. Accession numbers of
the various protein sequences: NP_036340.1: Homo sapiens; XP_517980.3: Pan troglodytes; XP_002808484.1: Macaca mulatta; XP_535214.3: Canis lupus;
NP_001091602.1: Bos taurus; NP_542367.1: Mus musculus; NP_001014034.2: Rattus norvegicus; XP_001231932.2: Gallus gallus; XP_002944317.2: Xenopus tropicalis.
 NOVEL HARS2 VARIANT IN PERRAULT SYNDROME 531

carried out using Nose/Poincare/Anderson equations of motion Table 1. Interpretation of the Identified Variant
(NPA algorithm), GB/VI 1:80, cutoff [8,10], rigid water and Using Various Databases
light bond constrains, and a time step of 0.002 ps. After mini-
mization, the system heating, equilibration, and data sampling Gene HARS2
were carried out for the mHARS2 and wtHARS2 model dimer. Mutation c.260G>A;
The heating system was gradually increased from 0 to 310K p.Arg87His
and equilibrated for 100 ps, followed by 2 ns simulation for data Isoform NM_012208.4
sampling at 310K (37C), respectively. The RMSD values for Previous reports of the mutation Novel
the backbone atoms were used to predict the protein flexibility. dbSNP rs369075888
ACMG Likely
Thermostability study. To better understand the thermo- pathogenic
stability of the dimer, we used MD simulations. MD simula- Allele frequency in gnomAD (Global) 0.00001060
tions were carried out (2 ns) for the mHARS2 and wtHARS2 Allele frequency in gnomAD 0.00005012
models at 310K. The RMSD and root-mean-square fluctua- (East Asian)
tion values for the backbone atoms were used to understand ClinVar Not reported
SIFT Damaging
the response behavior. Provean Deleterious
Results MutationTaster Disease causing
BayesDel addAF Damaging
Clinical report BayesDel noAF Damaging
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A four-generation Lebanese family with PS was recruited for
the present study. This consanguineous family presented an
affected female proband (IV-1), two unaffected females, and
her consanguineous unaffected parents (Fig. 1A). A detailed
physical examination and an audiometric assessment were
carried out on the affected member. The patient was a 31-year-
old girl and was diagnosed with profound bilateral HL at birth
(Fig. 1B). Pelvic ultrasonography revealed streak gonads. FSH
was 95.42 IU/I. The patient had menarche at the age of 10 years.
At the age of 11 years, she demonstrated irregular menstrual
periods. Currently, she has amenorrhea and chronic fatigue
with balance problems. Her height is 160 cm and her weight is
47 kg. A thyroid hormone test demonstrated normal values of
TSH, T3 and T4. For cerebellar signs, a clinical examination
revealed the presence of ataxia and weak patellar reflexes.
HARS2 missense variant identified by custom gene
panel sequencing
The variant HARS2 p.(Arg87His) in patient IV-1 is present
as a heterozygous variant in three individuals of 282900 se-
quenced (minor allele frequency, 0.00001060) in gnomAD
and has never been detected on a homozygous state. This low
carrier frequency is consistent with a variant causative of
rare autosomal recessive disease.
Segregation analysis confirmed that this variant segregates
with the disease since it was present at the homozygous state
in the affected female (IV-1) and at the heterozygous state in
both unaffected parents. In addition, a wild-type state was
observed in the unaffected sibling IV-3. For the second un-
affected sibling IV-2, unfortunately, DNA was not available.
HARS2 p.(Arg87His) is also predicted to be deleterious by
multiple in silico analysis tools (Table 1). The residue HARS2
Arg87 is an almost invariant residue conserved in eight
species (Fig. 1C).
The structure model of the HARS2 shows a two-compact
domain. The structure has globular shape with an a/b archi-
tecture consisting of a central b-sheet surrounded by a-helical
layers (Fig. 2A). The Arg87His substitution is located at the
end of the helices a1 on Nt-domain. The dimer mHARS2 and
wtHARS2 structures share (70, 72) interchain interactions
and (640, 643) intrachain interactions, respectively. The
Arg87 makes an h-bond with Ser83 and an ionic-bond with
DEOGEN2 Damaging
EIGEN Pathogenic
EIGEN PC Pathogenic
FATHMM-MKL Damaging
FATHMM-XF Damaging
MVP Pathogenic
gnomAD, genome Aggregation Database.
Glu198 for the intrachain interactions in addition to an
h-bond with Asp434, Gly436, Lys438, and Ala439 for the
interchain interactions. However, His87 makes an h-bond
with Ser83 for the intrachain interactions and an h-bond with
Gly436, Lys438, and Ala439 for the interchain interactions.
The Asp434, Gly436, Lys438, and Ala439 are located be-
tween a13-a14 on the Ct-domain (Fig. 2B). In addition, the
two-dimensional RMSD plot, where the RMSD of every
conformation to all other conformations of a simulation is
shown, demonstrated that the conformational space sampled
by HARS2 in the simulations was larger than the wtHARS2
model at 310K, which was 1.750 versus 1.616Å (Fig. 2C).
This is correlated to the number of total interactions 710
(mHARS2) and 715 (wtHARS2).
Discussion
In this article, we report a PS patient in a Lebanese fam-
ily carrying a novel missense variant in exon3 of HARS2
[c.260G>A; p.(Arg87His)]. All the variants reported in the
HARS2 gene are scattered over 10 exons out of 13 and there
are no hotspot variant regions (Table 2). Since 2011, only 19
patients (10 women, 9 men) with variants in HARS2 were
reported (Pierce et al., 2011; Lerat et al., 2016; Demain et al.,
2020; Karstensen et al., 2020; Yu et al., 2020; Zou et al.,
2020) (Table 2). Demain et al. (2020) reported two families
with HARS2 variants associated with nonsyndromic HL.
In addition, Zou et al. (2020) described the implication
of HARS2 in one nonsyndromic Chinese Han deaf patient.
Furthermore, Yu et al. (2020) reported one family with non-
syndromic HL harboring variants in HARS2 gene, whereas
the other reported families suffer from PS.
The p.(Arg87His) variant identified in this work is mis-
sense and most of the previously reported variants are mis-
sense (Table 2). However this does not exclude the exception
 532 SOUISSI ET AL.
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FIG. 2. Cartoon representation of the model structure of HARS2. (A) The Nt-domain and the Ct-domain are indicated.
The Arg87His is shown as ball and sticks. (B) Secondary structure of the dimer. Residues Ser83, Glu198, Asp434, Gly436,
Lys438 and Ala439 involved in the interchain and intrachain interaction with Arg87His are shown as ball and sticks and
indicated. (C) Molecular dynamics simulation. 2D-RMSD in simulation of wtHARS2 (left panel) and mHARS2 (right
panel). The root mean square deviation of each conformation to all other conformations of simulation, as a function of time
of back-bone atoms during a 2 ns simulation, is shown in the 2D-RMSD. 2D-RMSD, two dimensional-root mean square
deviation; MD, molecular dynamics.

that concerns one frameshift and one nonsense variants, in a
compound heterozygous state with two missense variants,
which was mentioned by Demain et al. (2020). This goes in
good consistency with the essential activity of HARS2 gene in
mitochondria, for which complete loss of function is likely to
be incompatible with life.
In accordance with the guidelines of the American
College of Medical Genetics and Genomics for sequence
variant interpretation (Richards et al., 2015), c.260G>A
(p.Arg87His) was interpreted as a likely pathogenic
variant.
In addition, it is noteworthy that the conservation of the
arginine 87 residue in 8 species (Fig. 1C), as well as the clas-
sification of p.(Arg87His) as deleterious by several in silico
analysis tools, is supporting arguments that further corrobo-
rate the pathogenicity of p.(Arg87His) changement (Table 1).
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Table 2. Identified HARS2 Variants Associated with Perrault Syndrome or Hearing Loss
Homozygous/
Variant Compound
Variant (cDNA) Exon (protein) heterozygous HARS2 domain Reference
c.598C>G 6 p.Leu200Val Compound N-terminal catalytic domain Pierce et al. (2011)
c.1102G>T 10 p.Val368Leu heterozygous N-terminal catalytic domain
c.1010A>G 10 p.Tyr337Cys Homozygous N-terminal catalytic domain Lerat et al. (2016)
c.172A>G 2 p.Lys58Glu Compound N-terminal catalytic domain Karstensen et al. (2020)
c.448C>T 5 p.Arg150Cys heterozygous N-terminal catalytic domain (conserved
dimer interface)
c.137T>A 2 p.Leu46Gln Compound N-terminal catalytic domain Karstensen et al. (2020)
c.259C>T 3 p.Arg87Cys heterozygous N-terminal catalytic domain (conserved
dimer interface)
c.980G>A 10 p.Arg327Gln Compound N-terminal catalytic domain (His A) Karstensen et al. (2020)
c.448C>T 5 p.Arg150Cys heterozygous N-terminal catalytic domain (conserved

533
dimer interface)
c.647G>A 7 p.Arg216Gln Compound N-terminal catalytic domain Zou et al. (2020)
c.697C>T 7 p.Arg233Cys heterozygous N-terminal catalytic domain
c.413G>A 5 p.Arg138His Compound N-terminal catalytic domain (conserved Demain et al. (2020)
c.1439G>A 12 p.Arg480His heterozygous dimer interface)
C-terminal anticodon binding domain
c.828delTinsGTATCCCTAGTATTTCTACTA 9 p.Gly277TyrfsTer3 Compound N-terminal catalytic domain Demain et al. (2020)
c.1439G>A 12 p.Arg480His heterozygous C-terminal anticodon binding domain
c.72C>A 1 p.Cys24Stop Compound N-terminal catalytic domain Demain et al. (2020)
c.1439G>A 12 p.Arg480His heterozygous C-terminal anticodon binding domain
c.908 T > C 9 p.Leu303Pro Compound N-terminal catalytic domain Yu et al. (2020)
c.349G>A 4 p.Asp117Asn heterozygous N-terminal catalytic domain (conserved
dimer interface)
c.260G>A 3 p.Arg87His Homozygous N-terminal catalytic domain (conserved This study
dimer interface)
Bold text indicates the results of this study.
 534
Moreover, according to UniProt, HARS2 is a mitochon-
drial aminoacyl-tRNA synthetase that catalyzes the ATP-
dependent ligation of histidine to the 3¢-end of its cognate
tRNA, via the formation of an aminoacyl-adenylate inter-
mediate (His-AMP). The variant residue, Arg87, is located
at the conserved dimer interface of the N-terminal catalytic
domain and makes an h-bond with Ser83 and an ionic-bond
with Glu198 for the intrachain interactions, in addition to an
h-bond with Asp434, Gly436, Lys438, and Ala439 for the
interchain interactions. However, His87 makes an h-bond
with Ser83 for the intrachain interactions and an h-bond with
Gly436, Lys438, and Ala439 for the interchain interactions.
The Asp434, Gly436, Lys438, and Ala439 are located be-
tween a13-a14 on the Ct-domain (Fig. 2). Therefore, the
Arg87His substitution may disturb correct folding or even
the stability of the protein, reducing subsequently its amino-
acylation efficiency.
Note that the change of the amino acid arginine to histidine
has also been reported twice previously in the HARS2 protein
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(Fig. 3 and Table 3). The first is p.(Arg138His) in the dimer
interface of the N-terminal catalytic domain and the second
one is p.(Arg480His) in the C-terminal anticodon binding
domain (Demain et al., 2020), which is suggestive that the
p.Arg87His substitution may have deleterious effects on the
function of HARS2.
We found a novel p.(Arg87His) variant that stands for the
second one identified in the homozygous state in the HARS2
gene. In previous studies, only the c.1010A>G (p.Tyr337-
Cys) variant was detected in the homozygous state in a
Moroccan family (Lerat et al., 2016). All other variants
were characterized in the compound heterozygous state
(Table 2).
Concerning Arab populations, five other variants have
been reported in Arab patients with PS with different clinical
characteristics originating from Morocco, Saudi Arabia, and
Palestine (Pierce et al., 2013; Ahmed et al., 2015; Lerat et al.,
2016; Al-Jaroudi et al., 2019). All these variants were present
at the homozygous state in the patients (Table 4). In these
countries, the offspring of consanguineous unions may be at
an increased risk for recessive disorders owing to the expres-
sion of autosomal recessive variants inherited from a com-
mon ancestor.
FIG. 3. Newly identified and previously reported HARS2 variants are marked along the schematic representation of
HARS2. The N-terminal catalytic domain spreads from amino acid 1 to aa 405. The catalytic domain containing the
highly conserved dimer interface (aa 65–177) and the histidine recognition and binding sites (HisA: aa 327–332 and
HisB: aa 361–365) are depicted. The C-terminal anticodon binding domain spreading from amino acid 406 to 506 is
also depicted.
SOUISSI ET AL.
In prior studies, Yu et al. (2020) argued that the cohort is
too small to elucidate a relationship between genotypes and
phenotypes, as variants of HARS2 have been associated with
HL or PS in 10 cases uniquely. Today and following our
and other studies, 20 patients with variants in HARS2 are
detected (Table 3). Some correlations between the phenotype
and genotype seem possible in PS patients with variants in
HARS2. The audiological evaluation of our patient as well as
the Moroccan patient, both carrying a homozygous variant
in HARS2 (Table 3), reveals profound HL. For the patients
carrying compound heterozygous variants, HL ranged from
mild to severe in 11 patients and from severe to profound in
5 patients. Furthermore, additional features described in the
two patients carrying homozygous variants in HARS2 ex-
hibited hypothyroidism in the Moroccan patient and loss of
balance, frequent falls, and chronic fatigue in our Lebanese
patient. Owing to the close anatomical relationship between
the cochlea and vestibulum, it is not surprising that children
with HL may have some degree of vestibular dysfunction,
which would be associated with balance deficits (Verbecque
et al., 2017). In addition, for cerebellar signs, a clinical ex-
amination revealed the presence of ataxia and weak patellar
reflexes. A brain scan was not undertaken because the patient
refused it. It is noteworthy that brain white matter changes
have been reported in some patients with PS (Dominguez-
Ruiz et al., 2019). Additional features such as problems with
tooth growth were observed only in two probands carrying
two compound heterozygous variants (Table 3).
At present, out of the 20 patients carrying variants in the
HARS2 gene, 11 patients are women. Regarding gonadal
dysfunction, different shapes have been observed. Three pa-
tients are asymptomatic, three patients had secondary amen-
orrhea, one patient had irregular menses, and four patients
had amenorrhea with streak gonads (Table 3). It is important
to point out that the examination age for the different women
varies from 6 to 32 years. Three asymptomatic patients were
examined at the age of 6, 7, and 13 years, which stands for
relatively early ages to know if there are irregular menses
or amenorrhea. In addition, according to Lerat et al. (2016),
ovarian dysgenesis cannot be diagnosed before the teenage
years. Therefore, it is difficult to enact genotype/phenotype
correlation for the ovarian phenotype.
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Table 3. Clinical Details and Genotypes of Reported Individuals with Variants in HARS2
Family Family 1 Family 2 Family 3 Family 4 Family 5

Origin European Moroccan Chinese Han European European

HARS2 M1 M2 M1 M2 M1 M2 M1 M2 M1 M2
Variants c.598C>G p.Leu200Val c.1102G>T c.1010A>G homozygous c.647G>A c.697C>T c.137 T > A c.259C>T c.172A>G p.Lys58Glu c.448C>T
p.Val368Leu p.Tyr337Cys p.Arg216Gln p.Arg233Cys p.Leu46Gln p.Arg87Cys p.Arg150Cys
Patient Sex P1 P2 P3 P4 P5 P1 P2 P1 P1 P1 P2 P3
F M M F F F F M F M F F
Age Of Onset 34 6 18 12 8 <3 <3 NR 25 5 26 0.3
of HL (years)
Age at Exam NR 22 NR 19 13 NR NR NR 32 18 16 13
(years)
Degree of HL Mild Moderate Moderate Mild to Moderate to Profound Profound NR Moderate Moderate Moderate Moderate
to severe to severe moderate profound to severe to severe to severe
Gonadal ovarian NA NA ovarian ovarian Secondary Secondary NA Secondary NA irregular asymptomatic
dysfunction dysgenesis, dysgenesis, dysgenesis, amenorrhea amenorrhea amenorrhea, cessation menses
with with with
amenorrhea amenorrhea amenorrhea
and streak and streak and streak
gonads gonads gonads
Age At POI NR NA NA NR NR 25 26 NO 24 NA 16 13
(years)
Neurological NO NO NO NO NO NO NO NO NO NO NO NO
features
Additional NO NO NO NO NO Hypothyroidism Hypothyroidism NO NO NO NO NO
features
Reference Pierce et al. (2011) Lerat et al. (2016) Zou et al., (2020) Karstensen et al. (2020) Karstensen et al. (2020)

535
Family Family 6 Family 7 Family 8 Family 9 Family 10 Family 11

Origin European European North American Proband Chinese Han Lebanese

HARS2 Variants M1 M2 M1 M2 M1 M2 c.1439G>A M1 M2 M1 M2 M1 M2
c.448C>T c.980G>A c.413G>A c.1439G>A c.828delTinsGTATCCC p.Arg480His c.72C>A c.1439G>A c.349G>A c.908 T > C c.260G>A; homozygous
p.Arg150Cys p.Arg327Gln p.Arg138His p.Arg480His TAGTATTTCTACTA p.Cys24Stop p.Arg480His p.Asp117Asn p.Leu303Pro p.Arg87His
p.Gly277TyrfsTer3

Patient Sex P1 P1 P1 P2 P1 P1 P2 P1
F F M M M M M F
Age Of Onset of 2 6 25 1 1.5 2.5 1 At birth
HL (years)
Age at Exam 7 6 14 11 4 7 10 31
(years)
Degree of HL Moderate to severe Moderate Severe to profound Severe to Profound Profound Severe to Profound
profound profound
Gonadal Asymptomatic Asymptomatic NA NA NA NA NA Amenorrhea and streak
dysfunction gonads
Age At POI (years) 7 NR NA NA NA NA NA 11
Neurological NO NO Difficulty with fine Tightness in the NO NO NO Ataxia and weak patellar
features motor movements muscles of reflexes
his lower
limbs
Additional features NO NO Problems with tooth Problems with NO NO NO Loss of balance, frequent falls,
growth tooth growth and chronic fatigue
Reference Karstensen et al. (2020) Demain et al. (2020) Demain et al. (2020) Demain et al. (2020) Yu et al. (2020) This study

F: female; M: male; P: patient; M1: mutation 1; M2: mutation 2.

HL, hearing loss; NA, not applicable; NR, not recorded; POI, premature ovarian insufficiency.
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Table 4. Clinical and Genetic Details of Reported Arab Patients with Perrault Syndrome
Age of
onset of Age at
Family Mutated Genetic Patient HL Degree Gonadal POI Neurological Additional
origin gene type Mutation sex (years) of HL dysfunction (years) features features Reference
Lebanon HARS2 2 c.260G>A; Patient 1:F At birth Profound Amenorrhea 11 Ataxia and weak loss of balance, This study
p.Arg87His and streak patellar reflexes frequent falls,
(homozygous) gonads and chronic
fatigue
Morocco HARS2 2 c.1010A>G Patient 1:F <3 Profound Secondary 25 NO Hypothyroidism Lerat et al.
p.Tyr337Cys amenorrhea (2016)
(homozygous) Patient 2:F <3 Profound Secondary 26 NO Hypothyroidism
amen
orrhea
Morocco LARS2 4 c.1565C>A; Patien 1:F 23 Moderate/ Small uterus, NR NR Marfanoid Zerkaoui
p.Thr522Asn profound ovaries not habitus et al.
(homozygous) visualized (2017)
Patient 16 Moderate/ NA NA NR Marfanoid
2:M profound habitus
Saudi LARS2 4 c. 457A>C; Patient 1:F At birth NR Hypoplastic NR Degeneration at the NR Al-Jaroudi
Arabia p.Asn153His uterus and L5-S1 level and et al.
(homozygous) both ovaries Tarlov cysts at (2019)
were not the S2 level

536
observed
Saudi CLPP 3 c.685T>G; Patient <2 Severe NA NA Severe spastic NO Ahmed
Arabia p.Tyr229Asp 1:M diplegia, et al.
(homozygous) hypertonia, deep (2015)
tendon reflexes
were brisk, loss
of white matter
volume
Patient 2:F <2 Severe NR NR lower limb spastic NO
diplegia with
reduced power
(3/5), deep
tendon reflexes
were brisk, and
the planter reflex
was upgoing;
mild to moderate
loss of white
matter volume
with reduced
myelination
Patient NR NR NA NA NR NR
3:M

(continued)
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Table 4. Continued
Age of
onset of Age at
Family Mutated Genetic Patient HL Degree Gonadal POI Neurological Additional
origin gene type Mutation sex (years) of HL dysfunction (years) features features Reference
Palestine LARS2 4 c.1565C>A; Patient 3–5 Severe to NA NA NO NO Pierce et al.
p.Thr522Asn 1:M profound (2013)
(homozygous) Patient 2:F 3–5 Right ear: severe Small uterus, NR NO NO
at lower ovaries not
frequencies, visualized
moderate at
higher
frequencies.

537
Left ear:
Moderate at
lower
frequencies,
mild at higher
frequencies
Patient3:M 3–5 Severe to NA NA NO NO
moderate at low
frequencies,
moderate to
mild at higher
frequencies
F female, M male, NA not applicable, NR not recorded.
 538
The reported patient carries a homozygous variant in the
highly conserved dimer interface of the N-terminal cata-
lytic domain of HARS2. Nonetheless, other patients harbor
variants in the N-terminal catalytic domain uniquely, rather
than in the highly conserved dimer interface, or in both the
N-terminal and the C-terminal domains (Table 2 and Fig. 3).
Our patient presents profound HL, amenorrhea, streak go-
nads, loss of balance, frequent falls, chronic fatigue, ataxia,
and weak patellar reflexes. Unfortunately, a comprehensive
clinical phenotype description is not available for all the
previously reported patients. Nevertheless, a careful assess-
ment of symptoms and signs of patients for whom these data
are available is indicative that variants in the highly conser-
ved dimer interface of HARS2 determine a more severe clin-
ical phenotype.
For PS, especially for women, the complete phenotypic
spectrum is absent from birth, but appears later during the
patient’s life, which further obstructs the correct diagnosis
of the disorder. It is worth noting that girls with PS are most
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likely clinically to be misdiagnosed and to be considered
patients with isolated HL. For males, the diagnosis of PS will
only be considered in a male with a sister with PS, or on
molecular diagnosis as all males will have nonsyndromic HL.
In the light of the above analysis, we assert that even though
an accurate clinical assessment can enable clinicians to
raise a particular suspect, only the identification of the mo-
lecular cause of the disease may shun any incomplete or even
incorrect diagnosis, permitting an appropriate genetic coun-
seling or a treatment. As far as HL is concerned, possible
interventions with hearing aids, vibrotactile, and cochlear
implantation can be undertaken (Newman et al., 1993).
Moreover, ovarian insufficiency can be treated in adolescents
displaying primary amenorrhea by induction of puberty with
incremental doses of estrogen and progesterone to mimic
menstrual cycle and trigger withdrawal bleeding. For gonadal
dysgenesis, assisted reproduction through in vitro fertiliza-
tion can be conducted (Newman et al., 1993).
Conclusion
This research centers on a further assessment of PS in a
patient with a novel HARS2 variant, consolidating previous
data about HARS2-associated phenotype. PS corresponds to
a rare disorder, but it is probably underdiagnosed. To better
identify genotype/phenotype correlations, a thorough and
exhaustive clinical phenotype description for previously re-
ported patients and more PS patients are needed. Early-onset
sensorineural HL in both sexes and ovarian dysfunction in
women suggest a wake-up call for consideration of HARS2
gene testing.
Authors’ Contributions
M.B.S. and F.F. wrote the article. A.M. performed the
collection of family members and the acquisition of clinical
data. I.E. and A.S. performed library preparation. M.B.S. and
A.S. interpreted the NGS data. F.F. performed the 3D
structural analysis. S.M. and M.B.S. have contributed to the
acquisition of the financial support for the project leading to
this publication. M.B.S., S.M., and A.M. commented and
substantively revised the article. All authors read and appro-
ved the final article.
SOUISSI ET AL.
Acknowledgments
Deep and sincere gratitude first and foremost goes to the
family members for their active contribution to the success of
this work. We extend our honorable thanks to the clinicians
who referred patients to this study.
Author Disclosure Statement
The authors have no conflicts of interest that are directly
relevant to the content of this article.
Funding Information
This work was supported by the VRR Tunisian grant, the
PAQ-ProMESsE Tunisian grant, and the Ministry of Higher
Education, Scientific Research of Tunisia (LR15-CBS-07).
References
Ahmed S, Jelani M, Alrayes N, et al. (2015) Exome analy-
sis identified a novel missense mutation in the CLPP gene in
a consanguineous Saudi family expanding the clinical spectrum
of Perrault Syndrome type-3. J Neurol Sci 353:149–154.
Al-Jaroudi D, Enabi S, AlThagafi MS (2019) Perrault syndrome
with amenorrhea, infertility, Tarlov cyst, and degenerative
disc. Gynecol Endocrinol 35:1037–1039.
Demain LA, Urquhart JE, O’Sullivan J, et al. (2017) Expanding
the genotypic spectrum of Perrault syndrome. Clin Genet 91:
302–312.
Demain LAM, Gerkes EH, Smith RJH, et al. (2020) A recurrent
missense variant in HARS2 results in variable sensorineural
hearing loss in three unrelated families. J Hum Genet 65:305–
311.
Dominguez-Ruiz M, Garcia-Martinez A, Corral-Juan M, et al.
(2019) Perrault syndrome with neurological features in a com-
pound heterozygote for two TWNK mutations: overlap of
TWNK-related recessive disorders. J Transl Med 17:290.
Karstensen HG, Rendtorff ND, Hindbaek LS, et al. (2020)
Novel HARS2 missense variants identified in individuals with
sensorineural hearing impairment and Perrault syndrome. Eur
J Med Genet 63:103733.
Lerat J, Jonard L, Loundon N, et al. (2016) An application of
NGS for molecular investigations in perrault syndrome: study
of 14 families and review of the literature. Hum Mutat 37:
1354–1362.
Newman WG, Friedman TB, Conway GS, et al. (1993) Perrault
Syndrome. Gene Reviews. University of Washington, Seattle
(WA).
Pierce SB, Chisholm KM, Lynch ED, et al. (2011) Mutations
in mitochondrial histidyl tRNA synthetase HARS2 cause ovar-
ian dysgenesis and sensorineural hearing loss of Perrault syn-
drome. Proc Natl Acad Sci U S A 108:6543–6548.
Pierce SB, Gersak K, Michaelson-Cohen R, et al. (2013)
Mutations in LARS2, encoding mitochondrial leucyl-tRNA
synthetase, lead to premature ovarian failure and hearing loss in
Perrault syndrome. Am J Hum Genet 92:614–620.
Richards S, Aziz N, Bale S, et al. (2015) Standards and
guidelines for the interpretation of sequence variants: a joint
consensus recommendation of the American College of
Medical Genetics and Genomics and the Association for
Molecular Pathology. Genet Med 17:405–424.
Souissi A, Ben Said M, Ben Ayed I, et al. (2021) Novel path-
ogenic mutations and further evidence for clinical relevance
of genes and variants causing hearing impairment in Tunisian
population. J Adv Res 31:13–24.
 NOVEL HARS2 VARIANT IN PERRAULT SYNDROME 539

Verbecque E, Marijnissen T, De Belder N, et al. (2017) Address correspondence to:

Vestibular (dys)function in children with sensorineural Mariem Ben Said, PhD
hearing loss: a systematic review. Int J Audiol 56:361– Laboratory of Molecular and Cellular
381. Screening Processes
Yu J, Jiang W, Cao L, et al. (2020) Two novel likely pathogenic Center of Biotechnology of Sfax
variants of HARS2 identified in a Chinese family with sen- University of Sfax
sorineural hearing loss. Hereditas 157:47. Rte. Sidi Mansour Km 6, BP ‘‘1177’’
Zerkaoui M, Demain LA, Cherkaoui Jaouad I, et al. (2017) Sfax 3018
Marfanoid habitus is a nonspecific feature of Perrault syn- Tunisia
drome. Clinical dysmorphology 26:200–204.
Zou S, Mei X, Yang W, et al. (2020) Whole-exome sequencing
identifies rare pathogenic and candidate variants in sporadic
Chinese Han deaf patients. Clin Genet 97:352–356. E-mail: mariem.bensaid@cbs.rnrt.tn
Downloaded by 41.228.57.218 from www.liebertpub.com at 08/23/21. For personal use only.

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