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Molecular Genetics and Metabolism Reports
journal homepage: www.elsevier.com/locate/ymgmr
Case Report
Mild form of Zellweger Spectrum Disorders (ZSD) due to variants in PEX1:
Detailed clinical investigation in a 9-years-old female
Maria Rosaria Barillari
a,1,⁎
, Marianthi Karali
b,c,1
, Valentina Di Iorio
d
, Maria Contaldo
e
,
Vincenzo Piccolo
f
, Maria Esposito
g
, Giuseppe Costa
a
, Giuseppe Argenziano
f
, Rosario Serpico
e
,
Marco Carotenuto
g
, Gerarda Cappuccio
b,h
, Sandro Banfi
b,c
, Paolo Melillo
d
, Francesca Simonelli
d
a
Division of Phoniatrics and Audiology, Department of Mental and Physical Health and Preventive Medicine, University of Campania "Luigi Vanvitelli", Via L. De Crecchio
4, 80138 Naples, Italy
b
Telethon Institute of Genetics and Medicine, Pozzuoli, Via Campi Flegrei 34, 80078 Pozzuoli, Italy
c
Department of Precision Medicine, University of Campania "Luigi Vanvitelli", Via L. De Crecchio 7, 80138 Naples, Italy
d
Eye Clinic, Multidisciplinary Department of Medical, Surgical and Dental Sciences, University of Campania Luigi Vanvitelli, Via Pansini 5, 80131 Naples, Italy
e
Dental Clinic, Multidisciplinary Department of Medical, Surgical and Dental Sciences, University of Campania “Luigi Vanvitelli”, Via L. De Crecchio 6, 80138 Naples, Italy
f
Pediatric Dermatology, Dermatology Unit, University of Campania Luigi Vanvitelli, Via Pansini 5, 80131 Naples, Italy
g
Clinic of Child and Adolescent Neuropsychiatry, Department of Mental Health, Physical and Preventive Medicine, University of Campania "Luigi Vanvitelli", Via Pansini 5,
80131 Naples, Italy
h
Department of Translational Medicine, Section of Paediatrics, Federico II University, Via Pansini 5, 80131 Naples, Italy
ARTICLE INFO
Keywords:
Sensorineural hearing loss
Retinitis pigmentosa
Enamel defects
PEX genes
Peroxisomal biogenesis disorders
Mild Zellweger syndrome
ABSTRACT
Peroxisomal biogenesis disorders (PBD) are rare autosomal recessive disorders with various degrees of severity
caused by hypomorphic mutations in 13 different peroxin (PEX) genes. In this study, we report the clinical and
molecular characterization of a 9-years-old female presenting an apparently isolated pre-lingual sensorineural
hearing loss (SNHL) and early onset Retinitis Pigmentosa (RP) that may clinically overlap with Usher syndrome.
Genetic testing by clinical exome sequencing identified two variants in PEX1: the missense variant c.274G > C;
p.(Val92Leu) that was already reported in a PBD patient, and the variant c.2140_2145dup; p.
(Ser714_Gln715dup) which is a novel, non-frameshift variant, absent in control databases. On the basis of the
molecular analysis, a thorough clinical examination revealed nail and dental abnormalities, a mild cognitive
impairment, learning disabilities and poor feeding, apart from the retinal and audiological features initially
identified. The clinical and molecular findings led us to the diagnosis of a mild form of PBD. This study further
emphasizes that mild forms of PBD can be a differential diagnosis of Usher syndrome and suggests that patients
with mild cognitive impairment associated to visual and hearing loss should perform a comprehensive mutation
screening that includes PEX genes.
1. Introduction
Peroxisomal biogenesis disorders (PBDs) are a group of autosomal
recessive disorders caused by mutations in one of the thirteen Peroxin
(PEX) genes. PBDs are caused by partial or generalized defects in per-
oxisome biogenesis [1]. These organelles are present in almost all eu-
karyotic cells and play an indispensable role in many biochemical
pathways including the metabolism of branched chain and very long
chain fatty acids, ether lipids, polyamines, amino acids and glyoxylate
[2,3]. Peroxisomal dysfunction leads to multisystem disease that in-
cludes neurological, visual and hearing symptoms [4].
PBDs are divided into two main types: the Rhizomelic
Chondrodysplasia Punctata type 1 and the Zellweger spectrum disorder
(PBD-ZSD) which ranges from severe (Zellweger Syndrome, ZS) to
https://doi.org/10.1016/j.ymgmr.2020.100615
Received 21 March 2020; Received in revised form 5 June 2020; Accepted 6 June 2020
Abbreviations: PBD, Peroxisomal biogenesis disorders; SNHL, sensorineural hearing loss; RP, retinitis pigmentosa; PEX, peroxin; ZSD, Zellweger spectrum disorder;
ZS, Zellweger Syndrome; HS, Heimler syndrome; BCVA, Best Corrected Visual Acuity; GVF, Goldmann Visual Field; ERG, full-field electroretinogram; OCT, optical
coherence tomography; FAF, color fundus and fundus autofluorescence; TEOAE, Transient-Evoked Otoacustic Emission; ABR, Auditory Brainstem Responses; PTA,
Pure Tone Average; WISC-IV, Wechsler Intelligence Scale for Children (4th Edition); CDI, Children’s Depression Inventory; VLCFA, Very Long Chain Fatty Acid
⁎
Corresponding author.
E-mail address: mariarosaria.barillari@unicampania.it (M.R. Barillari).
1
These authors contributed equally to this work.
Molecular Genetics and Metabolism Reports 24 (2020) 100615
2214-4269/ © 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/BY/4.0/).
T
intermediate (Neonatal Adreno-LeukoDystrophy) and mild (Infantile
Refsum Disease) phenotypes [5]. The PBD-ZSD is a clinically hetero-
geneous group with a continuum of disease severity. In particular, the
clinical presentation of the most severe form, namely ZS (OMIM:
614872), is characterized by craniofacial abnormalities, neuronal mi-
gration defects, leukodystrophy, absence of language, cognitive and
psychomotor delay, renal and liver diseases, hypotonia, hearing loss
and vision problems including cataracts and/or retinal abnormalities
[6]. Children with this condition do not develop properly and usually
die before one year of age [7], while patients with intermediate and
mild phenotypes can live into adulthood because their clinical features
(e.g. sensorineural hearing loss, retinal diseases, leukodystrophy and
cognitive delay) are less pronounced compared to those of ZS. Patho-
genic variants in PEX1 are the most common cause of PBD-ZSD and
have been associated with various degrees of disease severity [8–11].
On the other hand, the Heimler Syndrome (HS) is considered the
mildest end of PBD-ZSD spectrum of disorders [12]. HS is caused by
hypomorphic mutations in PEX1 and PEX6 genes and is characterized
by severe to profound bilateral sensorineural deafness, enamel defects
and nail abnormalities such as Beau's lines and punctate leukonychia
[12–15]. Macular dystrophy has also been reported in the context of the
HS
16
.
The clinical heterogeneity of PBD-ZSD renders patients' diagnosis
challenging, especially in the case of very mild, late-onset forms that
may overlap with other syndromic phenotypes, in particular Usher
syndrome. This may delay diagnosis and proper management of the
disease.
Here, we report the clinical and molecular characterization of a 9-
years-old female presenting an apparently isolated sensorineural
hearing loss (SNHL) and early onset atypical Retinitis Pigmentosa (RP).
Clinical exome sequencing identified two biallelic variants in the PEX1
gene. A thorough clinical reevaluation led to the diagnosis of a mild
form of PBD-ZSD. We therefore suggest that patients with mild cogni-
tive impairment associated to visual and hearing loss should perform a
comprehensive mutation screening that includes PEX genes.
2. Materials and methods
Written informed consent for research and publication was obtained
by the family prior to participation of the subject to the current study.
All the procedures of this study were in complete accordance with the
Declaration of Helsinki on ethical principles for medical research in-
volving human subjects (2014).
The patient underwent a multidisciplinary clinical examination. It
included ophthalmological and audiological tests as well as pediatric,
dental, dermatological and neuropsychological evaluation and standard
biochemical tests.
2.1. Ophthalmological evaluation
Best Corrected Visual Acuity (BCVA), slit lamp anterior segment
examination, Goldmann Visual Field (GVF), fundus examination, full-
field electroretinogram (ERG), optical coherence tomography (OCT),
color fundus and fundus autofluorescence (FAF) were performed.
2.2. Audiological evaluation
The audiological assessment was obtained by the use of Otoacustic
Emission (OAE), in terms of Transient-Evoked Otoacustic Emission
(TEOAE), Auditory Brainstem Responses (ABR), liminar pure tone
audiometry with evaluation of the Pure Tone Average (PTA) and
Impedance test composed by tympanometry and stapedial reflexes.
2.3. Neuropsychological evaluation
This clinical examination was performed by a trained child
psychiatrist and included an interview with the patient, clinical ob-
servation of her behavior and the administration of validated tests and
questionnaires such as SCARED and WISC-IV.
2.4. Dental evaluation
An extra- and intra-oral examination was performed to evaluate the
oral mucosal integrity and teeth status. An X-ray orthopantomography
was combined with the clinical evaluation to identify any dental
agenesis, dental malocclusions and other abnormalities in the shape
and/or structure of the teeth.
2.5. Dermatological examination
A total body examination was performed including inspection of
nails and hair. Further details about the above-mentioned tests are
provided in Supplementary Materials.
2.6. Biochemical analysis
Measurement of plasmatic Very Long Chain Fatty Acid (VLCFA) and
branched-chain fatty acid was performed by a specialized laboratory
using capillary gas chromatography/mass spectrometry of penta-
fluorobenzyl bromide fatty acid esters as described elsewhere [17,18].
2.7. Clinical exome sequencing and segregation analysis
The proband's genomic DNA sample underwent a panel-based Next
Generation Sequencing using the ClearSeq Inherited Disease Panel
(Agilent) that allows the screening of more than 2700 genes known to
cause human inherited disorders. Sequencing data were analysed as
previously described [19]. Selected variations were validated by Sanger
sequencing in the patient and parents to assess proper segregation.
Further details are provided in Supplementary Materials.
3. Results
The patient was born at 40 weeks of gestation to unrelated parents
with no prior pregnancies. The Apgar index at birth was 8/10. No
dysmorphic features were evident, and no low muscle tone was re-
ported.
At 3 months of age she presented a bilateral sensorineural hearing
loss, diagnosed by the use of TEOAE and ABR. The auditory deficit was
treated by digital hearing aids and speech therapy. Annual audiological
follow-ups were regularly performed.
The patient was clinically evaluated by our multidisciplinary team
for the first time at the age of 9 because of a suspicion of an inherited
retinal disease associated to SNHL.
3.1. General pediatric evaluation
In the last pediatric evaluation performed at 9 years of age the
subject had a height of 128.6 cm (−2.17 SD), an OFC of 49.3 cm (−3.7
SD) and weighed 22.9 Kg (−2.6 SD). Her low weight was associated
also with a significant food selectivity and poor feeding for which a
specific behavioral feeding program was indicated.
The values of the routine hematochemical analysis were within the
normal range. Liver function tests showed normal levels of glutamic
oxaloacetic transaminase (GOT) and serum glutamic-pyruvic transa-
minase (GPT) at 9 years, respectively of 21.58 U/L (N·V: < 42 U/L) and
21.23 U/L (N·V: < 41). Similarly, alkaline phosphatase, albumin,
creatinine and total protein values were normal. Total bile acids were
normal at the age of 9 while total bilirubin resulted weakly elevated at
the last check (1.49 mg%; N·V: < 1).
Abdominal ultrasounds revealed no major renal or liver abnormal-
ities.
M.R. Barillari, et al. Molecular Genetics and Metabolism Reports 24 (2020) 100615
2
3.2. Ophthalmological findings
At the anamnestic evaluation, the subject referred night-blindness in
the last year. BCVA was 20/120 in both eyes. Fundus examination re-
vealed a normal optic disk, punctiform lens opacities, dystrophy of
Retinal Pigmented Epithelium (RPE) with bone spicule-shaped pigment
deposits arranged within and beyond the vascular arcades with a
normal appearance of the far periphery and macular dystrophy
(Fig. 1a). GVF showed a constricted visual field. Specifically, the
average radius was 17° and 27° using the III4e target stimulus size and
30° and 40° using the V4e target stimulus size in the right and left eye,
respectively. OCT scans showed RPE dystrophy with loss of the EZ band
in both eyes and revealed multiple inner retinal cystoid spaces (with a
mean macular thickness of 260 μm and 293 μm in the right and left eye,
respectively) (Fig. 1b). FAF imaging revealed hyper- and hypo-auto-
fluorescent dots in the posterior pole and beyond the temporal vascular
arcades sparing the fovea in both eyes. Dark-adapted 0.01 ERG re-
sponses were below noise level, whereas dark-adapted 3.0 responses
were subnormal in both eyes with a b/a ratio < 1. Light-adapted ERG
responses were subnormal in both eyes. Given the presence of cystoid
spaces, the patient was prescribed treatment with oral acetazolamide
(250 mg/die) and was regularly followed up. Over the two-year follow-
up period, we recorded a reduction of cystic spaces (with mean macular
thickness of 185 μm and 208 μm in the right and left eye, respectively)
without significant changes in BCVA and in GVF (Fig. 1b). Taken to-
gether, the ophthalmological findings confirmed an ocular phenotype
compatible with RP complicated by macular cystoid edema.
OCT scans over a two-year follow-up showed an improvement of the
macular edema in the patient following treatment with acetazolamide.
3.3. Audiological findings
There was no history of sensorineural deafness on either side of the
family. The patient has one younger sister with a normal audiometric
threshold. Parents reported that the child underwent the universal
newborn hearing screening program (performed by law in Italy)
twenty-four hours after birth with normal results (a document reporting
the results of TEOAE was not available). At 3 months of age, the general
practitioner suspected hearing loss and recommended a specific
audiological evaluation which was performed by ABR. This test, con-
sidered as the gold standard for the early diagnosis of childhood deaf-
ness, showed an abnormal morphology and latency of the auditory
brain response waves. The latency of the I, III and V waves was in-
creased and only the V wave was observed up to 60 dB hearing level
bilaterally. This finding suggested an auditory deficit characterized by
severe SNHL in both ears.
The impedance test showed a Type “A”tympanogram bilaterally,
considered normal, and the presence of the stapedial reflexes (SR) in
ipsi and contra in the entire frequency range. The Metz recruitment test,
i.e. the gap between the acoustic reflex threshold and the pure-tone
audiometry hearing threshold level, was positive and indicated a co-
chlear SNHL. Subsequent checks performed at the age of 6 and
9 months by ABR confirmed the SNHL diagnosis prompting for an early
rehabilitation treatment by digital hearing aids and speech therapy that
started at 10 months of age. The patient has been regularly undergoing
an annual audiological check from 2008 to date. The most recent ones,
performed at the age of 9 and 10, confirmed the presence of bilateral
and symmetric SNHL of a severe degree (according to World Health
Organization - Grades of Hearing Impairment) that does not appear to
be progressive over time, with a “sloping audiogram”and a PTA
(without hearing device) around 70 dB HL (Fig. 2). Further annual
follow-ups are needed to monitor the hearing impairment over time.
Fig. 1. Fundus imaging (a) and OCT scans (b) of the reported case.
M.R. Barillari, et al. Molecular Genetics and Metabolism Reports 24 (2020) 100615
3
3.4. Phoniatric and neuropsychological findings
A phoniatric and neuropsychological assessment was performed as
learning disabilities were referred by the patient's teacher and parents.
The administration of ad hoc tests revealed a deficit of language skills
both in input and output. The obtained results were below the average
expected for this age (< 2 SD) and indicated a global immaturity of
speech abilities as well as inadequate capacities of phonological dis-
crimination, lexical and grammar understanding.
Phonological and metaphonological skills (particularly the analy-
tical ones) as well as the verbal comprehension were severely com-
promised compared to the other children in the same age-group This
suggests that the child had learning disabilities, especially with regards
to reading and writing. Working memory abilities and short-term verbal
memory abilities were compromised compared to average degrees. For
this reason, a further clinical examination was recommended in order
to evaluate the presence of an intellectual disability.
Neuropsychological evaluation, according to the results of beha-
vioral observation and clinical interviews, showed that the patient was
affected by Separation anxiety (revealed by SCARED score) that im-
pacts on school performance. The scores obtained by the administration
of the WISC-IV (IQ total score = 65) underline the presence of a mild
intellectual disability. In light of these findings, the learning disabilities
observed in the patient (i.e. in writing and reading) can be considered
secondary to the cognitive delay.
Magnetic resonance imaging (MRI) of the brain did not reveal any
signs of leukodystrophy or other significant brain abnormalities.
3.5. Molecular analysis identifying biallelic variants in PEX1
The genetic analysis revealed the presence of two variants of un-
certain significance (VUS) in PEX1 (NM_000466). The identified var-
iants were present in heterozygosity in the proband and their frequency
in population databases (e.g. gnomAD, the ExAC database, the 1000
Genomes project) was compatible with a possible pathogenic role. The
missense variant c.274G > C; p.(Val92Leu) is predicted to interfere
with a canonical splice site (cadd-13 score = 24.6) and was reported in
a homozygous state in a patient with atypical Zellweger syndrome [20].
The second variant c.2140_2145dup; p.(Ser714_Gln715dup) is a novel
non-frameshift duplication of six nucleotides predicted to introduce a
third copy of a Ser-Gln repeat at amino acid position 715 (UniProtKB:
O43933). Both variants were validated by Sanger sequencing in the
proband and segregated properly in the unaffected parents, confirming
their presence in trans in the patient (i.e. p.(Val92Leu) on the paternal
allele and p.(Ser714_Gln715dup) on the maternal one), consistent with
the recessive pattern of inheritance of PEX1 variants (Fig. 3).
PEX1 is responsible for ZSD-PBD and HS. Therefore, the identifica-
tion of these variants in PEX1 prompted us to reexamine the patient's
phenotype in search for clinical features that may have been missed at
first evaluation.
Sanger sequencing traces indicate compound heterozygosity of the
two variants in exon 3 and exon 13 in the patient (II:1) and hetero-
zygosity in the unaffected parents (I:1, I:2). In exon 13, the duplicated
bases of the c.2140_2145dup variant are connected with a dashed line
to the corresponding peaks in the proband's chromatogram. Arrow in-
dicates the proband. Ref; Reference sequence.
3.6. Laboratory analysis and VLCFA assay
Routine serum VLCFA and branched-chain fatty acid (phytanic and
pristanic acid) measurements were performed to check for defects in
the metabolism of fatty acids, given the role of peroxins in this process.
The proband was not on special diet and fasting serum samples were
collected. The majority of the values were within the normal range,
with C26:0 slight increase (Table 1)[
21].
VLCFA plasma concentration may vary, with subjects demonstrating
normal or only modest increases, especially in milder or atypical forms
of PBDs [22,23]; moreover normal or slightly elevated results of routine
serum VLFCA and phytanic acid in patients affected by mild PBDs have
already been described by other authors [20,24,25].
Anyway we have to consider that the value of C26:0 obtained in our
patient can be due to nonspecific factors and, for this, further im-
munocytochemical studies in cultured fibroblasts (considered a more
sensitive indicator for a mild PBD) would be performed in the future as
well as measuring plasma pipecolic acid and the bile acid intermediates
dihydroxycholestanoic (DHCA) and trihydroxycholestanoic (THCA)
may be helpful.
Fig. 2. Pure tone audiometry (air and bone conduction) in the right ear (in red)
and in the left ear (in blue) in the frequency range between 250 Hz - 8 KHz.
Down-sloping audiogram with severe bilateral sensorineural hearing loss is
more evident in the higher frequencies.
Fig. 3. Biallelic variants identified in PEX1.
M.R. Barillari, et al. Molecular Genetics and Metabolism Reports 24 (2020) 100615
4
3.7. Dental findings
At the extraoral examination, on the frontal plane, the patient
showed a symmetrical face both on transverse and vertical planes, with
competent lips. At sagittal examination, the profile was straight. The
intraoral examination revealed a mixed dentition, as expected at this
age [26]. In particular, the upper and lower permanent incisors and the
first permanent molar were present. The remaining dentition consisted
of primary teeth (canines and molars), except for the two first primary
inferior molars, which were exfoliated (as reported by the patient) and
replaced by barely visible, thin, erupting cusps. Several enamel and
structural defects of various degree affected the permanent teeth [27]
(Fig. 4a).
Specifically, the upper central incisors displayed white spots on the
vestibular surfaces. Moreover, the occlusal areas of the first permanent
molars and the mesial-vestibular margin of the inferior right second
incisor were affected by enamel hypoplasia. These appeared yellowish
and had an irregular shape at the cusps. The erupting first lower pre-
molars also seemed to have similar defects.
The X-ray orthopantomography (Fig. 4b) revealed no agenesis, since
all the permanent teeth were present at a variable degree of maturation
and root formation, including the four buds of the third molars. The
anatomy of the roots of the permanent teeth did not show any obvious
abnormalities. However, the enamel density of canines and premolars
appeared reduced and their cusps were slightly hypoplastic. The pulp
chambers were dimensionally enlarged as expected at that age, yet not
excessively big as in taurodontic teeth, as previously reported [28,29].
On the vertical plane, the occlusion was altered by the presence of an
anterior open bite due to mouth breathing reported by the patient. The
same behavior was responsible for the discrepancies observed on the
transversal plane, between maxilla and mandible, namely a reduced
bilateral overjet and a high-arched palate. Oral mucosae were healthy
and normochromic. Tongue and teeth were covered by a visible layer of
dental plaque, which was subsequently removed by professional oral
hygienist.
Overall, the structural and chromatic dental defects pointed towards
the diagnosis of enamel defects with hypomineralization, particularly
affecting incisors and molars.
3.8. Dermatological findings
Total body skin examination revealed a true leukonychia partialis
equally affecting all fingernails and minimally sparing the distal edges
of each nail. Leukonychia was confirmed by onychoscopy showing a
whitish discoloration of nail plates which did not disappear after
pressure on the nail matrix (Fig. 5a). Moreover, a careful examination
of scalp hair showed an alternation of light and dark bands of hair
shafts. This was confirmed by trichoscopy and corresponded to ab-
normal cavities in the cortex of the hair shaft compatible with the di-
agnosis of pili annulati (Fig. 5b).
4. Discussion
In this report, we employed a multidisciplinary approach to char-
acterize in detail the clinical phenotype of a pediatric patient with a
Table 1
Main results of serum standard biochemical markers in the patient.
Serum VLCFA Patient Normal control range
C22:0 57.9 μmol/L 26.5–75.3 μmol/L
C24:0 44.9 μmol/L 24.9–73.0 μmol/L
C26:0 1.130 μmol/L
a
0.460–0.960 μmol/L
C24:0/C22:0 0.77 0.62–1.01
C26:0/C22:0 0.019 0.008–0.026
Pristanic acid 0.13 μmol/L Traces - 1.50 μmol/L
Phytanic acid 1.76 μmol/L Traces - 7.0 μmol/L
Pristanic/Phytanic Ratio 0.07 0.01–0.60 μmol/L
a
Out-of-normal-range value.
Fig. 4. Main dental findings of the reported case.
a) High-arched palate and visible enamel hypoplasia of the first permanent
upper molars; further chromatic and structural enamel alterations of the first
permanent lower molars b) Orthopantomography reveal the presence of all the
permanent teeth expected, lack of taurodontism, nor agenesis. The cusps of the
permanent canines and premolars, particularly of the mandible, seem thinner
and, presumably hypoplastic.
Fig. 5. Dermatological findings of the reported case.
a) Leukonychia partialis of all fingernails sparing the distal margin of the nail
plates, better magnified by onychoscopy in the right caption b) Clinically de-
tectable banding of hair, confirmed by trichoscopy and light microscopy,
showing abnormal cavities in the cortex of the hair shaft typical of pili annulati.
M.R. Barillari, et al. Molecular Genetics and Metabolism Reports 24 (2020) 100615
5
mild form of PBD-ZSD.
The patient was referred to our clinic with an apparently isolated
pre-verbal SNHL and night blindness. The full ophthalmological eva-
luation revealed a pericentral form of RP complicated by bilateral
macular edema. The ocular and audiological phenotype along with the
absence of dysmorphic features pointed towards a preliminary clinical
diagnosis of Usher syndrome. However, the patient's phenotype of pre-
puberal RP and early onset, severe SNHL did not fit well in the spectrum
of Usher disease. This was because in Usher type 1 the presence of pre-
puberal RP is associated with congenital SNHL of a profound degree,
whereas in Usher type 2 a moderate-to-severe (generally congenital)
SNHL is normally associated with post-puberal RP.
In parallel, genetic testing by clinical exome sequencing did not
reveal variants in genes that cause Usher syndrome, but identified
biallelic variants in PEX1, a gene responsible for peroxisome biogenesis
disorders. The two identified variants were classified as VUS according
to the American College of Medical Genetics and Genomics (ACMG)
guidelines. The p.(Ser714_Gln715dup) variant is novel and absent from
reference databases. Its translation introduces a third copy of a Ser-Gln
pair in a small repeat region of PEX1. The second mutation is a missense
variant (p.(Val92Leu)) predicted in silico to interfere with a canonical
acceptor splice site. We believe that both variants are likely to be pa-
thogenic since they were found in trans (as confirmed by segregation
analysis) in a patient presenting a clinical phenotype compatible with a
peroxisome biogenesis disorder. Future in vitro studies should experi-
mentally verify the functional consequences of the identified variants.
In support of its potential causative role, the variant p.(Val92Leu) has
been previously described in homozygous state in a Turkish patient
with a non-classical ZS, although it was not specified whether zygosity
was confirmed by segregation in the parents [20]. This patient, contrary
to the case reported herein, presented several dysmorphic features (e.g.,
large fontanelle, wide sutures, high forehead, broad nasal bridge, ex-
ternal ear deformity and sickle foot), hypotonia, severe psychomotor
retardation but no ocular findings [20]. A possible explanation for the
absence of ocular findings could be attributed to the patient's age at
examination (i.e. 2 years old), also considering that our case reported
the first symptoms of visual impairment at the age of 8 years.
The similarity between Usher syndrome and mild phenotypes of
PBD-ZSD has been previously reported [21,30–32]. We, therefore,
pursued a comprehensive clinical assessment to differentiate between
the two possibilities. Neuropsychological assessment of the patient re-
vealed the presence of a mild cognitive impairment, a common feature
in the spectrum of peroxisome disorders. Brain MRI did not reveal
generalized defects. Moreover, the patient had a lower weight com-
pared with normal population associated with a significant food se-
lectivity and poor feeding, features that have been described in inter-
mediate-mild forms of ZSD [1,5]. Oral examination showed minor oral
abnormalities, in terms of enamel defects, as previously described in
cases of intermediate-milder PBD-ZSD [5]. The dermatological findings
present in the patient (i.e. leukonychia, Beau's lines and hair defects,)
are not considered as diagnostic criteria in the spectrum of PBD-ZSD.
Nevertheless, mutations in PEX1 and PEX6 genes may determine nail
and hair defects [31]. Taking into account both the clinical and genetic
findings, we formulated a diagnosis of a mild PBD-ZSD. Plasmatic
VLCFA levels were overall normal, except for a slight increase of C26:0.
This was in line with previous studies showing that individuals with
very mild/mild PBDs do not necessarily demonstrate significantly al-
tered values in VLCFA metabolic screening tests as observed for patients
with severe forms of ZSD [33–35]. Unfortunately, measurement of
C26:0-lysoPC, which was recently proposed as a novel, sensitive serum
biomarker for the diagnosis of mild PBD-ZSD [1], could not be per-
formed in the patient, as well as skin biopsy and immunocytochemical
studies in cultured fibroblasts as mentioned above.
5. Conclusions
In conclusion, we report here a case of mild form of PBD-ZSD
characterized by slight abnormalities of VLCFA, early onset SNHL,
atypical RP, enamel defects, nail abnormalities, minor feeding problems
and mild cognitive impairment in absence of hypotonia, dysmorphic
features and other major abnormalities. The association of hearing loss,
enamel defect and nail abnormalities, with or without macular dys-
trophy, has been described also in Heimler syndrome (HS), which is
considered the mildest form known to date in the spectrum of PBD
[12–15]. Although these clinical features are observed in our patient,
the presence of a mild cognitive impairment and feeding problems
renders her phenotype more compatible with a mild form of ZSD [1]
which has a slightly more severe clinical presentation compared to the
typical Heimler syndrome [13–16,22,31].
Based on the presented case, we recommend that patients with vi-
sual and hearing impairment perform a comprehensive mutation
screening that includes the PEX genes.The molecular analysis can be
instrumental for the early identification of patients with mild forms of
PBD-ZSD that may overlap with other syndromic diseases. Moreover,
the timely and proper diagnosis of these rare cases requires the com-
bined effort of a multidisciplinary team of clinicians. Following initial
diagnosis, periodic multidisciplinary follow-ups are required to monitor
disease progression and ensure appropriate disease management.
Declaration of Competing Interest
None of the authors has a conflict of interest.
Acknowledgement
This work was supported by grant from the Italian Fondazione
Roma (to S.B. and F.S.). This study was also funded by Ministero
dell'Istruzione, dell'Università e della Ricerca (MIUR) under PRIN 2015
(to S.B. and F.S.)
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ymgmr.2020.100615.
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