Middle interhemispheric variant of holoprosencephaly: First prenatal report of a ZIC2 missense mutation

Abstract

We present a case of a middle interhemispheric variant of antenatal discovery associated with a de novo missense variant (NM_007129.5: c.1109G>A p.(Cys370Tyr)) in the ZIC2 gene. Our case represents the first prenatal description of a ZIC2 missense mutation found in association with syntelencephaly. We present a case of a middle interhemispheric variant of antenatal discovery associated with a de novo missense variant (NM_007129.5: c.1109G>A p.(Cys370Tyr)) in the ZIC2 gene. Our case represents the first prenatal description of a ZIC2 missense mutation found in association with syntelencephaly.

Full text

Clin Case Rep. 2020;8:1287–1292.
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1
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INTRODUCTION
Holoprosencephaly (HPE; MIM# 236100) is the most recur-
rent congenital brain malformation (1/10 000 live births),
resulting from the incomplete midline division of the prosen-
cephalon between 18th and 28th day of gestation and affect-
ing the forebrain and the face.1,2 It is characterized by a wide
clinical spectrum, ranging from severe HPE (alobar form:
with a single cerebral ventricle and cyclopia) to clinically un-
affected carriers. According to the degree of brain separation
and whether the failure occurs ventrally or dorsally, two main
classes of HPE can be distinguished: classic and middle in-
terhemispheric variant (MIHV). In classic HPE, the lack of
separation is most severe ventrally. This leads to a spectrum
of classic HPE, of which alobar HPE is the most severe form,
followed by semilobar and lobar forms (in which most of the
cerebral hemispheres are separated except at the ventral level
of the frontal poles where the interhemispheric fissure re-
mains absent).2 A septopreoptic type, in which nonseparation
is restricted to the subcallosal cortex and/or the ventral preop-
tic region, is also described in small case series.3 On the other
hand, MIHV (otherwise known as syntelencephaly), firstly
described in 1993,4 is characterized by the failure of division
of posterior frontal and parietal regions of the cerebral hemi-
spheres along the dorsal midline. Nevertheless, this condition
entails the normal separation of the basal forebrain, anterior
frontal lobes, and occipital regions.
Similarly to its wide clinical spectrum, HPE etiology
is very heterogeneous.5-7 Environmental (consume of cu-
mulative tobacco with alcohol, insulin-dependent maternal
diabetes, retinoic acid and statins intakes) and infectious
causes (toxoplasmosis, cytomegalovirus, syphilis, rubella)
Received: 29 November 2019
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Accepted: 24 January 2020
DOI: 10.1002/ccr3.2896
CASE REPORT
Middle interhemispheric variant of holoprosencephaly: First
prenatal report of a ZIC2 missense mutation
CarolineGounongbé1
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MartinaMarangoni2
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VanessaGouder de Beauregard3
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MélanieDelaunoy2
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PatriceJissendi4
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MarieCassart1,4
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JulieDésir2,4
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium,
provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
© 2020 The Authors. Clinical Case Reports published by John Wiley & Sons Ltd.
Caroline Gounongbé and Martina Marangoni equally contributed to this work.
Marie Cassart and Julie Désir equally supervise this work.
1Department of Fetal Medicine, CHU Saint-
Pierre, Brussels, Belgium
2Center of Human Genetics, Hôpital
Erasme, Université Libre de Bruxelles,
Brussels, Belgium
3Department of Pediatrics, CHU Saint-
Pierre, Brussels, Belgium
4Department of Radiology, Hôpitaux Iris
Sud and CHU Saint-Pierre, Brussels,
Belgium
Correspondence
Caroline Gounongbé, Department of Fetal
Medicine, CHU St Pierre, Rue Haute 322,
1000 Brussels, Belgium.
Email: caroline_gounongbe@stpierre-bru.be
Abstract
We present a case of a middle interhemispheric variant of antenatal discovery associ-
ated with a de novo missense variant (NM_007129.5: c.1109G>A p.(Cys370Tyr))in
theZIC2gene. Our case represents the first prenatal description of a ZIC2 missense
mutation found in association with syntelencephaly.
KEYWORDS
middle interhemispheric variant of holoprosencephaly, missense mutation, syntelencephaly, ZIC2

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GOUNONGBÉ et al.
are known to cause HPE. Moreover, chromosomal anom-
alies (numeric: trisomy 13, 18, triploidy; structural: de-
letions or duplications involving various regions of 13q,
del(18p), del(7)(q36), dup(3)(p24-pter), del(2)(p21),
del(21)(q22.3)) are responsible of the majority of HPE
cases (up to 50%), while the remaining cases (including
both syndromic or nonsyndromic HPE) are associated to
pathogenic mutations. In nonchromosomal and nonsyn-
dromic cases, HPE is usually considered to be inherited
in an autosomal dominant mode.8-12 In particular, fourteen
genes have been implicated in nonsyndromic HPE (SHH,
ZIC2, TGIF1, SIX3, CDON, DISP1, DLL1, FGF8, GLI2,
FOXH1, GAS1, PTCH1, NODAL, TDGF1). In classic
HPE, the most commonly mutated genes are SHH (12%)
and ZIC2 (9%), which together account for ~85% of solved
cases.9-11,13 ZIC2, located on chromosome 13q32, was
firstly identified in patients with brain anomalies and har-
boring deletions involving the long arm of chromosome 13.
It belongs to the zinc finger protein of the cerebellum fam-
ily, encoding for a transcription factor that plays two dis-
tinct roles in the forebrain development. Interestingly, and
conversely to the classic HPE genes, ZIC2 mutations have
been found across the entire HPE phenotypic spectrum,
including MIHV. In particular, just few mutations (splice
variants, small deletions, and duplications) in ZIC2 have
been reported in patients displaying MIHV to date. Herein,
we describe a fetus presenting MIHV and harboring a de
novo ZIC2 missense mutation.
2
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CLINICAL REPORT
We present the sixth pregnancy of a 39-year-old woman
of Moroccan origin with no familial history of genetic dis-
eases. Although a notion of second-degree consanguinity
was known, the couple had already five healthy children.
The mother was not ill during the pregnancy and did not
take any medication, alcohol, or other toxic substances.
She was protected for toxoplasmosis and rubella. Her
serologies were negative for human immunodeficiency
virus, hepatitis B and C viruses, and syphilis. The first
trimester ultrasound performed at 13.6weeks of amenor-
rhea appeared normal. Noninvasive prenatal testing was
performed, and no chromosomal anomalies involving the
chromosomes 13, 18, and 21 were found. The morphologi-
cal ultrasound performed at 22weeks was of poor quality
and did not reveal any particular malformation. In addition,
the diabetes screening was normal. During the 3rd trimes-
ter ultrasound, we observed the absence of a septal cavity
with a fusion of the occipital horns of the lateral ventricles.
In sagittal section, the corpus callosum seemed to be pre-
sent in its anterior part but interrupted between the knee
and the body. The anterior cerebral artery had a deviating
forward path, with an absent pericallosal artery. The lateral
ventricles were moderately dilated (12mm). The cerebel-
lum was morphologically normal, and its measurement
was less than the 3rd percentile according to Hadlock. No
other anomalies were detected during the morphological
examination.
We decided to carry out fetal cerebral magnetic res-
onance imaging (fMRI) and amniocentesis in order to
perform array comparative genomic hybridization (ar-
ray-CGH). The fMRI confirmed the septal agenesis and
showed an interhemispheric parenchymal bridge connect-
ing the frontal lobes, wrongly interpreted on ultrasound
as the anterior part of the corpus callosum (Figure1). It
concluded in a complex midline malformation correspond-
ing to syntelencephaly. Moreover, fMRI confirmed the
cerebellar hypoplasia (Figure1C) and depicted a posterior
fossa arachnoid cyst (Figure 1A). On the other hand, as
array-CGH showed no pathogenic chromosomal abnor-
malities, clinical exome sequencing was proposed to the
couple. The analysis performed in duo (fetus and mother)
revealed a probably de novo variant c.1109G>A p.(Cy-
s370Tyr) in the exon 2 of the ZIC2 gene (NM_007129.5)
(Figure2A), predicted pathogenic and fitting with the brain
phenotype. Sanger sequencing of both parents confirmed
the de novo character (Figure2B), allowing classifying the
variant as probably pathogenic (Class IV). The couple de-
cided to continue the pregnancy. At 35.3weeks and after
a spontaneous and premature labor, the mother gave birth
to a girl of 2830 g, length of 46 cm, and head circum-
ference of 36cm. Her Apgar was 9-10-10 and, the pH at
the cord was 7.23 and -3 excess base. Immediate neona-
tal adaptation was excellent. The neurological assessment
on the third day of life was also reassuring, with a nor-
mal electroencephalogram and no dysmorphism. Cerebral
postnatal MRI performed at 1month of age confirmed the
hypoplasia of the falx cerebri (absent in its anterior part),
agenesis of the corpus callosum, and septum pellucidum. It
also allowed the visualization of gyration anomalies, focal
polymicrogyria as well as subependymal heterotopias of
gray matter at the left ventricular crossroads. At 3months,
the patient's follow-up was reassuring in terms of her neu-
rological evolution. At 9months, the girl was hypotonic
and a neurological physiotherapy support was set up once
a week. Then, at 11months the neurological evolution was
favorable.
3
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GENETIC ANALYSIS
The parents gave written consents for them and the index case
for the participation in this study (approved by local Hôpital
Erasme Ethical committee under P2016/236 reference). Array-
CGH was performed on a CytoSure™ Constitutional v3 8x60K

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GOUNONGBÉ et al.
array (Oxford Gene Technology) and analyzed with CytoSure
Analysis Software (Oxford Gene Technology). Clinical exome
sequencing was performed in duo (on fetal DNA extracted di-
rectly from amniotic fluid and maternal DNA [paternal DNA
was not available at that time]), using an in-house SeqCap EZ
choice XL capture (Roche Nimblegen) on a NovaSeq 6000
(Illumina) at the Brussels Interuniversity Genomics High
Throughput core (BRIGHTcore). Sequences were aligned to
the reference genome (hg19) and variants were called using
a BWA-mem Unified Genotyper-Haplotype Caller GATK
pipeline. Filtering of the variants was accomplished using
Highlander (http://sites.uclou vain.be/highl ander). Details on
pipeline and filtering are available on request. Sanger sequenc-
ing was performed for the validation of the detected ZIC2
variant. DNA was amplified using a standard PCR (primers
sequences are available upon request). PCR products were pu-
rified with BigDye® XTerminator™ Purification Kit (Applied
Biosystems, Thermo Fischer Scientific) and analyzed on a
3130XL Genetic Analyser (Biosystems).
4
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DISCUSSION
Here, we described the first MIHV case displaying a
ZIC2 missense mutation (NM_007129.5: c.1109G>A
p.(Cys370Tyr)). Our detected variant is not present in
frequency databases (gnomAD, Exome Variant Server) and
several prediction tools (SIFT, MutationTaster, FATHMM,
PolyPhen-2, DEOGEN14) agree to predict this change as del-
eterious. Like a considerable part of all ZIC2 mutations,15
our variant is localized into the well conserved zinc finger
domain of the protein. To date, only five variants (one splice
variant, one small deletion, and three small duplications) in
the ZIC2 gene have been described in MIHV (Figure2C).16,17
As a matter of fact, the majority of ZIC2 mutations gener-
ally result in classic HPE, and more often in severe structural
brain anomalies (alobar or semilobar presentations) which
account for 75% of the ZIC2-associated HPE cases in which
the phenotype is recorded.15 Moreover, our variant arose as
a de novo event in the fetus, similarly to the data provided
by several HPE cohorts showing that ZIC2 mutations appear
often de novo, with inherited mutations accounting for 27%-
30%.9,11,17 Focusing on the ZIC2-associated MIHV, ZIC2
mutations appeared de novo in three cases, while in the other
two the mutation was inherited (in one case from the father
and in the other from the mother, with an assumed germline
mosaicism).17
To date, our case represents the first prenatal report of
ZIC2-associated MIHV. Overall, only six prenatal reports
of MIHV, including 10 fetuses, are described in the liter-
ature. The presence of chromosomal anomalies solved six
cases (monosomy 13q and 21q were found in five and one
FIGURE 1 A, Midsagittal slice
showing the agenesis of the posterior
part of the corpus callosum, the fused
parenchyma on the midline (arrow), and the
retrocerebellar arachnoid cyst (arrow head).
B, Coronal slice through the third ventricle
showing the parenchymal bridge (arrow).
C, D, Axial slices showing the cerebellar
hypoplasia (C) and the parenchymal bridge
(D, arrow)
(A) (B)
(C) (D)

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case, respectively) and a normal karyotype was observed
in the remaining four cases and no other genetic tests were
performed. Among these 10 prenatal cases, medical termi-
nation of pregnancy has been carried out in seven cases.18-20
Pulitzer et al21 described a fetus carrying several extrace-
rebral malformations who died in utero. Robin et al22 re-
ported a fetus presenting an isolated syntelencephaly who
was born at term and with a normal neonatal development
at 2months. For the last prenatal case, the antenatal and
postnatal evolutions were not transmitted. Overall, our pa-
tient represents the eleventh case of antenatal MIHV di-
agnosis and the second case born alive. In particular, our
case displayed the classic radiological signs of MIHV and
its associated abnormalities, including posterior fossa mal-
formation (cerebellar hypoplasia and arachnoid cyst), sub-
ependymal heterotopias, and tight areas of polymicrogyria.
Nineteen percent of MIHV patients described in Simon's
report (21 children) have a malformation of the poste-
rior fossa, including one case of cerebellar hypoplasia.23
Interestingly, no arachnoid cysts have previously been
described in the literature. The co-occurrence of MIHV
and cerebellar malformations in patients with ZIC2 mu-
tations may be explained by its involvement in the neural
tube closure. Furthermore, malformations of the posterior
fossa (like Chiari [types 1 and 2] and cephalocele) are also
often being associated with MIHV.23 Foci of subependy-
mal heterotopia and polymicrogyria of gray matter appear
to be relatively common in MIHV cases. They have been
described in six postnatal case studies and 86% of the chil-
dren in Simon's study had cortical dysplasia.23 Regarding
the hypothalamic-pituitary axis, it appeared intact in our
report as well as in the six antenatal studies, whereas it
FIGURE 2 A, De novo c.1109G>A p.(Cys370Tyr) variant in exon 2 of the ZIC2 gene visualized in IGV. B, Presence of the mutation in
the fetus but none of the parent was confirmed by Sanger sequencing. C, Schematic representation of the ZIC2 gene and its protein (with the main
protein domains) along with the mutation described in MIHV until now (data collected from Ref.14,15). Our missense mutation is shown in red

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GOUNONGBÉ et al.
has been described as hypoplastic in two postnatal stud-
ies.24 In Simon's cohort, 23% of the children had pituitary
gland hypoplasia. Endocrine disorders are therefore also
less frequent in MIHV than in holoprosencephaly, where
midline abnormalities frequently initiate the development
of the hypothalamus and pituitary gland.1 Associated ex-
tracerebral abnormalities, including two types of cardiac
abnormalities (transposition of the great vessels, situs in-
versus with interventricular septum anomaly), have been
described in seven fetuses and two children (under 1year
of age).25,26 One patient showed diaphragmatic hernia with
vertebral malformations21 and another presented cleft pal-
ate, clubfoot, and hypospadias.27 In some reports, five fe-
tuses and 15 children displayed facial dysmorphia such as
cleft lip and palate and hypertelorism, limb abnormalities,
external genitalia, and renal hypoplasia.18,28 In these cases,
the genetic studies (karyotype) were normal, with the ex-
ception of five cases where a 13q monosomy was found.18
Moreover, craniofacial anomalies are common in HPE but
the majority of MIHV patients present moderate facial dys-
morphia or even a normal face,18,28 like in our case. The dif-
ferent embryological origin of MIHV and HPE explains this
clinical difference: MIHV arises as a consequence of failed
dorsal patterning, while HPE is associated with failed ven-
tral patterning. Thus, ZIC2 gene is believed to be responsible
for the more posterior defects presented in MIHV, being less
often associated with facial malformations. Interestingly, it
has been shown that even severe HPE linked to ZIC2 genes
have mild facial dysmorphic features. However, a typical fa-
cial phenotype can be associated in ZIC2-associated HPE, in-
cluding bitemporal narrowing, up slanting palpebral fissures,
flat nasal bridge, short nose with anteverted nares, broad and
deep philtrum, and large ears.17
Regarding the postnatal clinical outcome of our case,
the patient has no dysmorphism and she seems to pres-
ent subnormal neurological development at 11 months.
Previous studies identified spasticity as the main clin-
ical sign of MIHV (found in 86% of children inLewis's
study28), probably due to the fact that the abnormal fusion
zone of the cortex is close to the motor cortex. In addition,
57% of patients display some degree of hypotonia, over-
lapping with our patient who needed physiotherapy sup-
port for hypotonia at 9months. Although choreoathetosis
has been frequently found in semilobar holoprosencephaly,
no cases displaying this symptom have been described in
MIHV.28 This is probably due to the fact that, conversely
to HPE, caudate and lenticular nuclei are normal in MIHV.
In conclusion, our findings expand the mutational spec-
trum and delineate the prenatal phenotype found in ZIC2-
associated MIHV. As there are limited prenatal MIHV cases
harboring ZIC2 mutations, this work may be an important
support for the prenatal diagnosis and the subsequent man-
agement of MIHV cases.
ACKNOWLEDGMENTS
The authors thank the family who gave its written consent for
this publication. The authors acknowledge all the members of
the Molecular Genetics laboratory of Hôpital Erasme for their
valuable technical assistance. They also thank all the mem-
bers of the Saint-Pierre UHC fetal medicine team for their
participation in the management of this clinical case. The au-
thors acknowledge Guillaume Smits for critically reading the
manuscript. MM is supported by a FNRS-FRIA fellowship.
CONFLICT OF INTEREST
None declared.
AUTHOR CONTRIBUTION
CG and MM: wrote the article. VG: examined the patient.
MD and MM: analyzed the genetics data. PJ: served as the
author of the postnatal MRI. MC: served as the author of the
antenatal MRI. MC and JD: coordinated and approved the
final version of the manuscript.
ORCID
Caroline Gounongbé https://orcid.
org/0000-0002-7408-883X
Martina Marangoni https://orcid.
org/0000-0002-9416-2400
Marie Cassart https://orcid.org/0000-0003-2766-1805
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How to cite this article: Gounongbé C, Marangoni M,
Gouder de Beauregard V, et al. Middle interhemispheric
variant of holoprosencephaly: First prenatal report of a
ZIC2 missense mutation. Clin Case Rep. 2020;8:1287–
1292. https://doi.org/10.1002/ccr3.2896