4 | Discussion
This study used WES and CNV analyses to identify genetic causes of
microcephaly in Korean patients. The overall diagnostic rate was 47.5%;
with 12 cases (30%) identified by WES and seven cases by CNV analysis.
The nine of all variants found by WES, including five novel ones, werede novo and autosomal dominant traits. Of the 40 patients
included in this study, 34 patients (85%) had primary microcephaly. Six
showed various structural anomalies by brain MRI, whereas the others
showed normal brain structures despite microcephaly. Hypotonia and
developmental delay were the most frequent accompanying symptoms.
Seizure was the next most frequent symptom (n = 27, 65%), and 11
patients had intractable seizures.
Microcephaly can present as non-syndromic or with various hereditary
syndromic features (von der Hagen et al., 2014). According to the OMIM
database, there are over 900 OMIM phenotype and genes related to
microcephaly entered to date. In the present study, the diagnostic yield
of WES (PVs and LPVs) was 30% and was comparable to that found in
previous reports of genetic causes of microcephaly (Boonsawat et al.,
2019; Rump et al., 2016; Shaheen et al., 2019). In general, the
diagnostic yield of WES in neurodevelopmental disorders ranges from
17.5% to 29% (Marques Matos, Alonso, & Leao, 2019; Vissers et al.,
2017). The yield depends on several factors such as phenotypic
distinctiveness of the disease, genes associated with the phenotype,
sequence read depth, bioinformatic filtering, and/or level of clinical
medical review (Farwell et al., 2015). Some neurologic disorders are
more closely associated with muscle disease, ataxia, and epilepsy, which
are known to have heterogeneous genetic causes (Farwell et al., 2015;
Marques Matos, Alonso, & Leao, 2019). Thus, WES should be the
first-line test for complex diseases presumed extensive genetic causes,
based on considerations of diagnostic yield and economic cost (Marques
Matos, Alonso, & Leao, 2019; Vissers et al., 2017). Similar to those
identified in previous reports, the variants found in this study were
highly diverse. This study reconfirms the utility of WES in determining
the genetic causes of microcephaly.
In this study, the CNV analysis increased the diagnostic rate from 30%
to 47.5%. This rate was similar to that reported in a recent study by
Boonsawat et al (2019). The diagnostic rate of pathogenic CNVs in
neurodevelopmental disorders, including primary microcephaly, has been
reported to be 13.2 – 20.8% (Jang et al., 2019; Shoukier et al.,
2013). According to a previous study, combined WES and CNV analyses
better enable diagnosis of rare neurologic disorders, with a 47.8%
diagnostic rate comparing to that with WES alone (32.7%) (Jiao et al.,
2019), which is consistent with the results of this study. A recent
study showed 11 definite and 7 probable CNVs in 53 patients with primary
microcephaly (Tsoutsou et al., 2017). Therefore,
WES with CNV analysis may be the
mostly effective approach to diagnosing the underlying causes of
microcephaly. A diagnosis of microcephaly via WES and CNV analysis can
help determine the genetic mechanism of the disease and predict the
prognosis. In addition, it may provide information to patients for
future reproductive decisions and genetic counseling, including
opportunities for targeted therapies.
In the present study, most of the detected variants showed an autosomal
dominant inheritance pattern. Two variants were associated with
autosomal recessive and X-linked recessive inheritance respectively.
Previous studies dealt with congenital microcephaly in consanguineous
families and mainly focused on neurodevelopmental defects in the fetal
period (Darvish et al., 2010; Sajid Hussain et al., 2013; Shaheen et
al., 2019). They classified cases of microcephaly as autosomal recessive
primary microcephaly (MCPH), and 18 loci in MCPH genes have been
revealed (Jayaraman, Bae, & Walsh, 2018). In our patients, no mutations
were identified in MCPH1 , WDR62 , CDK5RAP2 ,CEP152 , ASPM , CENPJ , STIL , CEP63 ,CEP135 , CASC5 , PHC1 , CDK6 , CENPE ,SASS6 , MFSD2A , ANKLE2 , CIT , or WDFY3genes. Our results suggest that autosomal dominant disorders are
highly prevalent among Korean patients with microcephaly. The
microcephaly-associated genes identified in this study were diverse, and
no genes were found to be predominant causes of microcephaly. This
suggests that various genes may lead to microcephaly, as reported
previously (Shaheen et al., 2019). These genes are involved in neuronal
myelination, neurotransmission, or regulation of neuronal excitability,
rather than centrosome-related neurogenesis and DNA damage repair
process (Woods, & Parker, 2013).
In this study, 34 patients showed normal brain structures despite small
brain size. In the six patients (15%) with structural abnormalities
observed by brain MRI, only one patient was found to have a causative
genetic mutation in the CASK gene. The CASK gene encodes a
membrane-associated guanylate kinase and is involved in neurotransmitter
regulation, axon branching, and dendritic outgrowth. The patient with
this gene variant showed a wide range of X-linked intellectual
disabilities, including microcephaly with pontine and cerebellar
hypoplasia (MICPCH) syndrome, nystagmus, dysmorphic faces, and hypotonia
(Hayashi et al., 2017). Because this is located on the X chromosome,
most cases of CASK mutation are females, and affected males are
very rare and show severe manifestations (Cristofoli, Devriendt, Davis,
Van Esch, & Vermeesch, 2018). We found a CASK mutation through
CMA in a female patient with MICPCH syndrome. She was born at term
without perinatal complications. Her HC at birth was 32.2 cm, and the
fourth ventricle was extensively dilated according to a brain
ultrasonogram performed on the second day of life. She showed poor
growth in all body gauges and dysmorphic facial features such as arched
eyebrows, midline hypoplasia, long philtrum, and micrognathia. Her brain
MRI was consistent with the criteria for microcephaly and
pontocerebellar hypoplasia, and her achievement of developmental
milestones was assessed at the five-month level at the age of one year
(Figure 2).
Racially homogeneous Korean patients with microcephaly, with no
consanguineous family members, were enrolled in this study. This is the
first study delineating the genetic causes of microcephaly in East Asia.
The patients in this study showed the same clinical characteristics that
were reported in previous studies, including infantile hypotonia and
developmental delay. Epilepsy was observed in 65% of the patients, and
about half of these patients showed an intractable clinical course. In
addition, movement disorders, behavioral problems, and autism spectrum
disorder were noted, which was similar to features in a recent report
(Boonsawat et al., 2019). However, our study showed different genetic
peculiarities from those reported previously for patients in the Middle
East or Europe, in which autosomal recessive inheritance was frequent.
In our study, 75% of detected variants were autosomal dominant; only
one sibling pair had an SMC1A gene variant with X-linked
inheritance. These results suggest that the genetic spectrum of
microcephaly may differ between ethnic groups, even if clinical
characteristics are similar. Therefore, consideration of the racial
background might be helpful in interpreting WES results.
This study had several limitations. First, the number of patients in our
study was small to determine the detection rate for genetic causes of
microcephaly. Second, a functional study of detected VUSs was not
performed. Although not classified as PVs or LPVs, some of the VUSs
found in this study may be useful in determining the genetic cause of
microcephaly through additional studies to verify gene function. In
addition, we did not identify cases of somatic mosaicism and balanced
translocation, which it is difficult to confirm by WES or CMA. However,
somatic mosaicism and balanced translocation have rarely been reported
as genetic causes of microcephaly.
We conclude that conducting WES and CMA for patients with microcephaly
is useful and time/cost-effective for genetic diagnosis and treatment
considerations. Unlike other studies, autosomal recessive microcephaly
was rare; autosomal dominant was predominant mode of inheritance in
Korean patients. This study confirmed that microcephaly is a condition
with genotypically and phenotypically heterogeneous causes.
Interdisciplinary cooperation between molecular geneticists and
clinicians is important to facilitate diagnosis of the underlying causes
of microcephaly.