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.