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Mutation in CEP135 causing primary microcephaly and subcortical heterotopia

Center for Molecular Medicine Cologne (CMMC), Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Department of Pediatrics, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Department of Child Neurology, Justus-Liebig-University Giessen, Giessen, Germany Cologne Center for Genomics (CCG), University of Cologne, Cologne, Germany Institute of Biochemistry I, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany


To the Editor
Autosomal recessive primary microcephaly (MCPH) is characterized by congenital microcephaly (>2-3 standard deviations [SD] below the mean for age and gender) and intellectual disability without additional syndromic features (Alcantara & O'Driscoll, 2014). Up to now, 18 genes have been linked to MCPH, all of them involved in the neurogenesis of radial glia cells as the primary progenitor cells of the developing cerebral cortex (Jayaraman, Bae, & Walsh, 2018). Most of these genes encode centrosomal proteins involved in centriole biogenesis (WDR62, CDK5RAP2, CASC5, ASPM, CENPJ, STIL, CEP135, CEP152, SASS6) (Jayaraman et al., 2018). Others are involved in DNA replication and repair, kinetochore function, transmembrane or intracellular transport, autophagy, or cell polarity (Jayaraman et al., 2018).
Barkovic et al. provide a comprehensive review on malformations of cortical development (MCD), a large heterogenous group of defects in cerebral cortex formation, resulting from dysfunctional neurogenesis, neuronal migration, and postmigrational development (Barkovich, Guerrini, Kuzniecky, Jackson, & Dobyns, 2012). MCPH was classified as group IA, representing a reduced size of the cerebral cortex due to reduced proliferation, generally without gross morphological abnormalities (Barkovich et al., 2012). Nonetheless, cases with structural changes have been reported, for example, cortical malformations and subcortical heterotopia in WDR62 patients and periventricular heterotopia in MCPH1 (Nicholas et al., 2010;Trimborn et al., 2004;Yu et al., 2010).
Several MCD are predominantly characterized by clusters of neurons unable to migrate to their proper position in the cortex, referred to as heterotopic gray matter brain malformation (HET) (Oegema, Barkovich, Mancini, Guerrini, & Dobyns, 2019). Periventricular nodular heterotopia (PNH) is the most common subtype, formed by nodules in the wall of the lateral ventricles (Oegema et al., 2019). Recently, a new classification for the less common subcortical heterotopias (SUBH) was introduced (Oegema et al., 2019). SUBH are considered a different disease entity, defined as heterotopic gray matter located in the white matter between the lateral ventricles and the cortex (Oegema et al., 2019).
Mutations in the Centrosomal Protein 135 gene (CEP135) are a very rare cause of primary microcephaly (MCPH8, OMIM 614673), since only three families have been reported without brain-MRI, and detailed information on brain morphology have been lacking. Here, we report a patient presenting with epilepsy as new feature in CEP135 related disease and provide the first brain-MRI images identifying subcortical heterotopia as underlying cerebral malformation.
The female patient is the only child of healthy unrelated German parents. Microcephaly had already been diagnosed prenatally. The girl was born spontaneously at term without complications after an otherwise uneventful pregnancy. Head circumference at birth was 31 cm (1.5 cm <third percentile, −3.6 SD) and 46.5 cm (2.5 cm <third percentile, −5 SD) at age 8 years. Her early motor and speech development were delayed, but she finished regular school and her overall IQ was 79 (WISCI-IV at age 15 years). First complex-partial seizures started at age 10 years, which initially were well-controlled by Oxcarbazepine.
At age 18 years, she suffered from generalized tonic-clonic-seizures requiring hospitalization. Her head circumference was 48.5 cm (3.5 cm <third percentile, −6.5 SD), height 165.3 cm (33rd percentile), and weight 69.5 kg (87th percentile) at 18 years of age. Apart from a sloping forehead and a long thin nose, no dysmorphisms were apparent. Brain-MRI displayed marked bilateral nodular heterotopia in the peritrigonal regions, defined as group 1a according to the most recent HET classification of Oegema et al. (Figure 1) (Oegema et al., 2019).
No further structural abnormalities were apparent, especially gyral pattern and myelination were unremarkable.
The variant has not been reported in any database and was classified as pathogenic (PVS1, PM2, PM3) according to the ACMG classification (Richards et al., 2015). In addition, two heterozygous variants in the FAT4 gene (NM_024582.4), known to cause autosomal-recessive syndromic periventricular nodular heterotopia were apparent (Alders et al., 2014). But as both variants in FAT4 were inherited form the healthy mother and the reported FAT4-phenotypes including the pattern of brain malformation and pathognomonic facial features did not match our patient, they were not considered disease causing. A missense variant c.12778G>A (p.Asp4260Asn) was detected in HUWE1 (NM_031407.6), variants in HUWE1 are known to cause dominant Xlinked intellectual disability of the Turner Type (OMIM: 309590). For HUWE1 patients multiple brain-MRI reports are available, none of them depicting cortical brain malformations, and in animal models, no structural nervous system anomalies were reported (Moortgat et al., 2018;Vandewalle et al., 2013). Thus, we considered this variant to be a less probable cause of the subcortical nodular heterotopia compared to the CEP135 variant. Nonetheless, it may act as a disease modifier for the intellectual disability depending on the degree of Xchromosome inactivation. No further likely pathogenic variants in genes previously associated with MCPH or MCD were identified, nor were convincing novel candidate-genes found (see Supplementary Material). Still a modifying effect of the reported secondary variants on the phenotype together with other unknown genetic disease modifiers has to be considered.
Because we have a whole-exome and not a whole genome sequencing, it is not possible to exclude the low probability of a digenic inheritance of deep intronic variants or structural variants in a second gene modifying the phenotype (Posey et al., 2017).
Homozygous truncating mutations in CEP135 have first been identified in two Pakistani families with primary microcephaly, learning disability, speech impairment, and sloping forehead (Farooq et al., 2016;Hussain et al., 2012). In addition, a two-year-old boy was reported presenting with primordial dwarfism, spasticity, and developmental delay. His dysmorphic features included microcephaly, scoliosis, hypotelorism, sloping forehead, small face, and broad nose F I G U R E 1 MR images of the brain at age 18 years. Notice bilateral nodular heterotopia in the peritrigonal regions (arrows) in T2-weighted axial (b) and coronal (c) images. See also normal gyration and myelination as well as normal corpus callosum, cerebellum, and brainstem (a) (Shaheen et al., 2019). All of these cases presented severe microcephaly with a head circumference of −10 to −13 standard deviation (SD).
Our patient has milder primary microcephaly (−6.5 SD) and mild learning disabilities.
Noteworthy in our case are the novel features: epilepsy and subcortical heterotopia group 1a associated with CEP135 mutations.
CEP135 is involved in centrosomal microtubule assembly by serving as a linker protein directly connecting the central hub protein HsSAS-6 to the outer microtubule (Lin et al., 2013). It consists of an N-terminal microtubule interacting domain, a central CENPJ interacting domain, and a C-terminal HsSAS-6 interacting domain (Lin et al., 2013). Our stop mutation in CEP135 presumably results in a loss of the last sixth coiled-coil domains and is predicted to be deleterious as it covers part of the region responsible for interaction with HsSAS-6 (MCPH14, OMIM 616402). In addition, the transcript is predicted to undergo nonsense mediated decay (NMD) which would lead to a complete loss of function. The milder microcephaly and subcortical heterotopia could represent a hypomorphic allele manifestation in our case. The premature stop codon near the C-terminus of CEP135 might only lead to partial NMD with residual activity in contrast to the previously complete loss-of-function mutations causing a more severe microcephaly.
Here, we report the first patient with a homozygous CEP135 nonsense mutation and subcortical heterotopia; thus CEP135 mutations shall be added to the genetic differential diagnosis of subcortical nodular heterotopia of the peritrigonal region (SUBH 1a). Furthermore, our findings emphasize the effect of CEP135 related centrosomal disruption on neuronal migration in human brain development.

CONFLICT OF INTEREST
All authors declare that they have no conflict of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.