Novel mutations consolidate KCTD7 as a progressive myoclonus epilepsy gene

Abstract

Background: The progressive myoclonus epilepsies (PMEs) comprise a group of clinically and genetically heterogeneous disorders characterised by myoclonus, epilepsy, and neurological deterioration. This study aimed to identify the underlying gene(s) in childhood onset PME patients with unknown molecular genetic background.

Methods: Homozygosity mapping was applied on genome-wide single nucleotide polymorphism data of 18 Turkish patients. The potassium channel tetramerisation domain-containing 7 (KCTD7) gene, previously associated with PME in a single inbred family, was screened for mutations. The spatiotemporal expression of KCTD7 was assessed in cellular cultures and mouse brain tissue.

Results: Overlapping homozygosity in 8/18 patients defined a 1.5 Mb segment on 7q11.21 as the major candidate locus. Screening of the positional candidate gene KCTD7 revealed homozygous missense mutations in two of the eight cases. Screening of KCTD7 in a further 132 PME patients revealed four additional mutations (two missense, one in-frame deletion, and one frameshift-causing) in five families. Eight patients presented with myoclonus and epilepsy and one with ataxia, the mean age of onset being 19 months. Within 2 years after onset, progressive loss of mental and motor skills ensued leading to severe dementia and motor handicap. KCTD7 showed cytosolic localisation and predominant neuronal expression, with widespread expression throughout the brain. None of three polypeptides carrying patient missense mutations affected the subcellular distribution of KCTD7.

Discussion: These data confirm the causality of KCTD7 defects in PME, and imply that KCTD7 mutation screening should be considered in PME patients with onset around 2 years of age followed by rapid mental and motor deterioration.

Figures

Figure 1. Results of homozygosity analysis at the candidate locus on 7q11.1-q13

The x-axis shows the genomic position in Megabases (Mb) in NCBI build 37. The horizontal bars indicate the lengths and positions of the homozygous segments found in each of the eight patients. Asterisks at the left end of patients’ N4103 and L3 haplotype blocks indicate that these patients carry mutations in KCTD7. The segments for patients N4103 and N4603 extend beyond the illustrated region (designated by the two diagonal lines). The minimum overlap interval of 1.5 Mb is marked by the vertical black lines. The position of KCTD7 is indicated with an arrow.

Figure 2. KCTD7 gene mutations

The seven families in which mutations in KCTD7 have been identified are illustrated. For each family member the chromatogram showing the change identified is given below the corresponding pedigree symbol. For the deletion mutations identified in families N126 and N151 the control sequence is shown below the mutation sequence in the affected offspring (N12604, N12606 and N15103). A red line is used to highlight the mutations, except for mutations c.861_863delATG and c.594delC identified in families N126 and N151 respectively, where the red line shows the deleted bases in control sequence. At the mutation sites the reference nucleotides are given as superscript while the mutant nucleotides as subscript. The positions at which nucleotide deletions have been detected, are indicated by red arrows. All sequence chromatograms are shown in the forward orientation.

Figure 3. Schematic representation of the disease course in the nine patients carrying KCTD7 mutations

The x axis shows the age in years. The y axis shows the symptoms. Each bar represents one symptom. The length of each bar represents the age range between which the symptom develops. The black vertical lines on the bars show the mean age of onset of individual symptoms/findings. On the right side of the graph the number of patients (N) that showed the symptom is given.

Figure 4. Intracellular distribution and expression of wild-type and mutant KCTD7

(A-C) In BHK cells transfected with HAKCTD7wt, HAKCTD7p.R94W, and HAKCTD7p.D115Y the wild-type and mutant polypeptides showed a similar widespread distribution across the cell, compatible with cytosolic localization. (D-F) The signal obtained for KCTD7 when HA-tagged KCTD7 (D) constructs were co-transfected with the plasma membrane marker wtCD8 (E) in BHK cells and the cytosol was removed by saponin treatment is much weaker, suggesting that KCTD7 is a soluble cytoplasmic protein (D versus A). (D, F) The nuclear staining observed upon cytoplasm removal is considered to be unspecific and to have occurred through permeabilization of also the nuclear membrane. (G-L) Double-stainings in BHK cells for KCTD7 (G, J) and markers for endosomes (H), or ER (K) did not reveal co-localization. (I, L). The nucleus was stained blue with Hoechst. (M) In western blot analyses wild-type (HAKCTD7wt and KCTD7HAwt) and mutant KCTD7 (HAKCTD7p.R94W, HAKCTD7p.D115Y, and HAKCTD7p.N273I) was visualized as an apparently 37 kDa band of which 2 kDa correspond to the HA-tag. Comparison of the mutant constructs to the wild-type protein did not reveal differences in the levels of protein expression. A β–tubulin (load) was used to affirm that equal amounts of protein were loaded into each lane. The molecular weight of the wild-type and mutant KCTD7 proteins is given on the right. The scale bars correspond to 20 μm.

Figure 5. Expression of endogenous KCTD7 in mouse embryonic neuronal cultures and brain tissue

Endogenous KCTD7 was detected with the anti-KCTD7 antibody. (A-G) No co-localization was observed with either β–tubulin (A), pre- (B-D) or post-synaptic (E, F), or lysosomal markers (G). (C, D) Magnified images of the neurite varicosities (C, white arrows) and the tip of a growth cone (D, white arrows). (H-J) In brain sections from P5, P14 and 4 month-old mice, KCTD7 was prominently expressed in the cerebellar Purkinje cells. (H-J) The neuronal progenitor cells detected with Cdc47 (H), the astrocytes with GFAP (I), and some neuronal populations such as the PV-immunopositive interneurons of the cerebellar molecular layer (J), are not immunopositive for KCTD7. (H-J) KCTD7 resides in the soma of the neurons although expression in the neurites of Purkinje cells can also be seen (J). (H-J) White arrowheads point to Purkinje cells (H-J), and white arrows to cerebellar granule cells (H, I) or cerebellar interneurons (J). (K) Cell lysates were evaluated for expression of KCTD7 from embryonic hippocampal neurons, cerebellar neurons, astrocyte and microglia cell cultures, and cell lysates from homogenized cerebella of different postnatal time points (P5, P14, 1mo and 2mo). Endogenous KCTD7 is detected as a single band with a molecular weight of 35 kDa. KCTD7 expression is already seen in embryonic hippocampal as well as in postnatal cerebellar granular neurons (lanes 1-2). In contrast with the neuronal cells, KCTD7 does not seem to be expressed in the microglia (lane 3) or astrocytes (lane 4). The faint band detected in the astrocyte lysate is likely to be due to contamination of the astrocyte culture from neuronal cells. The KCTD7 expression levels in cerebellar lysates from P5, P14, 1mo, and 2mo-old mice remained unaltered across the evaluated time-points, suggesting constant and unaffected protein expression throughout the maturation (lanes 5-8). Immunostaining with β–actin was used as a control for protein loading. The molecular weight of KCTD7 is given on the left. The scale bar is corresponding to 20 μm.