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. 2015 Jul 1;24(13):3732-41.
doi: 10.1093/hmg/ddv117. Epub 2015 Apr 8.

Germline recessive mutations in PI4KA are associated with perisylvian polymicrogyria, cerebellar hypoplasia and arthrogryposis

Alistair T Pagnamenta  1 , Malcolm F Howard  1 , Eva Wisniewski  2 , Niko Popitsch  1 , Samantha J L Knight  1 , David A Keays  3 , Gerardine Quaghebeur  4 , Helen Cox  5 , Phillip Cox  6 , Tamas Balla  2 , Jenny C Taylor  1 , Usha Kini  7
Affiliations

Germline recessive mutations in PI4KA are associated with perisylvian polymicrogyria, cerebellar hypoplasia and arthrogryposis

Alistair T Pagnamenta et al. Hum Mol Genet. .

Abstract

Polymicrogyria (PMG) is a structural brain abnormality involving the cerebral cortex that results from impaired neuronal migration and although several genes have been implicated, many cases remain unsolved. In this study, exome sequencing in a family where three fetuses had all been diagnosed with PMG and cerebellar hypoplasia allowed us to identify regions of the genome for which both chromosomes were shared identical-by-descent, reducing the search space for causative variants to 8.6% of the genome. In these regions, the only plausibly pathogenic mutations were compound heterozygous variants in PI4KA, which Sanger sequencing confirmed segregated consistent with autosomal recessive inheritance. The paternally transmitted variant predicted a premature stop mutation (c.2386C>T; p.R796X), whereas the maternally transmitted variant predicted a missense substitution (c.5560G>A; p.D1854N) at a conserved residue within the catalytic domain. Functional studies using expressed wild-type or mutant PI4KA enzyme confirmed the importance of p.D1854 for kinase activity. Our results emphasize the importance of phosphoinositide signalling in early brain development.

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Figures

Figure 1.
Figure 1.
Pedigree, photographs, histology and MRI images of affected fetuses. (A) Pedigree showing three terminations of pregnancies (II-1 at 34 weeks, II-2 at 28 weeks and II-3 at 16 weeks) and three miscarriages. Shading indicates severe congenital abnormalities which were identified initially by prenatal ultrasound. WES was performed on the five individuals labelled, whereas SNP arrays were only performed on I-1, I-2 and II-2. (B) Photographs showing three female fetuses, all with arthrogryposis and micrognathia. (C) Macroscopic appearance of fixed brain of fetus II-1 with PMG and small cerebellum. (D) Histopathological appearances of the brain. (i) Dysplasia of the cerebellar dentate nucleus. The nucleus is irregular and fragmented rather than forming a single continuous undulating ribbon. ((ii) and (iii)) PMG of the cerebral cortex—abnormal folding of the cortex which is disorganised with fusion of the small cortical folds in II-1 and II-2, respectively. (E) Fetal MRI scans ((i) and (ii)) showing PMG and small cerebellum in II-1 at 34 weeks, ((iii) and (iv)) showing small cerebellum and delayed sulcation in II-2 at 28 weeks. Arrows indicate the regions where PMG and cerebellar atrophy are most apparent.
Figure 2.
Figure 2.
Genetic analysis. (A) Pairwise analysis for regions of identity-by-descent (IBD2). Absolute differences in allelic ratio are shown plotted against chromosome position. The largest five regions of shared IBD2 identified on chromosomes 1, 2, 4, 18 and 22 are labelled. In all cases, where IBD2 is detected in II-1 versus II-2 and II-1 versus II-3, IBD2 is also seen in II-2 versus II-3 (as expected). (B) Sanger sequencing confirming that the variants segregated consistent with recessive inheritance. Sequencing was performed in both directions. Both mutations were also detected in RNA from the respective parent. (C) Exome data from II-1 visualized in IGV. The arrows indicate two reads with an apparent CC>GT dinucleotide substitution, but which are really artefacts due to mismapping of pseudogene sequence. Reads that support the c.5560G>A variant (here shown on the +ve genomic strand as a C>T alteration) do not harbour the dinucleotide change, indicating that c.5560G>A is unlikely to be a mismapping artefact. (D) Image from the UCSC Genome Browser showing the positions of primers used to validate the missense variant. Sequences from PI4KAP1 and PI4KAP2 were mapped back to PI4KA using the BLAT tool such that any mismatches are shown by vertical red lines. The redesigned +ve strand primer has a 3′ mismatch with the pseudogene sequences, as shown by the asterisk. The −ve strand primer also has a mismatch in the middle. The position of the CC>GT dinucleotide mismatch is shown (by an arrow) at chr22:21 067 589-90. The window shown corresponds to chr22:21 067 417-21 067 723 (hg19).
Figure 3.
Figure 3.
Amino acid conservation and structural modelling. (A) Sequence alignment of human type III PI 4-kinases, PI 3-kinases and PI kinase-related kinases within their catalytic domains. Highly conserved residues are highlighted in yellow and those with conservative substitutions with blue. Residues conserved between at least some of the groups are highlighted with green. The affected aspartate residue is labelled by a red asterisk. (B) Sequence alignment of orthologues of PI4KA from different eukaryotes. (C) The structure of the ATP-binding pocket of PI4KB with the ATP competitive inhibitor, PIK93 based on 4D0L (33). Red and yellow colours represent the C- and N-terminal halves of the catalytic site, respectively, while light blue is the N-terminal helical domain. Green indicates the activation loop. The conserved Aspartate residue (D579) is coloured magenta. (D) Model of the catalytic domain of PI4KA with ATP bound form (29) showing very similar features.
Figure 4.
Figure 4.
Functional studies of enzyme activity and protein stability. (A) In vitro PI 4-kinase activity assay performed on immunoprecipitated human PI4KA enzymes expressed in COS-7 cells. Wild-type or p.D1854N mutant enzymes were prepared and assayed with the ADP-GLO kinase assay as described under Materials and Methods. Activities were expressed as per cent of wild-type. Results are the means ± S.E.M from three independent kinase preparations. Note that the activity of the mutant enzyme is indistinguishable from the two controls, one measured from immunoprecipitates of AT1a angiotensin receptors (AT1R) and the other is assaying the wild-type enzyme without its substrate, PI (w/o PI). (B) Western blot analysis showing comparable expression of the wild-type and mutant PI4KA enzymes. Left lanes show the expression from the cell lysate and right lanes from the immunoprecipitates used for the kinase assay. Results shown are representative of three similar observations. The presence of the AT1 receptor in the lysates is not showing well in this exposure setting but the immature form of the receptor is visible (shown by the arrow) and the fully glycosylated receptor appears as a faint smear between the 82 and 115 kDa markers.
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