A biallelic frameshift indel in PPP1R35 as a cause of primary microcephaly

Abstract

Protein phosphatase 1 regulatory subunit 35 (PPP1R35) encodes a centrosomal protein required for recruiting microtubule-binding elongation machinery. Several proteins in this centriole biogenesis pathway correspond to established primary microcephaly (MCPH) genes, and multiple model organism studies hypothesize PPP1R35 as a candidate MCPH gene. Here, using exome sequencing (ES) and family-based rare variant analyses, we report a homozygous, frameshifting indel deleting the canonical stop codon in the last exon of PPP1R35 [Chr7: c.753_*3delGGAAGCGTAGACCinsCG (p.Trp251Cysfs*22)]; the variant allele maps in a 3.7 Mb block of absence of heterozygosity (AOH) in a proband with severe MCPH (-4.3 SD at birth, -6.1 SD by 42 months), pachygyria, and global developmental delay from a consanguineous Turkish kindred. Droplet digital PCR (ddPCR) confirmed mutant mRNA expression in fibroblasts. In silico prediction of the translation of mutant PPP1R35 is expected to be elongated by 18 amino acids before encountering a downstream stop codon. This complex indel allele is absent in public databases (ClinVar, gnomAD, ARIC, 1000 genomes) and our in-house database of 14,000+ exomes including 1800+ Turkish exomes supporting predicted pathogenicity. Comprehensive literature searches for PPP1R35 variants yielded two probands affected with severe microcephaly (-15 SD and -12 SD) with the same homozygous indel from a single, consanguineous, Iranian family from a cohort of 404 predominantly Iranian families. The lack of heterozygous cases in two large cohorts representative of the genetic background of these two families decreased our suspicion of a founder allele and supports the contention of a recurrent mutation. We propose two potential secondary structure mutagenesis models for the origin of this variant allele mediated by hairpin formation between complementary GC rich segments flanking the stop codon via secondary structure mutagenesis.

Keywords: PPP1R35; centriole; centrosome; complex indel; microcephaly; primary microcephaly.

Figure 1. Perturbations in the Centriole Biogenesis Pathway are Known to Cause Primary Microcephaly. Adapted from (Sydor et al., 2018)

(A) Depiction of structural components of centriole in relation to PPP1R35. (B) Pathway utilized by centrosomal proteins functioning in centriole biogenesis. Proteins in green have been implicated in a known Mendelian disease (per OMIM) with a phenotype of microcephaly. Arrows indicate the progressive steps of centriole elongation. PLK4 phosphorylates STIL to interact with SAS6 and with CEP135 forms the centrosomal cartwheel. Following initial centriole formation, PPP1R35 and RTTN localize to the cartwheel where elongation will occur, and CPAP interacts with CEP120 and SPICE to regulate centriole elongation. CEP295 is recruited to the centriole and recruits POC1B and POC5 with subsequent addition of CEP152.

Figure 2. Pedigrees, genotyping, and phenotyping of BAB9269, M8600491 III:2, and M8600491 III:3 for candidate disease gene PPP1R35.

(A) Pedigree of Turkish family with PPP1R35 genotype indicated, proband identified by black arrow, and affected individuals shaded black. Sanger sequencing confirms autosomal recessive Mendelian segregation of the complex indel PPP1R35 [Chr7: c.753_*3delGGAAGCGTAGACCinsCG (p.Trp251Cysfs*22)]. (B) Microcephaly of proband BAB9269 shown at 1 week, 5 months, and 3 years and 5 months after birth. Microcephaly-related dysmorphic features including a sloped forehead, large and constricted ears, a smooth long philtrum, and micrognathia can be appreciated. (C) Mid-sagittal T2 sequence showing extremely small head size consistent with primary microcephaly, foreshortening and thinning of corpus callosum (red arrow) and mild to moderate cerebellar volume loss that is most prominent at the superior vermis. (D) Axial T2 sequence showing increased extra-axial CSF spaces, pachygyria with anterior posterior gradient (top green arrow points to left frontal lobe at the anterior aspect, and bottom green arrow points to left parietal lobe at the posterior aspect of the gradient), dysmorphic ventricular system and abnormal delayed myelination of the internal capsule. (E) Coronal T2 sequence showing generous extra-axial CSF spaces, pachygyria, lack of myelination for age cerebellar volume loss. Red arrow shows prominence of left cerebellar folia. (F) Plot of ratio of reads containing variants versus chromosome 7 genomic coordinates for BAB9269 with absence of heterozygosity (AOH) shaded in grey overlaps 3.7 Mb. Location of PPP1R35 variant contained within AOH region is displayed by the red horizontal bar. (G) Droplet digital PCR showed comparable gene expression of the mutant and wild-type alleles in fibroblasts of family members confirming expression of the mutant mRNA. (H) Pedigree of Iranian family with PPP1R35 genotype indicated, affected individuals shaded black. Sanger sequencing confirms autosomal recessive Mendelian segregation of the complex indel PPP1R35 [Chr7: c.753_*3delGGAAGCGTAGACCinsCG (p.Trp251Cysfs*22)]. (I) Microcephaly-related dysmorphic features including sloping forehead, large nose, maxillary hyperplasia, and misalignment of teeth can be appreciated.