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

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.     

Apically extruded debris of different file systems used with various kinematic movements during retreatment: An in vitro study

This study aimed to examine the amount of apically extruded debris and time during retreatment with five current file systems, which exhibit various kinematic movements. One hundred upper central incisors were shaped with manual files and filled using the thermoplastic injection method. The root canal fillings in each group (n = 20) were removed using the Genius (GN), ProTaper Next (PTN), Reciproc (RCP) Blue, Tango-Endo (TE) and Twisted File Adaptive (TFA) file systems. The apically extruded debris was collected in preweighed Eppendorf tubes. Time to reach working length and total time were also recorded. The PTN, RCP Blue and TFA instruments caused significantly less apically extruded debris and shorter total retreatment time than the GN and TE file systems (p < 0.05). The time to reach the working length was the shortest in the PTN group compared to the other groups (p < 0.05). All file systems extruded debris while removing the root canal filling.