Computational protocol: Fam83h null mice support a neomorphic mechanism for human ADHCAI

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Protocol publication

[…] Peripheral whole blood (5 mL) or saliva (2 mL) was obtained from recruited individuals and genomic DNA was isolated using the QIAamp DNA Blood Maxi Kit (51194; Qiagen, Valencia, CA) or Saliva DNA Collection, Preservation, and Isolation Kit (RU35700; Norgen Biotek Corporation; Thorold, Canada), respectively. The quality and quantity of the extracted DNA samples were determined by spectrophotometry at OD260 and OD280.Genomic DNA from the proband of Family 1 was characterized by whole‐exome sequencing (Yale Center for Genome Analysis, West Haven, CT, USA) in which the procedure was previously described (Choi et al. ). In brief, the genomic DNA was captured with NimblGen v2.0 exome capture reagent (Roche/NimblGen Incorporation; Madison, WI) and sequenced with Illumina HiSeq 2000for 75 base paired‐end reads. Reads were aligned to human reference genome hg19 using ELAND v2. Single nucleotide variants and short insertions and deletions (indels) were called using SAM tools. The called variants were annotated using an in‐house script. The annotated results were first inspected to search for potential disease‐causing sequence variations in the known candidate genes for syndromic and non‐syndromic AI. The identified FAM83H mutation was further validated by Sanger sequencing.Genomic DNAs from the probands of Family 2 and 3 were screened for potential FAM83H disease‐causing mutations by analyzing the FAM83H Exon 5. Specifically, the coding region of Exon 5 was amplified by using six pairs of primers (Fig. S37). The amplification products were purified and characterized by Sanger sequencing at the University of Michigan DNA Sequencing Core. The sequencing data were then compared to the human reference sequence, and sequence variants called and evaluated. FAM83H c.DNA and genomic changes were numbered with respect to the National Center for Biotechnology Information (NCBI) human FAM83H mRNA reference sequence: NM_198488.3 (numbered from the first nucleotide of the FAM83H translation initiation codon in exon 2) and genomic reference sequence NG_016652.1 (numbered from the first nucleotide of the reference sequence). [...] The procedures for tissue preparation and microhardness testing were previously described (Wang S‐K et al. ). In brief, hemimandibles from 7‐week‐old mice were cleaned free of soft tissue and embedded in Epon resin following graded acetone dehydration. After polymerization at 65°C, the incisors were cross‐sectioned at the level of the crest of the alveolar bone close to where the incisor erupts into the mouth, about 8 mm from the apex of the incisor. The sectioned hemimandibles were reembedded in Castolite AC (Eager Polymers, Chicago, IL) using 25‐mm SteriForm molds (Struers Inc., Westlake, OH), allowed to harden overnight, and polished.Microhardness testing was performed using a LM247AT microhardness tester (Leco Corp., St. Joseph, MI) with a load of 25 g for 10 sec with a Knoop tip to obtain a Knoop hardness number (KHN). Measurements were made at 500 × magnification. Indentations were placed in the outer, middle, and inner enamel as well as the dentin as a control reading for a total of four indentations per row. This series was performed three times in each animal, for a total of twelve points per animal. Hardness Data points were treated as independent, unweighted numbers and subjected to one‐way ANOVA than Tukey's HSD test using calculation spreadsheet at http://vassarstats.net/anova1u.html. Statistical significance was determined at P < 0.01. […]

Pipeline specifications

Software tools ELAND, VassarStats
Applications Miscellaneous, WES analysis
Organisms Mus musculus, Homo sapiens
Diseases Amelogenesis Imperfecta