A Rare Myelin Protein Zero (MPZ) Variant Alters Enhancer Activity In Vitro and In Vivo

expression. variants. that resides within a previously described SOX10 binding site is associated with decreased enhancer activity, and alters binding of nuclear proteins. Additionally, the genomic segment harboring this variant directs tissue-relevant reporter gene expression in zebrafish. variant within a cis-acting transcriptional regulatory element. While we were unable to implicate this variant in disease onset, our data suggests that similar non-coding sequences should be screened for mutations in patients with neurological disease. Furthermore, our multi-faceted approach for examining the functional significance of non-coding variants can be readily generalized to study other loci important for myelin structure and function

Additional file 1: Figure S1. of Replicate exome-sequencing in a multiple-generation family: improved interpretation of next-generation sequencing data

Systematic approach to study exome capture variability in exome-sequencing (A) Three-generation pedigree in which two individuals have an undiagnosed disease that segregated as an autosomal dominant disorder and a de novo variation arose in the second generation. (B) Model of individual subject sample blood DNA processing and sequencing. A sample of blood went through DNA isolation, and independent libraries (in triplicate) were sequenced to appropriate comparable depth and analyzed for various quality control parameters, target coverage, read depth and nucleotide variation detection. (C) Schematic illustration of sequencing read depth vs. targeted genomic region in relation to exome sequencing in replicate. Listed are also the main approaches taken in this study to analyzed exome replicate data. (D) Two main hypotheses tested using replicate exome data: (i) Biases in sequence capture resulting in poor coverage are addressable through repetition (ii) Library replication is beneficial to overall interpretation of sequence variation data. Figure S2. Titration of percentage targeted exome sequenced as a function of depth of sequencing thresholds in all three replicates per sample. Error bars show standard error for replicate sequencing. As expected, higher depth of sequencing thresholds (x-axis) result in higher-coverage (y-axis) variability in replicate exome data. Table S2.Titration of percentage target exome sequenced as a function of depth of sequencing thresholds (attached excel file). Table S3. Primers used for and results of Sanger sequencing analysis for resolution of replicate discordances in NGS data. Table S4. Primers used for and results of Sanger sequencing validation of de novo variants detected using NGS. (Concordant NGS and Sanger genotypes are highlighted in yellow). Figure S3. Box-plot of GC-content distribution in all first-exons (blue) and high-GC content (>70% GC; >=50 bp length). Table S5. Evaluation of coverage of targeted exons with high GC content (attached excel file). Table S6. Quote from Illumina for exome enrichment kits. Quotes in red indicate costs when the study was undertaken. Nextera prices, and other kit prices (in white) reflect current costs per sample (see last column). (DOC 1 mb

Additional file 2: Table S2. of Replicate exome-sequencing in a multiple-generation family: improved interpretation of next-generation sequencing data

Titration of percentage target exome sequenced as a function of depth of sequencing thresholds. (XLSX 59 kb

ATP6V1H Deficiency Impairs Bone Development through Activation of MMP9 and MMP13.

ATP6V1H is a component of a large protein complex with vacuolar ATPase (V-ATPase) activity. We identified two generations of individuals in which short stature and osteoporosis co-segregated with a mutation in ATP6V1H. Since V-ATPases are highly conserved between human and zebrafish, we generated loss-of-function mutants in atp6v1h in zebrafish through CRISPR/Cas9-mediated gene knockout. Homozygous mutant atp6v1h zebrafish exhibited a severe reduction in the number of mature calcified bone cells and a dramatic increase in the expression of mmp9 and mmp13. Heterozygous adults showed curved vertebra that lack calcified centrum structure and reduced bone mass and density. Treatment of mutant embryos with small molecule inhibitors of MMP9 and MMP13 significantly restored bone mass in the atp6v1h mutants. These studies have uncovered a new, ATP6V1H-mediated pathway that regulates bone formation, and defines a new mechanism of disease that leads to bone loss. We propose that MMP9/MMP13 could be therapeutic targets for patients with this rare genetic disease

Correction: ATP6V1H Deficiency Impairs Bone Development through Activation of MMP9 and MMP13.

[This corrects the article DOI: 10.1371/journal.pgen.1006481.]