Filtering of variants identified through exome sequencing of individuals in LGMD2359.

<p>Variant filtration was performed for all variants (SNVs and indels) identified in known muscle disease genes (A) and in all genes (B). The total number of variants identified in any of the three cases (i) was restricted to variants that were not on the X or Y chromosomes and were not identified in any of the control individuals (ii). This list was further reduced to variants at ≤1% frequency in the HapMap and 1000 Genomes Caucasian databases (iii), then to likely functional variants or variants within 10 bp of splice site (iv), and finally to variants that are shared in all 3 cases (v).</p

Filtering of variants identified through exome sequencing of individuals in LGMD2692.

<p>Variant filtration was performed for all variants (SNVs and indels) identified in known muscle disease genes (A) and in all genes (B). The total number of variants identified in either of the cases (i) was restricted to variants that were not on the X or Y chromosomes and were not identified in any of the control individuals (ii). This list was further reduced to variants at ≤1% frequency in the HapMap and 1000 Genomes Caucasian databases (iii), then to likely functional variants or variants within 10 bases of a splice site (iv), and finally to variants that are shared in both cases (v).</p

Known muscle disease genes in which ≥30% of CCDS+splice bases have <10X coverage and/or QUAL scores of <50 in at least one case.

*<p>Failed bases are defined as any base with <10 reads at that position and/or a GATK QUAL score of <50.</p>#<p>CCDS+splice bases = total number of bases in the gene region (consensus coding plus 10 bp on each side for splice sites).</p

Exome Analysis of Two Limb-Girdle Muscular Dystrophy Families: Mutations Identified and Challenges Encountered

<div><p>The molecular diagnosis of muscle disorders is challenging: genetic heterogeneity (>100 causal genes for skeletal and cardiac muscle disease) precludes exhaustive clinical testing, prioritizing sequencing of specific genes is difficult due to the similarity of clinical presentation, and the number of variants returned through exome sequencing can make the identification of the disease-causing variant difficult. We have filtered variants found through exome sequencing by prioritizing variants in genes known to be involved in muscle disease while examining the quality and depth of coverage of those genes. We ascertained two families with autosomal dominant limb-girdle muscular dystrophy of unknown etiology. To identify the causal mutations in these families, we performed exome sequencing on five affected individuals using the Agilent SureSelect Human All Exon 50 Mb kit and the Illumina HiSeq 2000 (2×100 bp). We identified causative mutations in <em>desmin</em> (IVS3+3A>G) and filamin C (p.W2710X), and augmented the phenotype data for individuals with muscular dystrophy due to these mutations. We also discuss challenges encountered due to depth of coverage variability at specific sites and the annotation of a functionally proven splice site variant as an intronic variant.</p> </div

Clinical description of individuals with skeletal muscle weakness and/or elevated creatine kinase in LGMD2359 and LGMD2692.

<p>Age onset is for skeletal muscle weakness NF = neck flexor THC = tight heel cords.</p><p>AP = age at pacemaker insertion PU = proximal upper extremities PC = pes cavus.</p>*<p> = questionable affection status DU = distal upper extremities PN = peripheral neuropathy.</p>∧<p> = DNA unavailable for Sanger PL = proximal lower extremities LE = lower extremity.</p><p>NL = normal DL = distal lower extremities UE = upper extremity.</p><p>CK = reported as ‘times upper limit of normal’ by gender (when abnormal).</p

LGMD2359 pedigree and results of Sanger sequencing for <i>DES</i> IVS3+3A>G.

<p>The pedigree shows the affection statuses, individual identifiers, and genotypes at <i>DES</i> IVS3+3. The genotypes obtained through Sanger sequencing of individuals with available high quality DNA are shown. The arrow indicates the proband. <b>*</b> indicates that DNA was sent for exome sequencing. Individuals with ? were of uncertain affection status.</p

Fetal hemoglobin levels during study period.

<p>Fetal hemoglobin levels were assessed with hemoglobin electrophoresis for the A) 50g, B) 100g, and C) 150g cohorts. Each individual patient is designated with letter, symbol, and color codes; several patients participated in multiple cohorts. Patient F was diagnosed with HbSß° thalassemia; all other patients were diagnosed with HbSS. Patients taking hydroxyurea during the study period are denoted with an asterisk.</p

Adverse Events.

<p>Adverse Events.</p

CONSORT flow diagram enrollment summary

<p>CONSORT flow diagram enrollment summary</p

Average percent change of mRNAs at end treatment relative to pre-treatment.

<p>Average percent change of mRNAs at end treatment relative to pre-treatment.</p