McDonald, Amber

Abstract A 2‐month‐old male patient harboring a duplication of DMD exons 1–7 classified as pathogenic by an outside institution presented with mildly elevated creatine phosphokinase (CK); molecular breakpoint analysis by our laboratory reclassified the duplication as likely benign. To date, proband continues to develop normally with decreased CK, further supporting our reclassification.

A 2-month-old male harboring a duplication of DMD exons 1-7 classified as pathogenic by an outside institution presented with mildly elevated CK levels; molecular breakpoint analysis by our laboratory reclassified the duplication as likely benign. To date, proband continues to develop normally with decreased CK levels, further supporting our reclassification.

Newborn screening for one or more lysosomal disorders has been implemented in several US states, Japan and Taiwan by multiplexed enzyme assays using either tandem mass spectrometry or digital microfluidics. Another multiplex assay making use of immunocapture technology has also been proposed. To investigate the potential variability in performance of these analytical approaches, we implemented three high-throughput screening assays for the simultaneous screening for four lysosomal disorders: Fabry disease, Gaucher disease, mucopolysaccharidosis type I, and Pompe disease. These assays were tested in a prospective comparative effectiveness study using nearly 100,000 residual newborn dried blood spot specimens. In addition, 2nd tier enzyme assays and confirmatory molecular genetic testing were employed. Post-analytical interpretive tools were created using the software Collaborative Laboratory Integrated Reports (CLIR) to determine its ability to improve the performance of each assay vs. the traditional result interpretation based on analyte-specific reference ranges and cutoffs. This study showed that all three platforms have high sensitivity, and the application of CLIR tools markedly improves the performance of each platform while reducing the need for 2nd tier testing by 66% to 95%. Moreover, the addition of disease-specific biochemical 2nd tier tests ensures the lowest false positive rates and the highest positive predictive values for any platform.

Abstract Background Deafness and hearing loss are common conditions that can be seen independently or as part of a syndrome and are often mediated by genetic causes. We sought to develop and validate a hereditary hearing loss panel (HHLP) to detect single nucleotide variants (SNVs), insertions and deletions (indels), and copy number variants (CNVs) in 166 genes related to nonsyndromic and syndromic hearing loss. Methods We developed a custom-capture next-generation sequencing (NGS) reagent to detect all coding regions, ±10 flanking bp, for the 166 genes related to nonsyndromic and syndromic hearing loss. Our validation consisted of testing 52 samples to establish accuracy, reproducibility, and analytical sensitivity. In addition to NGS, supplementary methods, including multiplex ligation-dependent probe amplification, long-range PCR, and Sanger sequencing, were used to ensure coverage of regions that had high complexity or homology. Results We observed 100% positive and negative percentage agreement for detection of SNVs (n = 362), small indels (1–22 bp, n = 25), and CNVs (gains, n = 8; losses, n = 17). Finally, we showed that this assay was able to detect variants with a variant allele frequency ≥20% for SNVs and indels and ≥30% to 35% for CNVs. Conclusions We validated an HHLP that detects SNVs, indels, and CNVs in 166 genes related to syndromic and nonsyndromic hearing loss. The results of this assay can be utilized to confirm a diagnosis of hearing loss and related syndromic disorders associated with known causal genes.

Abstract Mitochondrial diseases display pathological phenotypes according to the mixture of mutant versus wild-type mitochondrial DNA (mtDNA), known as heteroplasmy. We herein examined the impact of nuclear reprogramming and clonal isolation of induced pluripotent stem cells (iPSC) on mitochondrial heteroplasmy. Patient-derived dermal fibroblasts with a prototypical mitochondrial deficiency diagnosed as mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) demonstrated mitochondrial dysfunction with reduced oxidative reserve due to heteroplasmy at position G13513A in the ND5 subunit of complex I. Bioengineered iPSC clones acquired pluripotency with multilineage differentiation capacity and demonstrated reduction in mitochondrial density and oxygen consumption distinguishing them from the somatic source. Consistent with the cellular mosaicism of the original patient-derived fibroblasts, the MELAS-iPSC clones contained a similar range of mtDNA heteroplasmy of the disease-causing mutation with identical profiles in the remaining mtDNA. High-heteroplasmy iPSC clones were used to demonstrate that extended stem cell passaging was sufficient to purge mutant mtDNA, resulting in isogenic iPSC subclones with various degrees of disease-causing genotypes. On comparative differentiation of iPSC clones, improved cardiogenic yield was associated with iPSC clones containing lower heteroplasmy compared with isogenic clones with high heteroplasmy. Thus, mtDNA heteroplasmic segregation within patient-derived stem cell lines enables direct comparison of genotype/phenotype relationships in progenitor cells and lineage-restricted progeny, and indicates that cell fate decisions are regulated as a function of mtDNA mutation load. The novel nuclear reprogramming-based model system introduces a disease-in-a-dish tool to examine the impact of mutant genotypes for MELAS patients in bioengineered tissues and a cellular probe for molecular features of individual mitochondrial diseases.