Next-generation carrier screening.

Purpose: Carrier screening for recessive Mendelian disorders traditionally employs focused genotyping to interrogate limited sets of mutations most prevalent in specific ethnic groups. We sought to develop a next-generation DNA sequencing-based workflow to enable analysis of a more comprehensive set of disease-causing mutations.

Methods: We utilized molecular inversion probes to capture the protein-coding regions of 15 genes from genomic DNA isolated from whole blood and sequenced those regions using the Illumina HiSeq 2000 (Illumina, San Diego, CA).

Distinctive genetic and clinical features of CMT4J: a severe neuropathy caused by mutations in the PI(3,5)P₂ phosphatase FIG4.

Charcot-Marie-Tooth disease is a genetically heterogeneous group of motor and sensory neuropathies associated with mutations in more than 30 genes. Charcot-Marie-Tooth disease type 4J (OMIM 611228) is a recessive, potentially severe form of the disease caused by mutations of the lipid phosphatase FIG4. We provide a more complete view of the features of this disorder by describing 11 previously unreported patients with Charcot-Marie-Tooth disease type 4J.

Alu-specific microhomology-mediated deletion of the final exon of SPAST in three unrelated subjects with hereditary spastic paraplegia.

Purpose: Autosomal dominant spastic paraplegia, type 4 (SPG4), a debilitating disorder of progressive spasticity and weakness of the lower limbs, results from heterozygous mutations in the SPAST gene. The full spectrum of SPAST mutations causing SPG4 and their mechanisms of formation remain to be determined.

Methods: We used multiplex ligation-dependent probe amplification, locus-specific array comparative genomic hybridization, and breakpoint DNA sequencing to identify and describe genomic rearrangements in three patients with a clinical presentation of hereditary spastic paraplegia.

GJB1/Connexin 32 whole gene deletions in patients with X-linked Charcot-Marie-Tooth disease.

The X-linked form of Charcot-Marie-Tooth disease (CMTX) is the second most common form of this genetically heterogeneous inherited peripheral neuropathy. CMT1X is caused by mutations in the GJB1 gene. Most of the mutations causative for CMT1X are missense mutations.

Mechanisms for nonrecurrent genomic rearrangements associated with CMT1A or HNPP: rare CNVs as a cause for missing heritability.

Genomic rearrangements involving the peripheral myelin protein gene (PMP22) in human chromosome 17p12 are associated with neuropathy: duplications cause Charcot-Marie-Tooth disease type 1A (CMT1A), whereas deletions lead to hereditary neuropathy with liability to pressure palsies (HNPP). Our previous studies showed that >99% of these rearrangements are recurrent and mediated by nonallelic homologous recombination (NAHR). Rare copy number variations (CNVs) generated by nonrecurrent rearrangements also exist in 17p12, but their underlying mechanisms are not well understood.

The DNA replication FoSTeS/MMBIR mechanism can generate genomic, genic and exonic complex rearrangements in humans.

We recently proposed a DNA replication-based mechanism of fork stalling and template switching (FoSTeS) to explain the complex genomic rearrangements associated with a dysmyelinating central nervous system disorder in humans. The FoSTeS mechanism has been further generalized and molecular mechanistic details have been provided in the microhomology-mediated break-induced replication (MMBIR) model that may underlie many structural variations in genomes from all domains of life. Here we provide evidence that human genomic rearrangements ranging in size from several megabases to a few hundred base pairs can be generated by FoSTeS/MMBIR.

Puromycin-sensitive aminopeptidase limits MHC class I presentation in dendritic cells but does not affect CD8 T cell responses during viral infections.

Previous experiments using enzyme inhibitors, cell lysates, and purified enzyme have suggested that puromycin-sensitive aminopeptidase (PSA) plays a role in creating and destroying MHC class I-presented peptides although its precise contribution to these processes is unknown. To examine the importance of this enzyme in MHC class I Ag presentation, we have generated PSA-deficient mice and cell lines from these animals. PSA-deficient mice are smaller and do not reproduce as well as wild type mice.

Analysis of the role of bleomycin hydrolase in antigen presentation and the generation of CD8 T cell responses.

Long oligopeptides (>10 residues) are generated during the catabolism of cellular proteins in the cytosol. To be presented to T cells, such peptides must be trimmed by aminopeptidases to the proper size (typically 8-10 residues) to stably bind to MHC class I molecules. Aminopeptidases also destroy epitopes by trimming them to even shorter lengths.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims MHC class I-presented peptides in vivo and plays an important role in immunodominance.

CD8(+) T cells respond to short peptides bound to MHC class I molecules. Although most antigenic proteins contain many sequences that could bind to MHC class I, few of these peptides actually stimulate CD8(+) T cell responses. Moreover, the T cell responses that are generated often follow a very reproducible hierarchy to different peptides for reasons that are poorly understood.

Leucine aminopeptidase is not essential for trimming peptides in the cytosol or generating epitopes for MHC class I antigen presentation.

To detect viral infections and tumors, CD8+ T lymphocytes monitor cells for the presence of antigenic peptides bound to MHC class I molecules. The majority of MHC class I-presented peptides are generated from the cleavage of cellular and viral proteins by the ubiquitin-proteasome pathway. Many of the oligopeptides produced by this process are too long to stably bind to MHC class I molecules and require further trimming for presentation.