Human MLPA Probe Design (H-MAPD): a probe design tool for both electrophoresis-based and bead-coupled human multiplex ligation-dependent probe amplification assays

<p>Abstract</p> <p>Background</p> <p>Multiplex ligation-dependent probe amplification (MLPA) is an efficient and reliable technique for gene dosage analysis. Currently MLPA can be conducted on two platforms: traditional electrophoresis-based, and FlexMAP bead-coupled. Since its introduction in 2002, MLPA has been rapidly adopted in both clinical and research situations. However, MLPA probe design is a time consuming process requiring many steps that address multiple criteria. There exist only one or two commercial software packages for traditional electrophoresis-based MLPA probe design. To our knowledge, no software is yet available that performs bead-coupled MLPA probe design.</p> <p>Results</p> <p>We have developed H-MAPD, a web-based tool that automates the generation and selection of probes for human genomic MLPA. The software performs physical-chemical property tests using UNAFold software, and uniqueness tests using the UCSC genome browser. H-MAPD supports both traditional electrophoresis-based assays, as well as FlexMAP bead-coupled MLPA.</p> <p>Conclusion</p> <p>H-MAPD greatly reduces the efforts for human genomic MLPA probe design. The software is written in Perl-CGI, hosted on a Linux server, and is freely available to non-commercial users.</p

Differential binding of Escherichia coli McrA protein to DNA sequences that contain the dinucleotide m5CpG

The Escherichia coli McrA protein, a putative C5-methylcytosine/C5-hydroxyl methylcytosine-specific nuclease, binds DNA with symmetrically methylated HpaII sequences (Cm5CGG), but its precise recognition sequence remains undefined. To determine McrA’s binding specificity, we cloned and expressed recombinant McrA with a C-terminal StrepII tag (rMcrA-S) to facilitate protein purification and affinity capture of human DNA fragments with m5C residues. Sequence analysis of a subset of these fragments and electrophoretic mobility shift assays with model methylated and unmethylated oligonucleotides suggest that N(Y > R) m5CGR is the canonical binding site for rMcrA-S. In addition to binding HpaII-methylated double-stranded DNA, rMcrA-S binds DNA containing a single, hemimethylated HpaII site; however, it does not bind if A, C, T or U is placed across from the m5C residue, but does if I is opposite the m5C. These results provide the first systematic analysis of McrA’s in vitro binding specificity

Comparative isoschizomer profiling of cytosine methylation:the HELP assay

The distribution of cytosine methylation in 6.2 Mb of the mouse genome was tested using cohybridization of genomic representations from a methylation-sensitive restriction enzyme and its methylation-insensitive isoschizomer. This assay, termed HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR), allows both intragenomic profiling and intergenomic comparisons of cytosine methylation. The intragenomic profile shows most of the genome to be contiguous methylated sequence with occasional clusters of hypomethylated loci, usually but not exclusively at promoters and CpG islands. Intergenomic comparison found marked differences in cytosine methylation between spermatogenic and brain cells, identifying 223 new candidate tissue-specific differentially methylated regions (T-DMRs). Bisulfite pyrosequencing confirmed the four candidates tested to be T-DMRs, while quantitative RT-PCR for two genes with T-DMRs located at their promoters showed the HELP data to be correlated with gene activity at these loci. The HELP assay is robust, quantitative, and accurate and is providing new insights into the distribution and dynamic nature of cytosine methylation in the genome. ©2006 by Cold Spring Harbor Laboratory Press

High-resolution genome-wide cytosine methylation profiling with simultaneous copy number analysis and optimization for limited cell numbers

Many genome-wide assays involve the generation of a subset (or representation) of the genome following restriction enzyme digestion. The use of enzymes sensitive to cytosine methylation allows high-throughput analysis of this epigenetic regulatory process. We show that the use of a dual-adapter approach allows us to generate genomic representations that includes fragments of <200 bp in size, previously not possible when using the standard approach of using a single adapter. By expanding the representation to smaller fragments using HpaII or MspI, we increase the representation by these isoschizomers to more than 1.32 million loci in the human genome, representing 98.5% of CpG islands and 91.1% of refSeq promoters. This advance allows the development of a new, high-resolution version of our HpaII-tiny fragment Enrichment by Ligation-mediated PCR (HELP) assay to study cytosine methylation. We also show that the MspI representation generates information about copy-number variation, that the assay can be used on as little as 10 ng of DNA and that massively parallel sequencing can be used as an alternative to microarrays to read the output of the assay, making this a powerful discovery platform for studies of genomic and epigenomic abnormalities

NUBPL mitochondrial disease: new patients and review of the genetic and clinical spectrum

Background: The nucleotide binding protein-like (NUBPL) gene was first reported as a cause of mitochondrial complex I deficiency (MIM 613621, 618242) in 2010. To date, only eight patients have been reported with this mitochondrial disorder. Five other patients were recently reported to have NUBPL disease but their clinical picture was different from the first eight patients. Here, we report clinical and genetic findings in five additional patients (four families).  Methods: Whole exome sequencing was used to identify patients with compound heterozygous NUBPL variants. Functional studies included RNA-Seq transcript analyses, missense variant biochemical analyses in a yeast model (Yarrowia lipolytica) and mitochondrial respiration experiments on patient fibroblasts.  Results: The previously reported c.815-27T>C branch-site mutation was found in all four families. In prior patients, c.166G>A [p.G56R] was always found in cis with c.815-27T>C, but only two of four families had both variants. The second variant found in trans with c.815-27T>C in each family was: C.311T>C [p.L104P] in three patients, c.693+1G>A in one patient and c.545T>C [p.V182A] in one patient. Complex I function in the yeast model was impacted by p.L104P but not p.V182A. Clinical features include onset of neurological symptoms at 3-18 months, global developmental delay, cerebellar dysfunction (including ataxia, dysarthria, nystagmus and tremor) and spasticity. Brain MRI showed cerebellar atrophy. Mitochondrial function studies on patient fibroblasts showed significantly reduced spare respiratory capacity.  Conclusion: We report on five new patients with NUBPL disease, adding to the number and phenotypic variability of patients diagnosed worldwide, and review prior reported patients with pathogenic NUBPL variants

Progressive multifocal leukoencephalopathy genetic risk variants for pharmacovigilance of immunosuppressant therapies

BackgroundProgressive multifocal leukoencephalopathy (PML) is a rare and often lethal brain disorder caused by the common, typically benign polyomavirus 2, also known as JC virus (JCV). In a small percentage of immunosuppressed individuals, JCV is reactivated and infects the brain, causing devastating neurological defects. A wide range of immunosuppressed groups can develop PML, such as patients with: HIV/AIDS, hematological malignancies (e.g., leukemias, lymphomas, and multiple myeloma), autoimmune disorders (e.g., psoriasis, rheumatoid arthritis, and systemic lupus erythematosus), and organ transplants. In some patients, iatrogenic (i.e., drug-induced) PML occurs as a serious adverse event from exposure to immunosuppressant therapies used to treat their disease (e.g., hematological malignancies and multiple sclerosis). While JCV infection and immunosuppression are necessary, they are not sufficient to cause PML.MethodsWe hypothesized that patients may also have a genetic susceptibility from the presence of rare deleterious genetic variants in immune-relevant genes (e.g., those that cause inborn errors of immunity). In our prior genetic study of 184 PML cases, we discovered 19 candidate PML risk variants. In the current study of another 152 cases, we validated 4 of 19 variants in both population controls (gnomAD 3.1) and matched controls (JCV+ multiple sclerosis patients on a PML-linked drug ≥ 2 years).ResultsThe four variants, found in immune system genes with strong biological links, are: C8B, 1-57409459-C-A, rs139498867; LY9 (alias SLAMF3), 1-160769595-AG-A, rs763811636; FCN2, 9-137779251-G-A, rs76267164; STXBP2, 19-7712287-G-C, rs35490401. Carriers of any one of these variants are shown to be at high risk of PML when drug-exposed PML cases are compared to drug-exposed matched controls: P value = 3.50E-06, OR = 8.7 [3.7–20.6]. Measures of clinical validity and utility compare favorably to other genetic risk tests, such as BRCA1 and BRCA2 screening for breast cancer risk and HLA-B*15:02 pharmacogenetic screening for pharmacovigilance of carbamazepine to prevent Stevens-Johnson Syndrome and Toxic Epidermal Necrolysis.ConclusionFor the first time, a PML genetic risk test can be implemented for screening patients taking or considering treatment with a PML-linked drug in order to decrease the incidence of PML and enable safer use of highly effective therapies used to treat their underlying disease