Karyomapping: a universal method for genome wide analysis of genetic disease based on mapping crossovers between parental haplotypes.

The use of genome wide single nucleotide polymorphism (SNP) arrays for high resolution molecular cytogenetic analysis using a combination of quantitative and genotype analysis is well established. This study demonstrates that by Mendelian analysis of the SNP genotypes of the parents and a sibling or other appropriate family member to establish phase, it is possible to identify informative loci for each of the four parental haplotypes across each chromosome and map the inheritance of these haplotypes and the position of any crossovers in the proband. The resulting 'karyomap', unlike a karyotype, identifies the parental and grandparental origin of each chromosome and chromosome segment and is unique for every individual being defined by the independent segregation of parental chromosomes and the pattern of non-recombinant and recombinant chromosomes. Karyomapping, therefore, enables both genome wide linkage based analysis of inheritance and detection of chromosome imbalance where either both haplotypes from one parent are present (trisomy) or neither are present (monosomy/deletion). The study also demonstrates that karyomapping is possible at the single cell level following whole genome amplification and, without any prior patient or disease specific test development, provides a universal linkage based methodology for preimplantation genetic diagnosis readily available worldwide.

Human herpesvirus 6 rhombencephalitis in immunocompetent children.

This article describes the clinical presentation, diagnostic workup, and neurologic outcome of 3 immunocompetent pediatric patients diagnosed with human herpesvirus 6 (HHV6) rhombencephalitis. Presentation of HHV6 rhombencephalitis included new onset seizures, ataxia, encephalopathy, and opsoclonus-myoclonus. Neurologic examination revealed cranial neuropathies, cerebellar dysfunction, and extremity weakness. Magnetic resonance imaging abnormalities located in the cerebellum, basal ganglia/thalamus, and cerebral hemispheres were detected in 2 patients. Diagnosis of HHV6 encephalitis was made by real-time and nested polymerase chain reaction of serum and cerebrospinal fluid. The HHV6 variant A was detected in 2 patients by sequence analysis, and HHV6 protein was detected by immunomicroscopy in a patient who underwent biopsy secondary to progressive clinical and neuroradiographic findings. Therapy with intravenous ganciclovir did not correlate with resolution of neurologic symptoms, despite eventual non-detectable HHV6. Human herpesvirus 6 should be considered in the differential diagnosis of unexplained cases of rhombencephalitis in immunocompetent children. Features may be rapidly progressive and include profound encephalopathy, seizures, ataxia, and opsoclonus-myoclonus.

The accuracy of chromosomal microarray testing for identification of embryonic mosaicism in human blastocysts.

BACKGROUND: Most previous studies of chromosomal mosaicism in IVF embryos were performed by fluorescence in situ hybridization (FISH) methods. While there are reports implicating chromosome aneuploidy in implantation failure following transfer and pregnancy loss by spontaneous miscarriage, the significance of mosaicism for the developmental potential of growing embryos is unknown. However, the low prevalence of chromosomal mosaicism in chorionic villus sampling and amniotic fluid specimens suggests the presence of selection against mosaic embryos for implantation and early pregnancy. The absence of evidence for selective allocation of abnormal cells to the trophectoderm (TE) of mosaic blastocysts permits these cells to be a good proxy for embryonic mosaicism detection by chromosomal microarrays (CMA). The purpose of this study was to establish the limits of detection and the prevalence of chromosome mosaicism in day 5/6 human embryos using CMA with TE biopsies. RESULTS: From reconstitution experiments we established log2 ratio thresholds for mosaicism detection. These studies indicated that chromosomal mosaicism at levels as low as between 25-37% can be consistently identified. Follow-up studies by FISH on non-transferred abnormal embryos confirmed the diagnostic accuracy of CMA testing. The number of cells in a TE biopsy can influence mosaicism detection. CONCLUSIONS: Chromosomal microarrays can detect mosaicism in TE biopsies when present at levels as low as between 25-37% and the prevalence of day 5/6 blastocysts which were mosaic and had no other abnormalities reached 15% among a cohort of 551 embryos examined. Validated protocols for establishing detection thresholds for mosaicism are important to reduce the likelihood of transferring abnormal embryos.

Polymerase Chain Reaction molecular diagnostic technology for monitoring chronic osteomyelitis.

BACKGROUND: Osteomyelitis is a devastating condition whose treatment relies on the detection of bacteria. The current standard of microbiology culture may not be adequate. Molecular biology based diagnostic procedures for detecting bacteria in orthopaedic infections was previously established, but has not been applied to the setting of chronic osteomyelitis. We aim to determine the applicability of molecular diagnostic procedures for monitoring chronic osteomyelitis, and to evaluate if these procedures are superior to standard culture methods of osteomyelitis detection. METHODS: A rabbit experimental model of chronic osteomyelitis was used; infection was induced in the proximal, medial aspect of the tibia with Staphylococcus aureus at titers ranging from 1 × 10(2) to 1 × 10(6) colony forming units. At 28 days post-infection, animals were sacrificed, and the tibias were examined radiographically, harvested, and assayed for the presence of bacteria. Two bacterial detection methods were used: (1) standard microbiological culturing, and (2) polymerase chain reaction (PCR) based diagnostic method to detect bacterial genomic DNA. RESULTS: The molecular diagnostic method was highly sensitive and accurate, and detected low titer infections that were undetected by radiographic and microbiological methods. By using two sets of PCR primers, one for a universal bacterial gene (16S rRNA) and one for a species-specific gene (nuc), the molecular protocol allowed both the detection and speciation of the bacterial infection. CONCLUSIONS: The use of the PCR-based method was effective for high-sensitivity detection and identification of bacteria associated with chronic osteomyelitis in a rabbit model. Our findings illustrate the applicability of PCR for monitoring chronic osteomyelitis, which may be useful for improved detection of osteomyelitis organisms in humans.

Malignant precursor cells pre-exist in human breast DCIS and require autophagy for survival.

BACKGROUND: While it is accepted that a majority of invasive breast cancer progresses from a ductal carcinoma in situ (DCIS) precursor stage, very little is known about the factors that promote survival of DCIS neoplastic cells within the hypoxic, nutrient deprived intraductal microenvironment. METHODOLOGY AND PRINCIPAL FINDINGS: We examined the hypothesis that fresh human DCIS lesions contain pre-existing carcinoma precursor cells. We characterized these cells by full genome molecular cytogenetics (Illumina HumanCytoSNP profile), and signal pathway profiling (Reverse Phase Protein Microarray, 59 endpoints), and demonstrated that autophagy is required for survival and anchorage independent growth of the cytogenetically abnormal tumorigenic DCIS cells. Ex vivo organoid culture of fresh human DCIS lesions, without enzymatic treatment or sorting, induced the emergence of neoplastic epithelial cells exhibiting the following characteristics: a) spontaneous generation of hundreds of spheroids and duct-like 3-D structures in culture within 2-4 weeks; b) tumorigenicity in NOD/SCID mice; c) cytogenetically abnormal (copy number loss or gain in chromosomes including 1, 5, 6, 8, 13, 17) compared to the normal karyotype of the non-neoplastic cells in the source patient's breast tissue; d) in vitro migration and invasion of autologous breast stroma; and e) up-regulation of signal pathways linked to, and components of, cellular autophagy. Multiple autophagy markers were present in the patient's original DCIS lesion and the mouse xenograft. We tested whether autophagy was necessary for survival of cytogenetically abnormal DCIS cells. The lysosomotropic inhibitor (chloroquine phosphate) of autophagy completely suppressed the generation of DCIS spheroids/3-D structures, suppressed ex vivo invasion of autologous stroma, induced apoptosis, suppressed autophagy associated proteins including Atg5, AKT/PI3 Kinase and mTOR, eliminated cytogenetically abnormal spheroid forming cells from the organ culture, and abrogated xenograft tumor formation. CONCLUSIONS: Cytogenetically abnormal spheroid forming, tumorigenic, and invasive neoplastic epithelial cells pre-exist in human DCIS and require cellular autophagy for survival.