David S. Cram

Efflux‐mediated antiseptic resistance gene qacA from Staphylococcus aureus: common ancestry with tetracycline‐ and sugar‐transport proteins

Resistance to intercalating dyes (ethidium, acriflavine) and other organic cations, such as quaternary ammonium‐type antiseptic compounds, mediated by the Staphylococcus aureus ptasmid pSK1 is specified by an energy‐dependent export mechanism encoded by the qacA gene. From nucleotide sequence analysis, qacA is predicted to encode a protein of Mr 55017 containing 514 amino acids. The gene is likely to initiate with a CUG codon, and a 36bp palindrome immediately preceding qacA, along with an upstream reading frame with homology to the TetR repressors, may be components of a regulatory circuit. The putative polypeptide specified by qacA has properties typical of a cytoplasmic membrane protein, and is indicated to be a member of a transport protein family that includes proteins reponsible for export‐mediated resistance to tetracycline and methylenomycin, and uptake of sugars and quinate. The analysis suggests that N‐ and C‐terminal regions of these proteins are involved in energy coupling (proton translocation) and substrate transport, respectively. The last common ancestor of the qacA and related tet (tetracycline resistance) lineages is inferred to have been repressor controlled, as occurs for modern tet determinants from Gram‐negative, but not those from Gram‐positive, bacteria.

Maternal Mosaicism Is a Significant Contributor to Discordant Sex Chromosomal Aneuploidies Associated with Noninvasive Prenatal Testing

BACKGROUND In the human fetus, sex chromosome aneuploidies (SCAs) are as prevalent as the common autosomal trisomies 21, 18, and 13. Currently, most noninvasive prenatal tests (NIPTs) offer screening only for chromosomes 21, 18, and 13, because the sensitivity and specificity are markedly higher than for the sex chromosomes. Limited studies suggest that the reduced accuracy associated with detecting SCAs is due to confined placental, placental, or true fetal mosaicism. We hypothesized that an altered maternal karyotype may also be an important contributor to discordant SCA NIPT results. METHODS We developed a rapid karyotyping method that uses massively parallel sequencing to measure the degree of chromosome mosaicism. The method was validated with DNA models mimicking XXX and XO mosaicism and then applied to maternal white blood cell (WBC) DNA from patients with discordant SCA NIPT results. RESULTS Sequencing karyotyping detected chromosome X (ChrX) mosaicism as low as 5%, allowing an accurate assignment of the maternal X karyotype. In a prospective NIPT study, we showed that 16 (8.6%) of 181 positive SCAs were due to an abnormal maternal ChrX karyotype that masked the true contribution of the fetal ChrX DNA fraction. CONCLUSIONS The accuracy of NIPT for ChrX and ChrY can be improved substantially by integrating the results of maternal-plasma sequencing with those for maternal-WBC sequencing. The relatively high frequency of maternal mosaicism warrants mandatory WBC testing in both shotgun sequencing- and single-nucleotide polymorphism-based clinical NIPT after the finding of a potential fetal SCA.

Y chromosome analysis of infertile men and their sons conceived through intracytoplasmic sperm injection: vertical transmission of deletions and rarity of de novo deletions

OBJECTIVE To determine the prevalence and type of Yq microdeletions in 86 consecutive men that fathered 99 sons by intracytoplasmic sperm injection (ICSI) and to determine the incidence of vertical transmission and de novo deletions in these boys. DESIGN Prospective clinical observational study. SETTING Genetics laboratory associated with a university IVF unit. PATIENT(S) Eighty-six consecutive infertile men presenting to an IVF clinic and their 99 ICSI-conceived sons. Fifty of the 86 men (58%) had idiopathic seminiferous tubule failure (STF); the remainder had a variety of other clinical indications for ICSI. INTERVENTION(S) Collection of peripheral and cord blood samples. MAIN OUTCOME MEASURE(S) The Yq genetic status of fathers who underwent ICSI and of their sons by the presence or absence of 22 Y-specific markers covering the four azoospermia factor (AZF) subregions. RESULT(S) Yq deletions of the AZFd/c region were detected in two (6.9%) of 29 azoo- or severely oligospermic men with STF. Identical deletions were found in their respective sons. No de novo deletions were detected in the remaining 97 sons conceived by men without deletions. CONCLUSION(S) The detection of Yq deletions only in men with severe STF is consistent with previous studies, with the AZFd/c region being most commonly affected. This study demonstrates the vertical transmission of these Yq deletions through the use of ICSI and supports the notion that, in most cases, Yq deletions will be inherited by male offspring. The absence of de novo Yq deletions in the male offspring indicates that these events are rare following ICSI in men with both STF and other common male factor indications.

Gene expression profiling of human oocytes following in vivo or in vitro maturation

BACKGROUND Immature human oocytes matured in vitro, particularly those from gonadotrophin stimulated ovaries, are developmentally incompetent when compared with oocytes matured in vivo. This developmental incompetence has been explained as poor oocyte cytoplasmic maturation without any determination of the likely molecular basis of this observation. METHODS Replicate whole human genome arrays were generated for immature and mature oocytes (matured in vivo and in vitro, prior to exposure to sperm) recovered from women undertaking gonadotrophin treatment for assisted reproduction. RESULTS More than 2000 genes were identified as expressed at more than 2-fold higher levels in oocytes matured in vitro than those matured in vivo (P < 0.05, range 4.98 x 10(-2) -2.22 x 10(-4)) and 162 of these are expressed at 10-fold or greater levels (P < 0.05, range 4.98 x 10(-2)-1.38 x 10(-3)). Many of these genes are involved in transcription, the cell cycle and its regulation, transport and cellular protein metabolism. CONCLUSIONS Global gene expression profiling using microarrays and bioinformatics analysis has provided a molecular basis for differences in the developmental competence of oocytes matured in vitro compared with in vivo. The over-abundance of transcripts identified in immature germinal vesicle stage oocytes recovered from gonadotrophin stimulated cycles and matured in vitro is probably due to dysregulation in either gene transcription or post-transcriptional modification of genes. Either mechanism would result in an incorrect temporal utilization of genes which may culminate in developmental incompetence of any embryos derived from these oocytes.

Human embryonic stem cell models of Huntington disease

Huntington disease (HD) is an incurable late-onset neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of the HD gene (HTT). The major hallmark of disease pathology is neurodegeneration in the brain. Currently, there are no useful in-vitro human models of HD. Recently, two human embryonic stem cell (hESC) lines carrying partial (CAG(37)) and fully (CAG(51)) penetrant mutant alleles have been derived from affected IVF embryos identified following preimplantation genetic diagnosis (PGD). Fluorescence polymerase chain reaction (F-PCR) and Genescan analysis confirmed the original embryonic HD genotypes. Reverse transcription PCR (RT-PCR) analysis confirmed the expression of mutant transcripts and western blot analysis demonstrated expression of mutant huntingtin protein (HTT). After treatment with noggin, HD hESC formed neurospheres, which could be further differentiated into cells susceptible to neurodegeneration in HD, namely primary neurones and astrocytes. Small pool PCR analysis of neurosphere cells revealed instability of disease-length CAG repeats following differentiation. The presence of active HTT genes, neural differentiation capabilities and evidence of CAG repeat instability indicates these HD hESC lines may serve as valuable in-vitro human models of HD to better understand the mechanisms of neurodegeneration in patients, and for drug screening to identify new therapies for human clinical trials.

Two cases of placental T21 mosaicism: challenging the detection limits of non‐invasive prenatal testing

Key Laboratory of Molecular Medicine, Ministry of Education, Shanghai Medical College, Fudan University, Shanghai, China Maternity and Child Health Hospital of Anhui Province, The Maternal and Child Health Clinical College, Anhui Medical University, Hefei, China Prenatal Diagnostic Center, International Peace Maternal and Children’s Hospital, Shanghai Jiaotong University, Shanghai, China The First Affiliated Hospital of Nanjing Medical University, Nanjing, China Xijing Hospital, The Fourth Military Medical College, Xian, China Baoji Maternity and Child Health Hospital, Baoji, China Berry Genomics C., Limited, Beijing, China *Correspondence to: Weiwei Cheng. E-mail: 18017316001@163.com; David Cram. E-mail: david.cram@berrygenomics.com †These authors contributed equally to this work.

Expression of GFP in the mitochondrial compartment using DQAsome-mediated delivery of an artificial mini-mitochondrial genome.

ABSTRACTPurposeWe describe a novel strategy for expression of GFP in mammalian mitochondria.MethodsThe key components of the strategy were an artificially created mitochondrial genome pmtGFP and a DQAsome transfection system.ResultsUsing immunofluorescence and a combination of immunohistochemical and molecular based techniques, we show that DQAsomes are capable of delivering the pmtGFP construct to the mitochondrial compartment of the mouse macrophage cell line RAW264.7, albeit at low efficiency (1–5%), resulting in the expression of GFP mRNA and protein. Similar transfection efficiencies were also demonstrated in a range of other mammalian cell lines.ConclusionsThe DQAsome-transfection technique was able to deliver the exogenous DNA into the cellular mitochondria and the pmtGFP was functional. Further optimization of this strategy would provide a flexible and rapid way to generate mutant cells and useful animal models of mitochondrial disease.

Mesenchymal Stem Cells for Treatment of CNS Injury

Brain and spinal cord injuries present significant therapeutic challenges. The treatments available for these conditions are largely ineffective, partly due to limitations in directly targeting the therapeutic agents to sites of pathology within the central nervous system (CNS). The use of stem cells to treat these conditions presents a novel therapeutic strategy. A variety of stem cell treatments have been examined in animal models of CNS trauma. Many of these studies have used stem cells as a cell-replacement strategy. These investigations have also highlighted the significant limitations of this approach. Another potential strategy for stem cell therapy utilises stem cells as a delivery mechanism for therapeutic molecules. This review surveys the literature relevant to the potential of mesenchymal stem cells for delivery of therapeutic agents in CNS trauma in humans.

DNA identification of fetal cells isolated from cervical mucus: potential for early non‐invasive prenatal diagnosis

Objectives  To develop a reliable method to isolate fetal cells for genetic diagnosis.

Confined placental origin of the circulating cell free fetal DNA revealed by a discordant non-invasive prenatal test result in a trisomy 18 pregnancy

BACKGROUND Non-invasive prenatal testing (NIPT) by massively parallel sequencing is a useful clinical test for the detection of common fetal aneuploidies. While the accuracy of aneuploidy detection can approach 100%, results discordant with the fetus are occasionally reported. In this study we investigated the basis of a discordant T21 positive and T18 negative NIPT result associated with a T18 fetus confirmed by karyotyping. METHODS Massively parallel sequencing was used to detect fetal DNA in maternal circulating plasma. The parental origin and nature of the fetal and placental aneuploidies were investigated by quantitative fluorescent PCR of short tandem repeat (STR) sequences and by copy number variation (CNV) sequencing. RESULTS There was no evidence of T21 maternal mosaicism, T21 microchimerism or a vanishing twin to explain the discordant NIPT result. However, examination of multiple placental biopsies showed both T21 and T18 mosaicism, including one confined region with a significantly higher proportion of T21 cells. Based on fetal DNA fractions and average mosaicism levels, the effective T21 and T18 fetal DNA fractions should have been sufficient for the detection of both trisomies. CONCLUSIONS In this pregnancy, we speculate that confined placental region(s) with higher proportions of T21 cells were preferentially releasing fetal DNAs into the maternal circulation. This study highlights placental mosaicism as a significant risk factor for discordant NIPT results.