Generation of two human ISG15 knockout iPSC clones using CRISPR/Cas9 editing.

Interferon stimulated gene 15 (ISG15) is one of the most highly upregulated proteins in response to viral infection and is involved in numerous pathways with multiple mechanisms of actions. ISG15 deficiency has been reported to induce type I interferonopathy owing to defective negative regulation of IFN-I signalling as well as enhanced antiviral protection. Here, we have generated ISG15 knockout clones from human iPSCs, which provide useful cell resources to study mechanisms of ISG15 deficiency and gain more insight into the biological function of ISG15.

Wave microcarrier cultivation of MDCK cells for influenza virus production in serum containing and serum-free media.

A process for equine influenza virus vaccine production using a microcarrier system (Cytodex 1) in a 2 L Wave bioreactor is described. Growth of Madin Darby canine kidney (MDCK) cells in serum containing GMEM medium (SC) is compared to growth in serum-free Ex-Cell MDCK medium (SF) without washing steps and medium exchange before infection. Cultivations with microcarrier concentrations of 2 and 4 g/L for both media are shown. Metabolic data from carbon and amino acid metabolism are discussed. Additionally, in roller bottle experiments the influence of multiplicity of infection (moi) and trypsin concentration on the HA value was investigated. Analysis of HA and TCID(50) at 37 degrees C showed a stable HA of maximum 2.6 log HA/100 microL for 2 weeks. Peak TCID(50) titers of 10(7.7) viruses/mL were achieved 20h post infection, but infectivity was below detection limit after 150 h. Cell attachment onto microcarriers under serum-free conditions was improved by Ca(2+) addition and by cell harvesting without trypsin using only an EDTA/PBS solution. For the wave cultivation maximum virus titers of 2.3-2.6 log HA units/100 microL were reached from infection with a moi of 0.05. However, in SF medium pH dropped to less than pH 6.8 which resulted in lower HA titers of 1.7 log HA units/100 microL. For the higher microcarrier concentration (4 g/L) medium exchange steps (500 mL) were needed for both media. Omission of the washing step and medium exchange before infection in SF medium clearly simplified the influenza production process; however, for higher virus yields a better pH control of the wave bioreactor would be required. Higher cell densities (2.8 x 10(6) cells/mL for 2 g/L microcarrier) and better attachment compared to stirred tank bioreactors showed, that the wave bioreactor is a good alternative to stirred tank processes for expanding production capacities in case of a pandemic.

Twin sisters, monozygotic with the fragile X mutation, but with a different phenotype.

The absence of the fragile X mental retardation protein (FMRP) results in fragile X syndrome. All males with a full mutation in the FMR1 gene and an inactive FMR1 gene are mentally retarded while 60% of the females with a full mutation are affected. Here we describe monozygotic twin sisters who both have a full mutation in their FMR1 gene, one of whom is normal while the other is affected. Using molecular and protein studies it was shown that owing to preferential X inactivation in the affected female a minority of the cells expressed the normal FMR1 gene, while in her sister most cells expressed the normal FMR1 gene. This shows that X inactivation took place in the female twins after separation of the embryos and that for a normal phenotype FMR1 expression is necessary in the majority of cells.

Prenatal diagnosis of the fragile X syndrome: loss of mutation owing to a double recombinant or gene conversion event at the FMR1 locus.

The fragile X syndrome, an X linked mental retardation syndrome, is caused by an expanded CGG repeat in the first exon of the FMR1 gene. In patients with an expanded repeat the FMR1 promoter is methylated and, consequently, the gene is silenced and no FMR1 protein (FMRP) is produced, thus leading to the clinical phenotype. Here we describe a prenatal diagnosis performed in a female from a fragile X family carrying a large premutation. In chorionic villus DNA of the male fetus the normal maternal CGG allele and a normal pattern on Southern blot analysis were found in combination with the FRAXAC2 and DXS297 allele of the maternal at risk haplotype. A second chorionic villus sampling was performed giving identical results on DNA analysis and, in addition, expression of FMRP was shown by immunohistochemistry. We concluded that the male fetus was not affected with the fragile X syndrome. Subsequent detailed haplotype analysis showed a complex recombination pattern resembling either gene conversion or a double crossover within a 20 kb genomic region.

BRCA1 genomic deletions are major founder mutations in Dutch breast cancer patients.

To date, more than 300 distinct small deletions, insertions and point mutations, mostly leading to premature termination of translation, have been reported in the breast/ovarian-cancer susceptibility gene BRCA1. The elevated frequencies of some mutations in certain ethnic subpopulations are caused by founder effects, rather than by mutation hotspots. Here we report that the currently available mutation spectrum of BRCA1 has been biased by PCR-based mutation-screening methods, such as SSCP, the protein truncation test (PTT) and direct sequencing, using genomic DNA as template. Three large genomic deletions that are not detected by these approaches comprise 36% of all BRCA1 mutations found in Dutch breast-cancer families to date. A 510-bp Alu-mediated deletion comprising exon 22 was found in 8 of 170 breast-cancer families recruited for research purposes and in 6 of 49 probands referred to the Amsterdam Family Cancer Clinic for genetic counselling. In addition, a 3,835-bp Alu-mediated deletion encompassing exon 13 was detected in 4 of 170 research families, while an deletion of approximately 14 kb was detected in a single family [corrected]. Haplotype analyses indicated that each recurrent deletion had a single common ancestor.

A high proportion of novel mutations in BRCA1 with strong founder effects among Dutch and Belgian hereditary breast and ovarian cancer families.

We have identified 79 mutations in BRCA1 in a set of 643 Dutch and 23 Belgian hereditary breast and ovarian cancer families collected either for research or for clinical diagnostic purposes. Twenty-eight distinct mutations have been observed, 18 of them not previously reported and 12 of them occurring more than once. Most conspicuously, a 2804delAA mutation has been found 19 times and has never been reported outside the Netherlands. A common haplotype spanning > or = 375 kb could be identified for each of the nine examined recurrent mutations, indicating the presence of multiple BRCA1 founder mutations in the Dutch population. The 2804delAA mutation has been estimated to have originated approximately 32 generations ago. No specific breast or ovarian cancer phenotype could be assigned to any of the common mutations, and the ovarian cancer incidence among 18 families with the 2804delAA mutation was heterogeneous.

Rapid detection of BRCA1 mutations by the protein truncation test.

More than 75% of the reported mutations in the hereditary breast and ovarian cancer gene, BRCA1, result in truncated proteins. We have used the protein truncation test (PTT) to screen for mutations in exon 11, which encodes 61% of BRCA1. In 45 patients from breast and/or ovarian cancer families we found six novel mutations: two single nucleotide insertions, three small deletions (1-5 bp) and a nonsense mutation identified two unrelated families. Furthermore, we were able to amplify the remaining coding region by RT-PCR using lymphocyte RNA. Combined with PTT, we detected aberrantly spliced products affecting exons 5 and 6 in one of two BRCA1-linked families examined. The protein truncation test promises to become a valuable technique in detecting BRCA1 mutations.

Inversions in the factor VIII gene: improvement of carrier detection and prenatal diagnosis in Dutch haemophilia A families.

Haemophilia A is an X linked bleeding disorder caused by a heterogeneous spectrum of mutations in the factor VIII gene. It has recently been reported that about 50% of severe haemophilia A cases are the result of an iversion in the factor VIII gene. The inversion results from homologous recombination between the A gene located in intron 22 of the FVIII gene and one of the two distal A genes, thus disrupting the coding sequence of the factor VIII gene. The inversion can be detected by conventional Southern blotting and hybridisation techniques. Here we present an analysis of 177 unrelated Dutch haemophilia A cases for the presence of an inversion. In 57% of the patients with severe disease an inversion was found and also in at least one of the 26 patients with moderately severe disease. The majority of inversions (85%) involved the most distal A gene, while in a minority (15%) the more proximal A gene was involved. We show that direct mutation detection greatly improves the assessment of carrier status and prenatal diagnosis for haemophilia A, especially in families with an isolated patient. The inversion is predominantly of grandpaternal origin.