Genome-wide analysis in 756,646 individuals provides first genetic evidence that ACE2 expression influences COVID-19 risk and yields genetic risk scores predictive of severe disease.

SARS-CoV-2 enters host cells by binding angiotensin-converting enzyme 2 (ACE2). Through a genome-wide association study, we show that a rare variant (MAF = 0.3%, odds ratio 0.60, P=4.5×10-13) that down-regulates ACE2 expression reduces risk of COVID-19 disease, providing human genetics support for the hypothesis that ACE2 levels influence COVID-19 risk. Further, we show that common genetic variants define a risk score that predicts severe disease among COVID-19 cases.

Localization of hepatitis C virus RNA on human erythrocytes by RT in situ PCR technique.

BACKGROUND: Our previous results showed that hepatitis C virus (HCV) is detectable on erythrocytes by RT in situ PCR. The aims of the present study were to compare the sensitivity of this erythrocyte in situ PCR to routine serum solution phase HCV PCR as well as to obtain more data about the binding and cellular localization of HCV in the erythrocyte. METHODS: 105 previously HCV-infected patients and 20 control individuals were studied using RT in situ PCR on erythrocytes and solution phase RT-PCR from serum samples. Binding of HCV to erythrocytes was studied by in vitro inoculation. RT in situ PCR results were evaluated by fluorescence and confocal laser scanning microscopy. RESULTS: From 105 HCV cases, 78 gave positive, while 5-and all control cases-gave negative results by both PCR techniques. In 21 cases, only the in situ technique provided positive results, while in only I case did the solution phase method provide positive results. During in vitro inoculation, an early HCV-erythrocyte binding was detected followed by virus internalization. CONCLUSIONS: Erythrocyte-in situ PCR was found to show higher sensitivity for the detection of HCV compared to the generally applied serum PCR method. In vitro studies suggested a specific binding of HCV to erythrocyte and showed the virus to be capable of internalization.

Comparative study of the two more frequent HFE mutations (C282Y and H63D): significant different allelic frequencies between the North and South of Portugal.

An earlier study of reference values of iron parameters in Portugal showed significant differences between populations from northern and southern villages. This study addresses the question of the geographical distribution in Portugal of the two main mutations (C282Y and H63D) of the hereditary hemochromatosis gene, HFE. For that purpose, a stratified sample of 640 anonymous dried blood spot samples was randomly selected from the major regions of Portugal: North, Center, Lisbon and the Tagus Valley, Alentejo and Algarve. Differences in the geographical distribution of these two mutations were observed thus confirming the presumed differences between the age of the two mutations which is compatible with the postulated Celtic/Nordic origin of the C282Y mutation. The finding of a significantly higher allelic frequency of the C282Y mutation in the North (0.058) than in the South (0.009) could also point to an effect of differential selective forces acting in the different geographical areas of the country. Data on archaeological, ethnographic and linguistic records and on the North/South distribution of Portuguese cattle breeds of European or African origin have also been reported. In addition to their interest for population genetics, the results represent a reminder of the need to take into account regional differences in the design of strategies for population screening of hereditary hemochromatosis.

A reverse-hybridization assay for the rapid and simultaneous detection of nine HFE gene mutations.

Hereditary hemochromatosis (HH) is a very common autosomal recessive disorder of iron metabolism and frequently associated with mutations in the HFE gene. Molecular genetic testing for HFE mutations is considered valuable for carrier identification, as well as for early diagnosis of the disease, allowing simple treatment by phlebotomy and normal survival of patients. We have developed a reverse-hybridization assay for the routine diagnosis of eight previously described and one novel (E168Q) HFE point mutations. The test is based on multiplex DNA amplification and ready-to-use membrane teststrips, which contain oligonucleotide probes for each wild-type and mutated allele immobilized as an array of parallel lines. The procedure is rapid and accessible to automation on commercially available equipment, and by adding new probes the teststrip can easily be adapted to cover an increasing number of mutations.

HLA-B*27 typing by group-specific hybridization in microtiter plates.

We have developed and evaluated a test for HLA-B*27 based on PCR and DNA hybridization in microtiter plates. A region within exon 2 of the HLA-B gene is amplified and labeled by PCR and the amplification product is hybridized to a group-specific HLA-B*27 and a generic control oligonucleotide probe in two separate cavities of the plate. Bound sequences are detected using an ELISA-like protocol. The assay has been evaluated on 254 DNA samples routinely received for B27 testing in parallel with serological and SSP-PCR typing. Results were concordant in typing 102 HLA-B27-positive and 152 HLA-B27-negative individuals except for two samples containing HLA-B*73, which stained B27 positive in the microwell test. The new procedure is rapid and simple to perform, and the microwell format is particularly suitable for automation.

Quantitative detection of reverse transcriptase-PCR products by means of a novel and sensitive DNA stain.

We constructed a plasmid for the in vitro synthesis of a competitor RNA for use as an internal exogenous control during reverse transcriptase--PCR (RT-PCR) detection of epidermal growth factor receptor (EGFR) expression. The competitor RNA harbors a 32-base deletion compared with wild-type EGFR mRNA and generates a PCR product that is easily distinguished from the wild-type PCR product by agarose gel electrophoresis. We encountered the problem of heteroduplex formation during later stages of PCR, which could be solved by decreasing the PCR cycle number. This was accompanied by a significant loss of sensitivity. Sensitivity could be restored by using a novel and extremely sensitive DNA stain (SYBR Green I) instead of ethidium bromide.

Increased natural killer cell activity correlates with low or negative expression of the HER-2/neu oncogene in patients with breast cancer.

BACKGROUND: Increased expression of the HER-2/neu oncogene in breast cancer correlates with decreased estrogen receptor concentration and seems to be an important prognostic factor. The authors investigated whether there is a correlation between HER-2/neu expression and immunologic parameters representing tumor defense in patients with breast cancer. METHOD: A Western blot analysis was used to investigate HER-2/neu expression, whereas a chromium-release assay using the K562 cell line as target was used to measure natural killer (NK) cell activity. RESULTS: In patients with breast cancer, NK cell activity was significantly higher compared with patients with benign tumors (P = 0.006) or healthy control subjects (P = 0.002). Moreover, 23.3% of patients with breast cancer showed an overexpression of HER-2/neu protein. Within this group of patients, NK cell activity was significantly lower (45.6 +/- 16.1%) compared with the group with no HER-2/neu overexpression (57.3 +/- 11.0%). NK cell activity did not increase in patients with HER-2/neu overexpression. Thus, there was a statistically significant correlation of cytolytic effector cell function with HER-2/neu expression of the tumor (P = 0.003), and HER-2/neu overexpression correlated with a negative estrogen receptor status (P = 0.005). CONCLUSION: These data add further evidence to previous observations from the authors' laboratory that certain tumor characteristics may be associated with reactions of the host with breast cancer.

Rapid quantitative analysis of differential PCR products by high-performance liquid chromatography.

We describe the use of high-performance liquid chromatography (HPLC) for the rapid quantitative analysis of short DNA fragments generated in differential PCR, where a target gene and a control gene are in vitro coamplified in one single reaction. Using an anion-exchange nonporous column, both separation and quantitation of the differential PCR products are achieved in about 5 min per sample. The performance of this technique proved to be superior to that of conventional gel electrophoresis and subsequent analysis by a laser densitometer, a solid-state scanner and a charge coupled device video camera imaging system. The usefulness for clinical testing is described in the example of the quantitative analysis of the c-erbB-2 oncogene copy number of human breast carcinomas by differential PCR. The combined use of differential PCR and automated HPLC analysis of the PCR products may well substitute for classical Southern blot hybridization in routine clinical analysis of oncogene amplification.

EGF-R and steroid receptors in breast cancer: a comparison with tumor grading, tumor size, lymph node involvement, and age.

In this study epidermal growth factor receptor (EGF-R), estrogen receptor (ER), and progesterone receptor (PR) status was evaluated in 326 primary breast carcinomas. Nineteen percent of samples were EGF-R positive, 63% were positive for ER, and 54% for PR. In 46% of the tumors both ER and PR were positive. These data are presented together, with grading, size of tumor, lymph node involvement, histological subtype, and age. Sixty-nine percent of EGF-R negative tumors were ER-positive and 51% were positive for ER as well as PR. In particular, negative correlation between EGF-R and steroid receptor status was found. A quantitative correlation was also shown. A combination of negative steroid receptor and positive EGF-R was found more often in the population of poorly differentiated tumors. Tumors bigger than 5 cm were related to a positive EGF-R status. No correlation between nodal status and any receptor status was found. Intraductal carcinomas were more often EGF-R positive than infiltrating ductal (NOS) or infiltrating lobular lesions. The age of patients correlated with the concentration of ER only. In our study we reaffirmed the negative correlation between steroid receptor status and the overexpression of EGF-R; furthermore the combination of EGF-R+ and ER- tumors was observed more often in histological high-risk tumors. Patient outcome did not show statistically significant differences concerning the EGF-R status, but was associated with the steroid receptor status.

A simple method for isolation of DNA from blood clots suited for use in PCR.

We describe a rapid, simple and inexpensive method for the isolation of DNA from blood clots suited for use in PCR. Our method is based on the lysing and nuclease-inactivating properties of guanidine thiocyanate together with the nucleic acid-binding properties of silica particles. Isolated DNA can be used for in vitro amplification as shown for a retinoblastoma gene PCR system.

[Epidermal growth factor receptor in human breast cancer: correlation with steroid receptors, tumor stage, grading and menopausal status]. / Epidermaler Wachstumsfaktor-Rezeptor im humanen Mammakarzinom: Korrelation mit Steroidrezeptoren, Tumorstadium, Grading und Menopausestatus.

In this study, correlations between the epidermal growth factor receptor (EGF-R), steroid receptors, and other prognostic parameters (grading, pTNM-status, menopausal status) were analysed in 326 primary breast carcinomas. 19% of the tumour samples were EGF-R positive, 63% were estrogen receptor (ER) and 54% progesteron receptor (PR) positive. Both steroid receptors were positive in 46% of all samples. We found a highly significant inverse correlation between EGF-R and steroid receptors. 88% of the ER positive tumours were EGF-R negative (p less than 5 x 10(-5)), 90% of the PR positive tumours were EGF-R negative (p less than 5 x 10(-5)) and 91% of the ER plus PR positive tumours were ERF-R negative (p less than 1 x 10(-6)). Grading was available in 170 cases. Six (4%) of the carcinomas were highly differentiated (G1), 82 (48%) were classified as G2, and another 82 (48%) were poorly differentiated (G3). A combination of negative ER and positive EGF-R was found more often in the population of G3 tumours. EGF-R was also positively correlated to tumour size. With regard to receptor status, we did not find a correlation with lymph node involvement. The ER correlated negatively (p less than 1.3 x 10(-5) and the EGF-R positively (p less than 0.042) with menopausal status. Thus, EGF-R overexpression seems to be a marker of morphological and functional dedifferentiation which is associated with a loss of steroid dependency and an increase of an autostimulatory-paracrine growth control. These changes seem to be related to poor prognosis.

[C-erbB-2 oncogene amplification in breast cancer in correlation to steroid and epidermal growth factor receptor]. / C-erbB-2-Onkogen-Amplifikation bei Mammakarzinom in Korrelation zum Steroid- und epidermalen Wachstumsfaktor-Rezeptor.

The amplification grade of oncogene c-erbB-2 was examined by the polymerase-chain-reaction-method in DNA's of 56 primary mammary carcinomas. 26 (46.4%) of these showed the amplified oncogene c-erbB-2. In the strongly amplified cases, the expression of the c-erbB-2 oncoprotein was verifiable immunohistochemically. Between the progesterone receptor status (PR) and the amplified c-erbB-2 oncogene there was a statistically proven dependency. No correlation was observed between the amplified c-erbB-2 oncogene and the epidermal growth-factor receptor (EGFR).

Determination of HER-2/neu amplification and expression in tumor tissue and cultured cells using a simple, phenol free method for nucleic acid isolation.

A rapid, simple and non-toxic procedure for the simultaneous isolation of DNA and RNA from tumor tissue and cells grown in vitro is described. Guanidinium isothiocyanate was used for homogenization of tumor tissue and for cell lysis. Separation of proteins, DNA and RNA was carried out by isopycnic centrifugation in cesium trifluoroacetate. DNA was further purified by salting out residual protein. Nucleic acids prepared by this method from 47 primary human carcinomas and 17 human cell lines were analysed for amplification and expression of the HER-2/neu proto-oncogene. 2- to 10-fold amplification of HER-2/neu was noted in 7/22 mammary carcinomas (32%) and in 4/14 ovarian carcinomas (28%). No amplification of the proto-oncogene was found in 4 laryngeal carcinomas, 1 pharyngeal carcinoma, 2 retrolingual carcinomas, 3 gastric carcinomas and 1 kidney carcinoma. HER-2/neu overexpression was observed in 6/22 of mammary carcinomas (27%) and 7/14 of ovarian carcinomas (50%). No overexpression was found in all other carcinomas studied. Concordance between amplification and overexpression was noted in 3 mammary and 4 ovarian carcinomas, respectively. 3 mammary and 3 ovarian carcinomas showed overexpression without amplification. 5 human mammary carcinoma cell lines showed both amplification and overexpression of HER-2/neu. In two mammary carcinoma cell lines (MDA MB-453 and ZR 75-1) overexpression was noted without amplification of the proto-oncogene. These data combine to suggest that mechanisms other than gene amplification may also lead to overexpression of the HER-2/neu protooncogene in cancer cells.

HER-2 oncogene amplification and overall survival of breast carcinoma patients.

DNA was extracted from tumour samples of 77 patients with primary breast carcinoma and HER-2 proto-oncogene amplification was assessed. Prognostic indices such as number of positive lymph nodes, tumour size and histological grading were strongly associated with overall survival. No statistically significant correlation between amplification of HER-2 and overall survival was observed. In addition, prognostic indices, HER-2 amplification and disease-free interval was not correlated. Analysis of HER-2 amplification alone is not a useful guide in the management of patients with mammary carcinoma.

HER-2 amplification, steroid receptors and epidermal growth factor receptor in primary breast cancer.

Amplification of HER-2 oncogene was analysed in DNAs obtained from 291 primary human mammary carcinomas. 52/291 (18%) were found to contain amplified HER-2 oncogene. Moderate amplification (2- to 5-fold) was noted in 36/291 (12%). Thirteen tumors (4.5%) had a copy number of 5 to 10. A 10- to 20-fold and greater than 20-fold amplification was observed in 2 and 1 patient, respectively. Sample sizes allowed the determination of estrogen receptor (ER) and progesterone receptor (PgR) levels in 253/291 primary breast cancers. HER-2 gene amplification was noted in 14% of ER+ patients and in 28% of ER- patients, respectively (P = 0.02). Similarly a significantly greater number of PgR- primary mammary carcinoma exhibited an amplification of the HER-2 gene compared to PgR+ cases (22% vs. 16%, P = 0.01). Although statistically not significant, tumors with HER-2 gene amplification were found to have lower levels of ER and PgR. No association of HER-2 amplification with the androgen receptor and EGF receptor was observed. Present data combine to suggest that tumor progression is more stringently controlled by the oncogene upon loss of hormone dependency. Differences found in HER-2 amplification between steroid receptor positive and negative tumors could be helpful to define a specific subset of women to whom adjuvant therapy should be directed.