Dominant-negative heterozygous mutations in AIRE confer diverse autoimmune phenotypes.

Autoimmune polyendocrine syndrome type 1 (APS-1) is an autosomal recessive disease characterized by severe and childhood onset organ-specific autoimmunity caused by mutations in the autoimmune regulator (AIRE) gene. More recently, dominant-negative mutations within the PHD1, PHD2, and SAND domains have been associated with an incompletely penetrant milder phenotype with later onset familial clustering, often masquerading as organ-specific autoimmunity. Patients with immunodeficiencies or autoimmunity where genetic analyses revealed heterozygous AIRE mutations were included in the study and the dominant-negative effects of the AIRE mutations were functionally assessed in vitro. We here report additional families with phenotypes ranging from immunodeficiency, enteropathy, and vitiligo to asymptomatic carrier status. APS-1-specific autoantibodies can hint to the presence of these pathogenic AIRE variants although their absence does not rule out their presence. Our findings suggest functional studies of heterozygous AIRE variants and close follow-up of identified individuals and their families.

Male origin microchimerism and brain cancer: a case-cohort study.

BACKGROUND: Despite considerable research effort, causes of brain cancer are largely unknown. Male brain cancer predominance and reduced brain cancer risk with increasing parity among women, however, support a favourable role of pregnancy. We set out to determine whether fetal-origin microchimerism, namely the presence and long-term persistence of fetal cells, likely obtained via natural trafficking across the placenta during pregnancy, associates with reduced risk of brain cancer in women. METHODS: Using a case-cohort design, we sampled 505 middle-aged women randomly at baseline in the Diet, Cancer and Health cohort (controls), and 73 women with incident brain cancer diagnosed during follow-up in the Danish Cancer Registry (cases). Male origin microchimerism was determined by presence of Y chromosome sequences in female blood samples. Data were analysed using weighted proportional Hazards regression, yielding Hazard Ratios with 95% confidence intervals. RESULTS: Compared with male origin microchimerism negative women, positive women had half the risk of developing brain cancer (Hazard Ratio = 0.50 [0.33-0.77]). Sensitivity analyses support that our findings are unlikely due to bias or chance. CONCLUSION: Here, for the first time, we demonstrate half the risk of brain cancer in male origin microchimerism positive compared with negative women. Our findings resemble those of previous studies of male origin microchimerism and other female cancers.

Male origin microchimerism and ovarian cancer.

BACKGROUND: Reduced risk of ovarian cancer is commonly ascribed to reduced exposure to endogenous hormones during pregnancy, using oral contraceptives or not using hormone replacement therapy. However, exposure to hormones alone account for less than half of all cases. Many women carry small amounts of male cells-known as male origin microchimerism-in their circulation and remarkable impacts of these cells on women's health are being published. Here, we pursue the possibility that male origin microchimerism has a role in reducing ovarian cancer risk. METHODS: We conducted a prospective case-cohort study using blood samples and questionnaire data from 700 women participating in the Danish Diet, Cancer, and Health cohort. Blood samples were analysed for Y chromosome presence as a marker of male microchimerism. We evaluated the association between male microchimerism and ovarian cancer, using weighted Cox regression models reporting hazard ratios (HRs) and corresponding 95% confidence intervals (CIs). RESULTS: Male microchimerism was detected in 46% of cases and 65.9% of controls. Women testing positive for male microchimerism had a reduced hazard rate of ovarian cancer compared with women testing negative (HR = 0.44, 95% CI: 0.29-0.68). We found no evidence of interaction with measures of hormonal exposures (P = 0.50). CONCLUSIONS: For the first time we report that women who test positive for male microchimerism in their circulation have reduced rates of ovarian cancer compared with women who test negative. Although the underlying mechanisms are presently unknown, we believe male microchimerism is potent in preventing ovarian cancer.

Impact of Male-Origin Microchimerism on Cardiovascular Disease in Women: A Prospective Cohort Study.

Increasing parity is associated with an increased risk of ischemic heart disease (IHD) and stroke in women. This is probably attributable to biological responses of pregnancy. Male cells of presumed fetal origin are commonly present in women years after pregnancy-a phenomenon termed male-origin microchimerism (MOM). In this study, we investigated whether MOM was associated with risk of IHD and ischemic stroke in women. We evaluated the association between MOM and ischemic events in a cohort of 766 Danish women enrolled in the Diet, Cancer and Health cohort during 1993-1997 when aged 50-64 years. Of these women, 545 (71.2%) tested positive for MOM through targeting of the Y chromosome (DYS14 DNA sequence) in their blood. Multiple Cox regression models were used to calculate hazard ratios with 95% confidence intervals. We found that MOM was associated with a significantly reduced rate of IHD (hazard ratio = 0.44, 95% confidence interval: 0.23, 0.83) but not ischemic stroke (hazard ratio = 0.80, 95% confidence interval: 0.46, 1.41). Our findings show that microchimerism positivity is associated with a lower rate of later IHD development in women. Although the underlying mechanisms are presently unknown, MOM may be relevant in women's cardiovascular health. More studies are needed to confirm these findings.

Association between neutropenia and IgG antineutrophil antibodies in a case of CD40LG deficiency due to two novel mutations.

This case suggests a mechanistic rationale for the clinical efficacy of intravenous immunoglobulins (IVIG) in treating CD40 ligand (CD40L) deficiency associated neutropenia as it is the first reported instance of free and cell-bound antineutrophil antibodies in a case of CD40L deficiency, accompanied by a prolonged and clinically severe neutropenia.

Next Generation Sequencing-Based Fetal ABO Blood Group Prediction by Analysis of Cell-Free DNA from Maternal Plasma.

INTRODUCTION: ABO blood group incompatibility between a pregnant woman and her fetus as a cause of morbidity or mortality of the fetus or newborn remains an important, albeit rare, risk. When a pregnant woman has a high level of anti-A or anti-B IgG antibodies, the child may be at risk for hemolytic disease of the fetus and newborn (HDFN). Performing a direct prenatal determination of the fetal ABO blood group can provide valuable clinical information. OBJECTIVE: Here, we report a next generation sequencing (NGS)-based assay for predicting the prenatal ABO blood group. MATERIALS AND METHODS: A total of 26 plasma samples from 26 pregnant women were tested from gestational weeks 12 to 35. Of these samples, 20 were clinical samples and 6 were test samples. Extracted cell-free DNA was PCR-amplified using 2 primer sets followed by NGS. NGS data were analyzed by 2 different methods, FASTQ analysis and a grep search, to ensure robust results. The fetal ABO prediction was compared with the known serological infant ABO type, which was available for 19 samples. RESULTS: There was concordance for 19 of 19 predictable samples where the phenotype information was available and when the analysis was done by the 2 methods. For immunized pregnant women (n = 20), the risk of HDFN was predicted for 12 fetuses, and no risk was predicted for 7 fetuses; one result of the clinical samples was indeterminable. Cloning and sequencing revealed a novel variant harboring the same single nucleotide variations as ABO*O.01.24 with an additional c.220C>T substitution. An additional indeterminable result was found among the 6 test samples and was caused by maternal heterozygosity. The 2 indeterminable samples demonstrated limitations to the assay due to hybrid ABO genes or maternal heterozygosity. CONCLUSIONS: We pioneered an NGS-based fetal ABO prediction assay based on a cell-free DNA analysis from maternal plasma and demonstrated its application in a small number of samples. Based on the calculations of variant frequencies and ABO*O.01/ABO*O.02 heterozygote frequency, we estimate that we can assign a reliable fetal ABO type in approximately 95% of the forthcoming clinical samples of type O pregnant women. Despite the vast genetic variations underlying the ABO blood groups, many variants are rare, and prenatal ABO prediction is possible and adds valuable early information for the prevention of ABO HDFN.

Correction: Genetic susceptibility to angiotensin-converting enzyme-inhibitor induced angioedema: A systematic review and evaluation of methodological approaches.

[This corrects the article DOI: 10.1371/journal.pone.0224858.].

Genetic susceptibility to angiotensin-converting enzyme-inhibitor induced angioedema: A systematic review and evaluation of methodological approaches.

Angiotensin-converting enzyme (ACE) converts angiotensin I to angiotensin II which causes vasoconstriction. ACE inhibitors reduce blood pressure by inhibiting ACE. A well-known adverse drug reaction to ACE inhibitors is ACE inhibitor-induced angioedema (ACEi-AE). Angioedema is a swelling of skin and mucosa, which can be fatal if the airway is compromised. We have performed a systematic review of the evidence suggesting that genetic polymorphisms are associated with ACEi-AE and evaluated the methodological approaches of the included studies. The Cochrane Database of Systematic Reviews, Google Scholar, and PubMed were searched. Studies investigating the association between genetic markers and ACEi-AE were included. The Q-genie tool was used to evaluate the quality of the study methodologies. Seven studies were included. With the exception of one whole genome study, all of the included studies were candidate gene association studies. Study quality assessment scores ranged from 36 to 55. One study was found to be of good quality, suggesting that the detected associations may be unreliable. The inferior quality of some studies was due to poor organization, lack of analyses and missing information. Polymorphisms within XPEPNP2, BDKRB2-9/+ 9 and neprilysin genes, were reported to be associated with increased risk of ACEi-AE. However, due to low quality, these associations need to be confirmed in larger studies.

Acquired complement C1 esterase inhibitor deficiency in a patient with a rare SERPING1 variant with unknown significance.

Angioedema (AE) is caused by a wide range of diseases and pharmaceuticals; it can become life-threatening when located to the airways. Patients with deficiency or malfunction of complement C1 esterase inhibitor (hereditary or acquired) experience recurrent AE due to an accumulation of the vasoactive mediator bradykinin (BK). Complement C1 inhibitor normally decreases BK production, so a reduced function hereof causes increased levels. The diagnosis of hereditary or acquired AE can be difficult due to similarities to allergic reactions (swelling, abdominal pain, rash). We describe a 35-year-old man presenting with upper-airway AE progressing rapidly and promptly required cricothyroidotomy. Complement and autoantibody screening together with sequencing of SERPING1 were performed and gave the diagnosis of acquired complement C1 esterase inhibitor deficiency. The patient is unusual to have this disease before the age of 40 years. No associated comorbidities were found. It is important to know that antiallergic medication is not effective in BK-mediated AE.

Hereditary angioedema: a mother diagnosing her child using Google as a diagnostic aid.

Hereditary angioedema (HAE), due to C1-inhibitor deficiency, is a rare autosomal dominant and potentially life-threatening disease characterised by recurrent oedema attacks of skin, mucosa and viscera. Due to the rarity and the fact that symptoms of HAE imitate other forms of angioedema and other conditions, HAE may be misdiagnosed, especially in emergency settings. Consequently, patients with HAE may experience significant delays in diagnosis. Without an accurate diagnosis patients with HAE may not receive proper treatment. At times 'Doctor Google' may be an important tool in establishing the diagnosis. The aim of this case report is to emphasise the importance of listening to patients and relatives and being humble to 'Doctor Google'. Furthermore, the aim is to remind all healthcare personal of HAE and the importance of considering the rare differential diagnoses to common symptoms.

[Review of a new subtype of hereditary angio-oedema with normal complement C1-inhibitor].

Hereditary angio-oedema (HAE) is a rare, potentially fatal disease characterized by recurrent swelling of skin and mucosa. Besides HAE with quantitative (type I) or qualitative (type II) deficiency of complement C1-inhibitor (C1-INH), a new subtype of HAE is now described with normal levels of C1-INH. This subtype is possibly underdiagnosed, and a treatment regimen and general knowledge about the condition is still in its infancy. The purpose of this article is to inform Danish doctors about the disease to identify more Danish patients.

Microchimerism of male origin in a cohort of Danish girls.

Male microchimerism, the presence of a small number of male cells, in women has been attributed to prior pregnancies. However, male microchimerism has also been reported in women with only daughters, in nulliparous women and prepubertal girls suggesting that other sources of male microchimerism must exist. The aim of the present study was to examine the presence of male microchimerism in a cohort of healthy nulliparous Danish girls aged 10-15 y using DNA extracted from cells from whole blood (buffy coats) and report the association with potential sources of male cells. A total of 154 girls were studied of which 21 (13.6%) tested positive for male microchimerism. There was a tendency that girls were more likely to test positive for male microchimerism if their mothers previously had received transfusion, had given birth to a son or had had a spontaneous abortion. Furthermore, the oldest girls were more likely to test positive for male microchimerism. However, less than half of microchimerism positivity was attributable to these factors. In conclusion, data suggest that male microchimerism in young girls may originate from an older brother either full born or from a discontinued pregnancy or from transfusion during pregnancy. We speculate that sexual intercourse may be important but other sources of male cells likely exist in young girls.

Testosterone treatment increases androgen receptor and aromatase gene expression in myotubes from patients with PCOS and controls, but does not induce insulin resistance.

Polycystic ovary syndrome (PCOS) is associated with insulin resistance and increased risk of type 2 diabetes. Skeletal muscle is the major site of insulin mediated glucose disposal and the skeletal muscle tissue is capable to synthesize, convert and degrade androgens. Insulin sensitivity is conserved in cultured myotubes (in vitro) from patients with PCOS, but the effect of testosterone on this insulin sensitivity is unknown. We investigated the effect of 7days testosterone treatment (100nmol/l) on glucose transport and gene expression levels of hormone receptors and enzymes involved in the synthesis and conversion of testosterone (HSD17B1, HSD17B2, CYP19A1, SRD5A1-2, AR, ER-α, HSD17B6 and AKR1-3) in myotubes from ten patients with PCOS and ten matched controls. Testosterone treatment significantly increased aromatase and androgen receptor gene expression levels in patients and controls. Glucose transport in myotubes was comparable in patients with PCOS vs. controls and was unchanged by testosterone treatment (p=0.21 PCOS vs. controls). These results suggest that testosterone treatment of myotubes increases the aromatase and androgen receptor gene expression without affecting insulin sensitivity and if testosterone is implicated in muscular insulin resistance in PCOS, this is by and indirect mechanism.

Report of the first nationally implemented clinical routine screening for fetal RHD in D- pregnant women to ascertain the requirement for antenatal RhD prophylaxis.

BACKGROUND: A combination of antenatal and postnatal RhD prophylaxis is more effective in reducing D immunization in pregnancy than postnatal RhD prophylaxis alone. Based on the result from antenatal screening for the fetal RHD gene, antenatal RhD prophylaxis in Denmark is given only to those D- women who carry a D+ fetus. We present an evaluation of the first national clinical application of antenatal RHD screening. STUDY DESIGN AND METHODS: In each of the five Danish health care regions, blood samples were drawn from D- women in Gestational Week 25. DNA was extracted from the maternal plasma and analyzed for the presence of the RHD gene by real-time polymerase chain reaction targeting two RHD exons. Prediction of the fetal RhD type was compared with serologic typing of the newborn in 2312 pregnancies, which represented the first 6 months of routine analysis. RESULTS: For the detection of fetal RHD, the sensitivity was 99.9%. The accuracy was 96.5%. The recommendation for unnecessary antenatal RhD prophylaxis for women carrying a D- fetus was correctly avoided in 862 cases (37.3%), while 39 women (1.7%) were recommended for antenatal RhD prophylaxis unnecessarily. Two RHD+ fetuses (0.087%) were not detected, and antenatal RhIG was not given. CONCLUSION: These data represent the first demonstration of the reliability of routine antenatal fetal RHD screening in D-, pregnant women to ascertain the requirement for antenatal RhD prophylaxis. Our findings should encourage the implementation of such screening programs worldwide, to reduce the unnecessary use of RhIG.

Detection of non-DeltaGT NCF-1 mutations in chronic granulomatous disease.

AIMS: Chronic granulomatous disease (CGD) is a rare inherited disorder caused by mutations in the subunits of the NADPH oxidase complex, leaving phagocytes unable to produce superoxide and thereby unable to kill invading microorganisms. A subgroup of CGD patients (approximately 20%) is reported to have mutations in NCF-1 encoding p47-phox, which is part of the cytosolic component of NADPH oxidase. More than 94% of these patients share the same mutation, a 2 bp GT deletion in the GTGT dinucleotide repeat in the start of exon 2. The presence of two pseudogenes more than 98% homologous to the functional NCF-1 has complicated the identification of other mutations in the gene. The aim of this study was to find a general technique for detection of non-GT deletion mutations in the coding region of NCF-1. RESULTS: A technique involving GeneScan analysis followed by amplification of cDNA with intact dinucleotide repeat was set up and used to identify a novel mutation in exon 7 of NCF-1 in a patient with autosomal recessive CGD, explaining the disease by changing a UGG codon to a premature UGA STOP codon. CONCLUSION: The method is generally applicable for the detection of NCF-1 mutations in patients with suspected CGD.

Postmortem detection of hepatitis B, C, and human immunodeficiency virus genomes in blood samples from drug-related deaths in Denmark*.

Blood-borne viral infections are widespread among injecting drug users; however, it is difficult to include these patients in serological surveys. Therefore, we developed a national surveillance program based on postmortem testing of persons whose deaths were drug related. Blood collected at autopsy was tested for anti-HBc, anti-HBs, anti-hepatitis C virus (HCV), or anti-human immunodeficiency virus (HIV) antibodies using commercial kits. Subsets of seropositive samples were screened for viral genomes using sensitive in-house and commercial polymerase chain reaction (PCR) assays. Hepatitis B virus (HBV) DNA was detected in 20% (3/15) of anti-HBc-positive/anti-HBs-negative samples, HCV RNA was found in 64% (16/25) of anti-HCV-positive samples, and HIV RNA was detected in 40% (6/15) of anti-HIV-positive samples. The postmortem and antemortem prevalences of HBV DNA and HCV RNA were similar. Postmortem HIV RNA testing was less sensitive than antemortem testing. Thus, postmortem PCR analysis for HBV and HBC infection is feasible and relevant for demonstrating ongoing infections at death or for transmission analysis during outbreaks.