Genotype-phenotype correlations in RHOBTB2-associated neurodevelopmental disorders.

PURPOSE: Missense variants clustering in the BTB domain region of RHOBTB2 cause a developmental and epileptic encephalopathy with early-onset seizures and severe intellectual disability. METHODS: By international collaboration, we assembled individuals with pathogenic RHOBTB2 variants and a variable spectrum of neurodevelopmental disorders. By western blotting, we investigated the consequences of missense variants in vitro. RESULTS: In accordance with previous observations, de novo heterozygous missense variants in the BTB domain region led to a severe developmental and epileptic encephalopathy in 16 individuals. Now, we also identified de novo missense variants in the GTPase domain in 6 individuals with apparently more variable neurodevelopmental phenotypes with or without epilepsy. In contrast to variants in the BTB domain region, variants in the GTPase domain do not impair proteasomal degradation of RHOBTB2 in vitro, indicating different functional consequences. Furthermore, we observed biallelic splice-site and truncating variants in 9 families with variable neurodevelopmental phenotypes, indicating that complete loss of RHOBTB2 is pathogenic as well. CONCLUSION: By identifying genotype-phenotype correlations regarding location and consequences of de novo missense variants in RHOBTB2 and by identifying biallelic truncating variants, we further delineate and expand the molecular and clinical spectrum of RHOBTB2-related phenotypes, including both autosomal dominant and recessive neurodevelopmental disorders.

No evidence for intervention-associated DNA methylation changes in monocytes of patients with posttraumatic stress disorder.

DNA methylation patterns can be responsive to environmental influences. This observation has sparked interest in the potential for psychological interventions to influence epigenetic processes. Recent studies have observed correlations between DNA methylation changes and therapy outcome. However, most did not control for changes in cell composition. This study had two aims: first, we sought to replicate therapy-associated changes in DNA methylation of commonly assessed candidate genes in isolated monocytes from 60 female patients with post-traumatic stress disorder (PTSD). Our second, exploratory goal was to identify novel genomic regions with substantial pre-to-post intervention DNA methylation changes by performing whole-genome bisulfite sequencing (WGBS) in two patients with PTSD. Equivalence testing and Bayesian analyses provided evidence against physiologically meaningful intervention-associated DNA methylation changes in monocytes of PTSD patients in commonly investigated target genes (NR3C1, FKBP5, SLC6A4, OXTR). Furthermore, WGBS yielded only a limited set of candidate regions with suggestive evidence of differential DNA methylation pre- to post-therapy. These differential DNA methylation patterns did not prove replicable when investigated in the entire cohort. We conclude that there is no evidence for major, recurrent intervention-associated DNA methylation changes in the investigated genes in monocytes of patients with PTSD.

One test for all: whole exome sequencing significantly improves the diagnostic yield in growth retarded patients referred for molecular testing for Silver-Russell syndrome.

BACKGROUND: Silver-Russell syndrome (SRS) is an imprinting disorder which is characterised by severe primordial growth retardation, relative macrocephaly and a typical facial gestalt. The clinical heterogeneity of SRS is reflected by a broad spectrum of molecular changes with hypomethylation in 11p15 and maternal uniparental disomy of chromosome 7 (upd(7)mat) as the most frequent findings. Monogenetic causes are rare, but a clinical overlap with numerous other disorders has been reported. However, a comprehensive overview on the contribution of mutations in differential diagnostic genes to phenotypes reminiscent to SRS is missing due to the lack of appropriate tests. With the implementation of next generation sequencing (NGS) tools this limitation can now be circumvented. MAIN BODY: We analysed 75 patients referred for molecular testing for SRS by a NGS-based multigene panel, whole exome sequencing (WES), and trio-based WES. In 21/75 patients a disease-causing variant could be identified among them variants in known SRS genes (IGF2, PLAG1, HMGA2). Several patients carried variants in genes which have not yet been considered as differential diagnoses of SRS. CONCLUSIONS: WES approaches significantly increase the diagnostic yield in patients referred for SRS testing. Several of the identified monogenetic disorders have a major impact on clinical management and genetic counseling.

Parathyroid Hormone in Pregnancy: Vitamin D and Other Determinants.

We aimed to assess the parathyroid hormone (PTH) concentration in pregnant women at the beginning of pregnancy (1st trimester) and within days before delivery (3rd trimester) and evaluate its determinants. From September 2014 through December 2015 in a cross-sectional study, 204 women in the 1st trimester of pregnancy and 203 women in the 3rd trimester of pregnancy were recruited. Blood samples were collected to measure PTH and circulating 25-hydroxy-vitamin D (25(OH)D) concentrations. Lifestyle and demographic data were collected using a questionnaire. Serum 25(OH)D and PTH were inversely correlated in both early and late pregnancy. Our analyses suggest that in the 3rd trimester of pregnancy, a 25(OH)D level of 18.9 ng/mL (47.3 nmol/L) could serve as an inflection point for the maximal suppression of PTH. Statistically significant determinants of PTH concentrations in multiple regression were 25(OH)D concentrations, season, multiparity and education of the partner (all p < 0.05) in early pregnancy. In late pregnancy, 25(OH)D concentrations and country of origin were statistically significant determinants of PTH concentrations (all p < 0.05). These factors and their effect on PTH appear to be vastly determined by 25(OH)D; however, they might also affect PTH through other mechanisms besides 25(OH)D.

The adult phenotype of Schaaf-Yang syndrome.

BACKGROUND: MAGEL2-associated Schaaf-Yang syndrome (SHFYNG, OMIM #615547, ORPHA: 398069), which was identified in 2013, is a rare disorder caused by truncating variants of the paternal copy of MAGEL2, which is localized in the imprinted region on 15q11.2q13. The phenotype of SHFYNG in childhood partially overlaps with that of the well-established Prader-Willi syndrome (PWS, OMIM #176270). While larger numbers of younger individuals with SHFYNG have been recently published, the phenotype in adulthood is not well established. We recruited 7 adult individuals (aged 18 to 36) with molecularly confirmed SHFYNG and collected data regarding the clinical profile including eating habits, sleep, behavior, personal autonomy, psychiatric abnormalities and other medical conditions, as well as information about the respective phenotypes in childhood. RESULTS: Within our small cohort, we identified a range of common features, such as disturbed sleep, hypoactivity, social withdrawal and anxiety, but also noted considerable differences at the level of personal autonomy and skills. Behavioral problems were frequent, and a majority of individuals displayed weight gain and food-seeking behavior, along with mild intellectual disability or borderline intellectual function. Classical symptoms of SHFYNG in childhood were reported for most individuals. CONCLUSION: Our findings indicate a high variability of the functional abilities and social participation of adults with SHFYNG. A high prevalence of obesity within our cohort was notable, and uncontrollable food intake was a major concern for some caregivers. The phenotypes of PWS and SHFYNG in adulthood might be more difficult to discern than the phenotypes in childhood. Molecular genetic testing for SHFYNG should therefore be considered in adults with the suspected diagnosis of PWS, if testing for PWS has been negative.

Further evidence for POMK as candidate gene for WWS with meningoencephalocele.

BACKGROUND: Walker-Warburg syndrome (WWS) is a rare form of alpha-dystroglycanopathy characterized by muscular dystrophy and severe malformations of the CNS and eyes. Bi-allelic pathogenic variants in POMK are the cause of a broad spectrum of alpha-dystroglycanopathies. POMK encodes protein-O-mannose kinase, which is required for proper glycosylation and function of the dystroglycan complex and is crucial for extracellular matrix composition. RESULTS: Here, we report on male monozygotic twins with severe CNS malformations (hydrocephalus, cortical malformation, hypoplastic cerebellum, and most prominently occipital meningocele), eye malformations and highly elevated creatine kinase, indicating the clinical diagnosis of a congenital muscular dystrophy (alpha-dystroglycanopathy). Both twins were found to harbor a homozygous nonsense mutation c.640C>T, p.214* in POMK, confirming the clinical diagnosis and supporting the concept that POMK mutations can be causative of WWS. CONCLUSION: Our combined data suggest a more important role for POMK in the pathogenesis of meningoencephalocele. Only eight different pathogenic POMK variants have been published so far, detected in eight families; only five showed the severe WWS phenotype, suggesting that POMK-associated WWS is an extremely rare disease. We expand the phenotypic and mutational spectrum of POMK-associated WWS and provide evidence of the broad phenotypic variability of POMK-associated disease.

Mutation in ITCH Gene Can Cause Syndromic Multisystem Autoimmune Disease With Acute Liver Failure.

Pediatric intractable autoimmune hepatitis is rare and may be responsible for acute liver failure. Mutations in the itchy E3 ubiquitin protein ligase (ITCH) gene (located on chromosome 20q11.22) can lead to a deficiency of the encoded protein, resulting in increased T-cell activity with lack of immune tolerance and manifestation of a complex systemic autoimmune disease. A 1-year-old girl of consanguineous parents received a liver transplant (LT) because of acute liver failure attributed to a drug-induced hypereosinophilic syndrome with positive liver-kidney-mikrosome-2 antibodies. Notable findings were syndromic features, dystrophy, short stature, psychomotor retardation, and muscular hypotonia. Later, we saw corticosteroid-sensitive rejections as well as a systemic autoimmune disease with detection of specific antibodies (de novo autoimmune hepatitis, thyroiditis with exophthalmos, diabetes mellitus type 1, and immune neutropenia). Histologically, liver cirrhosis with lobular inflammatory infiltrates, giant-cell hepatitis, and ductopenia was verified in chronic cholestasis. Shortly after a second LT, a comparable liver histology could be detected, and viral, bacterial, and mycotic infections deteriorated the general health condition. Because of refractory pancytopenia related to portal hypertension and hypersplenism, a posttransplant lymphoproliferative disorder was excluded. One year after the second LT, epidural and subdural bleeding occurred. Three months afterward, the girl died of sepsis. Postmortem, whole-exome sequencing revealed a homozygous mutation in the ITCH gene. A biallelic mutation in ITCH can cause a severe syndromic multisystem autoimmune disease with the above phenotypic characteristics and acute liver failure because of autoimmune hepatitis. This case reveals the importance of ubiquitin pathways for regulation of the immune system.

Vitamin D status and its determinants in healthy pregnant women living in Switzerland in the first trimester of pregnancy.

OBJECTIVES: Our study aimed at assessing the prevalence and determinants of vitamin D deficiency (25-hydroxy-vitamin D [25(OH)D] < 20 ng/mL) in pregnant women in the first trimester living in Switzerland. METHODS: From September 2014 through December 2015, 204 pregnant women were conveniently recruited during their first clinical appointment at the Clinic of Obstetrics of the University Hospital Zurich (between week 6 and 12 of pregnancy). Blood samples were collected and a questionnaire focusing on lifestyle and skin colour was completed face-to-face with the responsible physician. Logistic regression analyses were performed with vitamin D status as dependent variable. RESULTS: 63.2% of the participating women were vitamin D deficient, and the median vitamin D concentration in the overall sample was 17.1 ng/mL [Q1, Q3: 9.78, 22.3]. The highest proportions of vitamin D deficiency were detected in women originating from Africa and Middle East (91.4% deficient, median vitamin D concentration of 10.7 ng/mL [Q1, Q3: 6.55, 14.45]) and from South-East Asia/Pacific (88.5% deficient, median vitamin D concentration of 8.4 ng/mL [Q1, Q3: 6.10, 14.88]). Multivariable logistic regression showed that significant risk factors of vitamin D deficiency were country of origin (women born in Switzerland and Germany had a lower risk than women born in other countries), smoking status (lower risk for former smokers) and intake of vitamin D supplements. CONCLUSIONS: Our results confirm a high prevalence of vitamin D deficiency in this Swiss cohort, in particular in women coming from Asian and African countries, and underline the importance of appropriate counseling and vitamin D supplementation in early pregnancy.

Obesity and risk of respiratory tract infections: results of an infection-diary based cohort study.

BACKGROUND: Respiratory tract infections (RTIs) are a major morbidity factor contributing largely to health care costs and individual quality of life. The aim of the study was to test whether obesity (BMI ≥ 30 kg/m2) is one of the risk factors underlying frequent RTIs in the German adult population. METHODS: We recruited 1455 individuals between 18 to 70 years from a cross-sectional survey on airway infections in Germany and invited them to self-report in diaries incident RTIs experienced during three consecutive winter/spring seasons. RTIs reported in these 18 months and summary measures adding-up individual RTIs were the outcomes of interest. RESULTS: Compared to individuals with normal weight, obese individuals reported a consistently higher frequency of upper and lower RTIs and predominantly fell in the upper 10% group of a diary sumscore adding-up 10 different RTI symptoms over time. Obesity was associated both with lower RTIs (adjustedOR = 2.02, 95%CI = 1.36-3.00) and upper RTIs (adjustedOR = 1.55, 95%CI = 1.22-1.96). Adjusting for demographic and lifestyle variables did only marginally affect ORs. Stratified analyses suggested a stronger association for women and effect modifications by sports activity and dietary habits. CONCLUSIONS: We confirm the association of obesity with infection burden and present evidence for putative interaction with sports activity and dietary patterns.

Mycobacteria exploit nitric oxide-induced transformation of macrophages into permissive giant cells.

Immunity to mycobacteria involves the formation of granulomas, characterized by a unique macrophage (MΦ) species, so-called multinucleated giant cells (MGC). It remains unresolved whether MGC are beneficial to the host, that is, by prevention of bacterial spread, or whether they promote mycobacterial persistence. Here, we show that the prototypical antimycobacterial molecule nitric oxide (NO), which is produced by MGC in excessive amounts, is a double-edged sword. Next to its antibacterial capacity, NO propagates the transformation of MΦ into MGC, which are relatively permissive for mycobacterial persistence. The mechanism underlying MGC formation involves NO-induced DNA damage and impairment of p53 function. Moreover, MGC have an unsurpassed potential to engulf mycobacteria-infected apoptotic cells, which adds a further burden to their antimycobacterial capacity. Accordingly, mycobacteria take paradoxical advantage of antimicrobial cellular efforts by driving effector MΦ into a permissive MGC state.

Reduced PRF1 enhancer methylation in children with a history of severe RSV bronchiolitis in infancy: an association study.

BACKGROUND: Acute lower respiratory tract infection is the commonest disease affecting children under five worldwide. Respiratory syncytial virus (RSV) is among the most common causative pathogens. Epidemiological data suggest an association between severe viral respiratory infections in infancy and increased incidence of childhood wheeze and asthma. DNA methylation is involved in immune cell differentiation and identity. It provides an avenue for environmental influences on the genome and therefore has potential as a marker for sustained effects of infectious insults. In this study we investigated the association between DNA methylation patterns in the perforin gene (PRF1) in childhood and a history of hospitalisation for severe RSV disease in the first two years of life. METHODS: In this retrospective study, we explored patterns of whole blood DNA methylation at a methylation sensitive region of the proximal PRF1 enhancer in a group of children with a record of hospitalisation for severe RSV disease during infancy (n = 43) compared to healthy controls matched for age and sex with no similar hospitalisation history, no allergy and no persistent wheeze (n = 43). Univariate and bivariate conditional logistic regression analyses were conducted to test the association between PRF1 enhancer methylation and record of hospitalisation for RSV disease. RESULTS: Children with a record of hospitalisation for severe RSV bronchiolitis demonstrated markedly lower levels of DNA methylation at two cytosine-phosphate-guanine dinucleotide (CpG) loci of the PRF1 proximal enhancer, corresponding to a signal transducer and activator of transcription 5 (STAT5) responsive element, compared to controls, adjusted odds ratios of 0.82 (95% confidence interval [CI] 0.71, 0.94) and 0.73 (95% CI 0.58, 0.92) for each 1% increase in DNA methylation. Smoking in the household showed a significant influence on DNA methylation at the assayed positions. CONCLUSIONS: Our findings support an association between childhood DNA methylation patterns in PRF1 and a record of severe RSV infection in infancy. Longitudinal studies are required to establish the utility of PRF1 methylation as a marker of severe RSV disease.

Smoking-associated DNA methylation markers predict lung cancer incidence.

BACKGROUND: Newly established blood DNA methylation markers that are strongly associated with smoking might open new avenues for lung cancer (LC) screening. We aimed to assess the performance of the top hits from previous epigenome-wide association studies in prediction of LC incidence. In a prospective nested case-control study, DNA methylation at AHRR (cg05575921), 6p21.33 (cg06126421), and F2RL3 (cg03636183) were measured by pyrosequencing in baseline whole blood samples of 143 incident LC cases identified during 11 years of follow-up and 457 age- and sex-matched controls without diagnosis of LC until the end of follow-up. The individual and joint associations of the 3 markers with LC risk were estimated by logistic regression, adjusted for potential confounders including smoking status and cigarette pack-years. The predictive performance was evaluated for both the individual markers and their combinations derived from multiple algorithms. RESULTS: Pronounced demethylation of all 3 markers was observed at baseline among cases compared to controls. Risk of developing LC increased with decreasing DNA methylation levels, with adjusted ORs (95% CI) of 15.86 (4.18-60.17), 8.12 (2.69-4.48), and 10.55 (3.44-32.31), respectively, for participants in the lowest quartile of AHRR, 6p21.33, and F2RL3 compared to participants in the highest 2 quartiles of each site among controls. The individual 3 markers exhibited similar accuracy in predicting LC incidence, with AUCs ranging from 0.79 to 0.81. Combination of the 3 markers did not improve the predictive performance (AUC = 0.80). The individual markers or their combination outperformed self-reported smoking exposure particularly in light smokers. No variation in risk prediction was identified with respect to age, follow-up time, and histological subtypes. CONCLUSIONS: AHRR, 6p21.33, and F2RL3 methylation in blood DNA are predictive for LC development, which might be useful for identification of risk groups for further specific screening, such as CT examination.

Disturbed B-lymphocyte selection in autoimmune lymphoproliferative syndrome.

Fas is a transmembrane receptor involved in the maintenance of tolerance and immune homeostasis. In murine models, it has been shown to be essential for deletion of autoreactive B cells in the germinal center. The role of Fas in human B-cell selection and in development of autoimmunity in patients carrying FAS mutations is unclear. We analyzed patients with either a somatic FAS mutation or a germline FAS mutation and somatic loss-of-heterozygosity, which allows comparing the fate of B cells with impaired vs normal Fas signaling within the same individual. Class-switched memory B cells showed: accumulation of FAS-mutated B cells; failure to enrich single V, D, J genes and single V-D, D-J gene combinations of the B-cell receptor variable region; increased frequency of variable regions with higher content of positively charged amino acids; and longer CDR3 and maintenance of polyreactive specificities. Importantly, Fas-deficient switched memory B cells showed increased rates of somatic hypermutation. Our data uncover a defect in B-cell selection in patients with FAS mutations, which has implications for the understanding of the pathogenesis of autoimmunity and lymphomagenesis of autoimmune lymphoproliferative syndrome.

Cord blood PRF1 methylation patterns and risk of lower respiratory tract infections in infants: findings from the Ulm Birth Cohort.

Lower respiratory tract infections (LRTIs) are a major cause of morbidity in children. DNA methylation provides a mechanism for transmitting environmental effects on the genome, but its potential role in LRTIs is not well studied. We investigated the methylation pattern of an enhancer region of the immune effector gene perforin-1 (PRF1), which encodes a cytolytic molecule of cytotoxic T lymphocytes (CTLs) and natural killer cells (NK), in cord blood DNA of children recruited in a German birth cohort in association with LRTIs in the first year of life.Pyrosequencing was used to determine the methylation levels of target cytosine-phosphate-guanines (CpGs) in a 2-stage case-control design. Cases were identified as children who developed ≥2 episodes of physician-recorded LRTIs during the first year of life and controls as children who had none. Discovery (n = 87) and replication (n = 90) sets were arranged in trios of 1 case and 2 controls matched for sex and season of birth.Logistic regression analysis revealed higher levels of methylation at a CpG that corresponds to a signal transducer and activator of transcription 5 (STAT5) responsive enhancer in the discovery (odds ratio [OR] per 1% methylation difference 1.24, 95% confidence interval [CI] 1.03-1.50) and replication (OR per 1% methylation difference 1.25, 95% CI 1.04-1.50) sets. Adjustment for having siblings <5 years old in the discovery and replication sets produced ORs of 1.19 (95% CI 0.98-1.45) and 1.25 (95% CI 1.04-1.50), respectively. Adjustment for gestational age in the replication set had no influence on the results. Methylation levels at adjacent CpGs varied with maternal age, smoking, education, and having siblings <5 years old.Our data support an association between cord blood PRF1 enhancer methylation patterns and subsequent risk of LRTIs in infants. Methylation levels at specific CpGs of the PRF1 enhancer varied according to maternal and family environmental factors suggesting a role for DNA methylation in mediating environmental influences on gene function.

Cord blood 25(OH)D levels and the subsequent risk of lower respiratory tract infections in early childhood: the Ulm birth cohort.

Lower respiratory tract infections (LRTIs) are a major cause of hospitalization in infants. Research suggests that immunomodulatory properties of vitamin D may influence LRTI risk. This study's objective was to examine whether 25-hydroxyvitamin D [25(OH)D] concentrations in cord blood influenced susceptibility to LRTI in the first year of life. Data was analyzed from a prospective birth cohort of 777 mother-infant pairs based in Ulm, Germany. Relative risks (RRs) of LRTI in relation to 25(OH)D cord blood levels were estimated by log-binomial regression after adjustment for potential confounders. To account for seasonal variation in both vitamin D levels and infections, we examined the association in different seasons. Analyses were conducted using clinical predefined cutpoints, quartiles, and season-standardized 25(OH)D quartiles. We observed a statistically significant association between 25(OH)D status in cord blood and risk of LRTI across the year using clinical cutpoints. The adjusted RR of LRTI for individuals with vitamin D deficiency (<25 nmol/L) in comparison to the referent category (>50 nmol/L) was 1.32 [95 % confidence interval (CI) 1.00, 1.73]. The association differed by maternal allergy status; children born to mothers without allergy demonstrated a RR of 1.45 (95 % CI 1.03, 2.03). The effect was largely driven by a strong association between 25(OH)D and LRTI in infants born in fall with a RR of 3.07 (95 % CI 1.37, 6.87). Our findings suggest that vitamin D deficiency at birth is associated with increased risk of LRTI particularly in infants born to mothers without allergy. The association seems strongest in infants born in fall.

Epstein Barr virus: a prime candidate of breast cancer aetiology in Sudanese patients.

Breast cancer is the commonest cancer in Sudanese women. Reported genetic alterations in the form of mutations in tumor suppressors are low in frequencies and could not explain the peculiarities of the diseases including its focal nature. Potential contributors disease aetiology include oncogenic viruses such as Epstein-Barr virus (EBV), an established culprit of nasopharyngeal carcinoma, one of the most frequent cancers in Sudan.In this study, DNA was extracted from malignant tissue samples and healthy tumour-free tissue from the same breast. Polymerase chain Reaction (PCR) was used to amplify two genes encoding for EBV viral proteins. The presence of Epstein-Barr virus and its cellular localization was confirmed by in situ hybridization (ISH) for Epstein-Barr encoded small RNAs (EBERs). Given the reported low frequency of mutations in BRCA1 and BRCA2 in Sudanese breast cancer patients, the methylation status of six tumor suppressor genes was investigated using methylation specific PCR. EBV genome was detected in 55.5% (n = 90) of breast cancer tissues as compared to 23% in control tissue samples (p = 0.0001). Using ISH, EBV signal was detected in all 18 breast cancer biopsies examined while all five normal breast tissue biopsies tested were negative for EBV. Of six tumour suppressor genes investigated BRCA1, BRCA2, and p14 appeared to be under strong epigenetic silencing.In conclusion, we present evidence of a strong association between EBV and breast carcinoma in Sudanese patients, and considerable epigenetic silencing of tumor suppressors that may likely be an outcome or an association with viral oncogenesis.