A silencing pathway to induce H3-K9 and H4-K20 trimethylation at constitutive heterochromatin

Histone lysine methylation is a central modification to mark functionally distinct chromatin regions. In particular, H3-K9 trimethylation has emerged as a hallmark of pericentric heterochromatin in mammals. Here we show that H4-K20 trimethylation is also focally enriched at pericentric heterochromatin. Intriguingly, H3-K9 trimethylation by the Suv39h HMTases is required for the induction of H4-K20 trimethylation, although the H4 Lys 20 position is not an intrinsic substrate for these enzymes. By using a candidate approach, we identified Suv4-20h1 and Suv4-20h2 as two novel SET domain HMTases that localize to pericentric heterochromatin and specifically act as nucleosomal H4-K20 trimethylating enzymes. Interaction of the Suv4-20h enzymes with HP1 isoforms suggests a sequential mechanism to establish H3-K9 and H4-K20 trimethylation at pericentric heterochromatin. Heterochromatic H4-K20 trimethylation is evolutionarily conserved, and in Drosophila, the Suv4-20 homolog is a novel PEV modifier to regulate position-effect variegation. Together, our data indicate a function for H4-K20 trimethylation in gene silencing and further suggest H3-K9 and H4-K20 trimethylation as important components of a repressive pathway that can index pericentric heterochromatin.     

A 10-Mb paracentric inversion of chromosome arm 2p inactivates MSH2 and is responsible for hereditary nonpolyposis colorectal cancer in a North-American kindred

Genomic deletions of the MSH2 gene are a frequent cause of hereditary nonpolyposis colorectal cancer (HNPCC), a common hereditary predisposition to the development of tumors in several organs including the gastrointestinal and urinary tracts and endometrium. The mutation spectrum at the MSH2 gene is extremely heterogeneous because it includes nonsense and missense point mutations, small insertions and deletions leading to frameshifts, and larger genomic deletions, the latter representing approximately 25% of the total mutation burden. Here, we report the identification and molecular characterization of the first paracentric inversion of the MSH2 locus known to cause HNPCC. Southern blot analysis and inverse PCR showed that the centromeric and telomeric breakpoints of the paracentric inversion map within intron 7 and to a contig 10 Mb 3' of MSH2, respectively. Pathogenicity of the paracentric inversion was demonstrated by conversion analysis. The patient's lymphocytes were employed to generate somatic cell hybrids to analyze the expression of the inverted MSH2 allele in an Msh2-deficient rodent cellular background. The inversion was shown to abolish MSH2 expression by both northern and western analysis. This study confirms that Southern blot analysis still represents a useful and informative tool to screen for and identify complex genomic rearrangements in HNPCC. Moreover, monoallelic expression analysis represents an attractive approach to demonstrate pathogenicity of unusual mutations in autosomal dominant hereditary conditions.     

Bi-allelic loss of function variants in SLC30A5 as cause of perinatal lethal cardiomyopathy

Perinatal mortality is a heavy burden for both affected parents and physicians. However, the underlying genetic causes have not been sufficiently investigated and most cases remain without diagnosis. This impedes appropriate counseling or therapy. We describe four affected children of two unrelated families with cardiomyopathy, hydrops fetalis, or cystic hygroma that all deceased perinatally. In the four patients, we found the following homozygous loss of function (LoF) variants in SLC30A5 NM_022902.4:c.832_836del p.(Ile278Phefs*33) and NM_022902.4:c.1981_1982del p.(His661Tyrfs*10). Knockout of SLC30A5 has previously been shown a cardiac phenotype in mouse models and no homozygous LoF variants in SLC30A5 are currently described in gnomAD. Taken together, we present SLC30A5 as a new gene for a severe and perinatally lethal form of cardiomyopathy.Subject terms: Cardiovascular diseases, Development, Medical genetics, Medical genomics     

Epidemiological and clinical features of Croatian children and adolescents with a PCR-confirmed coronavirus disease 2019: differences between the first and second epidemic wave

MethodsData on patients aged ≤19 years with a positive SARS-CoV-2 PCR test recorded in the period March 12-May 12 (first wave) and June 19-July 19, 2020 (second wave) were retrospectively analyzed. The periods were separated by several weeks with no incident cases.ResultsWe analyzed data on 289 children and adolescents (6.5% of all cases; incidence rate [IR] = 3.54, 95% confidence interval [CI] 3.14-3.97/million person-days), 124 in the first wave (IR = 2.27) and 165 in the second wave (IR = 6.37): IRR second/first = 2.71 (2.13-3.44). During the first wave, the incidence was highest in infants (IR = 3.48), while during the second wave it progressively increased to IR = 7.37 in 15-19-year olds. Family members were the key epidemiological contacts (72.6% cases), particularly during the first wave (95.8% vs 56.3%). Overall, 41.3% patients were asymptomatic, 25.3% in the first and 52.6% in the second wave. Age 15-19 years (vs younger) was associated with a higher (RR = 1.26, 1.02-1.54) and infection in the second wave with a lower probability (RR = 0.66, 0.53-0.81) of being symptomatic. The most common symptoms were fever, cough, and rhinorrhea. In children aged ≥7 years, headache, anosmia/ageusia, and sore throat were also recorded. Only one child suffered a severe disease. All but 18 (7.8%) children were treated only symptomatically, and all fully recovered.ConclusionA large proportion of SARS-CoV-2 PCR-positive children/adolescents were asymptomatic. The associated disease was predominantly mild, comparably so in the first and second pandemic wave.

Since the late December 2019, coronavirus disease 2019 (COVID-19) caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread quickly worldwide and as of early December accounts for more than 65 million cases diagnosed in more than 200 countries (1). At this point, the most affected countries in Europe are Russia, Spain, France, United Kingdom (UK), and Italy with consequently the highest mortality rates. The first case in Croatia was reported in the late February 2020, and within the next two months the infection expanded nationwide. During this first epidemic wave, Croatia was under a one-month lockdown, which rapidly decreased the disease incidence, and only a few newly diagnosed cases were reported between May 25 and June18, 2020. Easing of restrictions increased the incidence in late June, causing a second wave of COVID-19 in Croatia, with >147 000 cases reported so far (1,2).Over the last two decades, there were two other coronavirus outbreaks. Severe acute respiratory syndrome coronavirus appeared in 2002, affecting around 8000 people, with 10% mortality. Children (4 months-17 years) accounted for <0.02% of total cases, and there was no reported death in this age group. During the outbreak of the Middle East respiratory syndrome coronavirus, around 2300 people were infected, and children (<19 years of age) were rarely affected as well (2% of total cases; 2 reported deaths) (3,4). COVID-19 has exhibited a similar epidemiological pattern. Although early reports from China, Italy, and the United States (US) suggested that children and adolescents accounted for only 1%-2% of the overall COVID-19 cases (5-7), later reports around the world indicated a higher proportions of pediatric cases, between 1%-8% (8-10). Children of all ages can be affected by SARS-CoV-2 infection, but in contrast to other respiratory viruses, they usually suffer a mild or asymptomatic infection. Compared with adults, severe infections and fatal outcomes in children are rare, and several immunopathological mechanisms could be responsible for such differences in disease severity (11). Although many studies have reviewed the features of adults with COVID-19, overall data regarding pediatric cases are scarce, and most of them are reports from China and the US, with only a few studies describing disease in children from European countries.We aimed to describe epidemiological and clinical features of children and adolescents with COVID-19 confirmed by the polymerase chain reaction (PCR) test for SARS-CoV-2 in Croatia and to assess potential differences between the first (March-May 2020) and second (on-going) pandemic wave (June-July 2020).     

Induction of intervertebral disc-like cells from adult mesenchymal stem cells

The potential of adult mesenchymal stem cells (MSCs) to differentiate towards cartilage, bone, adipose tissue, or muscle is well established. However, the capacity of MSCs to differentiate towards intervertebral disc (IVD)-like cells is unknown. The aim of this study was to compare the molecular phenotype of human IVD cells and articular chondrocytes and to analyze whether mesenchymal stem cells can differentiate towards both cell types after transforming growth factor beta (TGF beta)-mediated induction in vitro. Bone marrow-derived MSCs were differentiated in spheroid culture towards the chondrogenic lineage in the presence of TGF beta(3) dexamethasone, and ascorbate. A customized cDNA-array comprising 45 cartilage-, bone-, and stem cell-relevant genes was used to quantify gene expression profiles. After TGF beta-mediated differentiation, MSC spheroids turned positive for collagen type II protein and expressed a large panel of genes characteristic for chondrocytes, including aggrecan, decorin, fibromodulin, and cartilage oligomeric matrix protein, although at levels closer to IVD tissue than to hyaline articular cartilage. Like IVD tissue, the spheroids were strongly positive for collagen type I and osteopontin. MSC spheroids expressed more differentiation markers at higher levels than culture-expanded IVD cells and chondrocytes, which both dedifferentiated in monolayer culture. In conclusion, mesenchymal stem cells adopted a gene expression profile that resembled native IVD tissue more closely than native joint cartilage. Thus, these cells may represent an attractive source from which to obtain IVD-like cells, whereas modification of culture conditions is required to approach the molecular phenotype of chondrocytes in hyaline cartilage.     

Presence and mechanism of macrolide-lincosamide resistance in Enterococcus columbae strains belonging to the intestinal flora of pigeons.

Faecal samples from 50 pigeons all originating from different lofts were screened for the presence of macrolide and lincosamide (ML)-resistant isolates of Streptococcus gallolyticus and Enterococcus columbae by plating the samples onto selective media. Sixty-eight ML-resistant E. columbae strains were recovered from the faecal samples of 29 animals. Two of these samples also harboured ML-resistant S. gallolyticus strains. The erm(B) gene was detected in 58 E. columbae and in five S. gallolyticus isolates. Four of these E. columbae isolates also carried the mef(A) gene. Five E. columbae strains possessed the mef(A) gene in the absence of erm(B). On the basis of the sequence of the complete erm(B) gene, 10 E. columbae isolates clustered together in six groups. In two of these isolates, the erm(B) gene sequence was identical to that of S. gallolyticus strains, indicating that exchange of resistance genes might occur between pathogenic and non-pathogenic bacterial species belonging to the pigeon's intestinal flora.     

Neonatal outcome in a Danish national cohort of 3438 IVF/ICSI and 10,362 non-IVF/ICSI twins born between 1995 and 2000

BACKGROUND: In Denmark, one-third of twin pregnancies are the result of IVF/ICSI treatment. Limited data on neonatal outcome in IVF/ICSI twins are available in the literature. METHODS: A register study was conducted on neonatal morbidity and mortality in a complete national twin cohort including all 3438 (3393 live-born) IVF/ICSI and 10,362 (10,239 live-born) non-IVF/ICSI twins born between 1995 and 2000. Twins were identified in the National Medical Birth Registry and dichotomized into IVF/ICSI and non-IVF/ICSI by cross-reference with the Danish IVF Registry. Data on neonatal morbidity and mortality were retrieved from the Danish Patient Registry and the Danish Registry of Causes of Deaths. In order to exclude monozygotic twins, sub-analyses on unlike-sex twins were conducted. RESULTS: A birth weight discordance of >20% was observed in 20.6% of IVF/ICSI versus 15.7% of control twin pairs (P < 0.001). The risk of discordant birth weight >20% was OR 1.29 (95% CI 1.04-1.58) in unlike-sex IVF/ICSI twins versus control twins. The risk of delivery at <37 completed weeks and birth weight <2500 g was similar in the two cohorts; however, in unlike-sex IVF/ICSI versus control twins the risk of delivery at <37 weeks and birth weight <2500 g was OR 1.22 (95% CI 1.09-1.38) and OR 1.25 (1.11-1.40) respectively. After stratification for maternal age and parity, these risks disappeared. IVF/ICSI twins carried a higher risk of admittance to a neonatal intensive care unit (NICU) than control twins (OR 1.18, 95% CI 1.09-1.27), and this was even more pronounced in unlike-sex twins [OR 1.34 (95% CI 1.19-1.51)]. No differences were observed in malformation or mortality rates between the two cohorts. CONCLUSIONS: Despite higher birth weight discordance and more NICU admissions among IVF/ICSI twins, neonatal outcome in IVF/ICSI twins seems to be comparable with that of non-IVF/ICSI twins, when only dizygotic twins were considered in the comparisons.