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Abstract Background We describe an outbreak of Bullous Impetigo (BI), caused by a (methicillin susceptible, fusidic acid resistant) Staphylococcus aureus (SA) strain, spa-type t408, at the neonatal and gynaecology ward of the Jeroen Bosch hospital in the Netherlands, from March-November 2011. Methods We performed an outbreak investigation with revision of the hygienic protocols, MSSA colonization surveillance and environmental sampling for MSSA including detailed typing of SA isolates. Spa typing was performed to discriminate between the SA isolates. In addition, Raman-typing was performed on all t408 isolates. Results Nineteen cases of BI were confirmed by SA positive cultures. A cluster of nine neonates and three health care workers (HCW) with SA t408 was detected. These strains were MecA-, PVL-, Exfoliative Toxin (ET)A-, ETB+, ETAD-, fusidic acid-resistant and methicillin susceptible. Eight out of nine neonates and two out of three HCW t408 strains yielded a similar Raman type. Positive t408 HCW were treated and infection control procedures were reinforced. These measures stopped the outbreak. Conclusions We conclude that treatment of patients and HCW carrying a predominant SA t408, and re-implementing and emphasising hygienic measures were effective to control the outbreak of SA t408 among neonates.

Abstract Background In The Netherlands the largest human Q fever outbreak ever reported in the literature is currently ongoing with more than 2300 notified cases in 2009. Pregnant women are particularly at risk as Q fever during pregnancy may cause maternal and obstetric complications. Since the majority of infected pregnant women are asymptomatic, a screening strategy might be of great value to reduce Q fever related complications. We designed a trial to assess the (cost-)effectiveness of a screening program for Q fever in pregnant women living in risks areas in The Netherlands. Methods/design We will conduct a clustered randomized controlled trial in which primary care midwife centres in Q fever risk areas are randomized to recruit pregnant women for either the control group or the intervention group. In both groups a blood sample is taken around 20 weeks postmenstrual age. In the intervention group, this sample is immediately analyzed by indirect immunofluorescence assay for detection of IgG and IgM antibodies using a sensitive cut-off level of 1:32. In case of an active Q fever infection, antibiotic treatment is recommended and serological follow up is performed. In the control group, serum is frozen for analysis after delivery. The primary endpoint is a maternal (chronic Q fever or reactivation) or obstetric complication (low birth weight, preterm delivery or fetal death) in Q fever positive women. Secondary aims pertain to the course of infection in pregnant women, diagnostic accuracy of laboratory tests used for screening, histo-pathological abnormalities of the placenta of Q fever positive women, side effects of therapy, and costs. The analysis will be according to the intention-to-screen principle, and cost-effectiveness analysis will be performed by comparing the direct and indirect costs between the intervention and control group. Discussion With this study we aim to provide insight into the balance of risks of undetected and detected Q fever during pregnancy. Trial registration ClinicalTrials.gov, protocol record NL30340.042.09.

Abstract Background A Q-fever outbreak occurred in an urban area in the south of the Netherlands in May 2008. The distribution and timing of cases suggested a common source. We studied the spatial relationship between the residence locations of human cases and nearby small ruminant farms, of which one dairy goat farm had experienced abortions due to Q-fever since mid April 2008. A generic geographic information system (GIS) was used to develop a method for source detection in the still evolving major epidemic of Q-fever in the Netherlands. Methods All notified Q-fever cases in the area were interviewed. Postal codes of cases and of small ruminant farms (size >40 animals) located within 5 kilometres of the cluster area were geo-referenced as point locations in a GIS-model. For each farm, attack rates and relative risks were calculated for 5 concentric zones adding 1 kilometre at a time, using the 5-10 kilometres zone as reference. These data were linked to the results of veterinary investigations. Results Persons living within 2 kilometres of an affected dairy goat farm (>400 animals) had a much higher risk for Q-fever than those living more than 5 kilometres away (Relative risk 31.1 [95% CI 16.4-59.1]). Conclusions The study supported the hypothesis that a single dairy goat farm was the source of the human outbreak. GIS-based attack rate analysis is a promising tool for source detection in outbreaks of human Q-fever.

The major spliceosome mediates pre-mRNA splicing by recognizing the highly conserved sequences at the 5' and 3' splice sites and the branch point. More than 150 proteins participate in the splicing process and are organized in the spliceosomal A, B, and C complexes. FRA10AC1 is a peripheral protein of the spliceosomal C complex and its ortholog in the green alga facilitates recognition or interaction with splice sites. We identified biallelic pathogenic variants in FRA10AC1 in five individuals from three consanguineous families. The two unrelated Patients 1 and 2 with loss-of-function variants showed developmental delay, intellectual disability, and no speech, while three siblings with the c.494_496delAAG (p.Glu165del) variant had borderline to mild intellectual disability. All patients had microcephaly, hypoplasia or agenesis of the corpus callosum, growth retardation, and craniofacial dysmorphism. FRA10AC1 transcripts and proteins were drastically reduced or absent in fibroblasts of Patients 1 and 2. In a heterologous expression system, the p.Glu165del variant impacts intrinsic stability of FRA10AC1 but does not affect its nuclear localization. By co-immunoprecipitation, we found ectopically expressed HA-FRA10AC1 in complex with endogenous DGCR14, another component of the spliceosomal C complex, while the splice factors CHERP, NKAP, RED, and SF3B2 could not be co-immunoprecipitated. Using an in vitro splicing reporter assay, we did not obtain evidence for FRA10AC1 deficiency to suppress missplicing events caused by mutations in the highly conserved dinucleotides of 5' and 3' splice sites in an in vitro splicing assay in patient-derived fibroblasts. Our data highlight the importance of specific peripheral spliceosomal C complex proteins for neurodevelopment. It remains possible that FRA10AC1 may have other and/or additional cellular functions, such as coupling of transcription and splicing reactions., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The sodium (Na + ):multivitamin transporter (SMVT), encoded by SLC5A6, belongs to the sodium:solute symporter family and is required for the Na + -dependent uptake of biotin (vitamin B7), pantothenic acid (vitamin B5), the vitamin-like substance α-lipoic acid, and iodide. Compound heterozygous SLC5A6 variants have been reported in individuals with variable multisystemic disorder, including failure to thrive, developmental delay, seizures, cerebral palsy, brain atrophy, gastrointestinal problems, immunodeficiency, and/or osteopenia. We expand the phenotypic spectrum associated with biallelic SLC5A6 variants affecting function by reporting five individuals from three families with motor neuropathies. We identified the homozygous variant c.1285 A > G [p.(Ser429Gly)] in three affected siblings and a simplex patient and the maternally inherited c.280 C > T [p.(Arg94*)] variant and the paternally inherited c.485 A > G [p.(Tyr162Cys)] variant in the simplex patient of the third family. Both missense variants were predicted to affect function by in silico tools. 3D homology modeling of the human SMVT revealed 13 transmembrane helices (TMs) and Tyr162 and Ser429 to be located at the cytoplasmic facing region of TM4 and within TM11, respectively. The SLC5A6 missense variants p.(Tyr162Cys) and p.(Ser429Gly) did not affect plasma membrane localization of the ectopically expressed multivitamin transporter suggesting reduced but not abolished function, such as lower catalytic activity. Targeted therapeutic intervention yielded clinical improvement in four of the five patients. Early molecular diagnosis by exome sequencing is essential for timely replacement therapy in affected individuals., (© 2022. The Author(s).)

Golgi-associated retrograde protein (GARP) and endosome-associated recycling protein (EARP) complexes are membrane-tethering heterotetramers located at the trans-Golgi network and recycling endosomes, respectively. GARP and EARP share the three subunits VPS51, VPS52 and VPS53, while VPS50 is unique to EARP and VPS54 to GARP. Retrograde transport of endosomal cargos to the trans-Golgi network is mediated by GARP and endocytic recycling by EARP. Here we report two unrelated individuals with homozygous variants in VPS50, a splice variant (c.1978-1G>T) and an in-frame deletion (p.Thr608del). Both patients had severe developmental delay, postnatal microcephaly, corpus callosum hypoplasia, seizures and irritability, transient neonatal cholestasis and failure to thrive. Light and transmission electron microscopy of liver from one revealed the absence of gamma-glutamyltransferase at bile canaliculi, with mislocalization to basolateral membranes and abnormal tight junctions. Using patient-derived fibroblasts, we identified reduced VPS50 protein accompanied by reduced levels of VPS52 and VPS53. While the transferrin receptor internalization rate was normal in cells of both patients, recycling of the receptor to the plasma membrane was significantly delayed. These data underscore the importance of VPS50 and/or the EARP complex in endocytic recycling and suggest an additional function in establishing cell polarity and trafficking between basolateral and apical membranes in hepatocytes. Individuals with biallelic hypomorphic variants in VPS50, VPS51 or VPS53 show an overarching neurodegenerative disorder with severe developmental delay, intellectual disability, microcephaly, early-onset epilepsy and variable atrophy of the cerebellum, cerebrum and/or brainstem. The term 'GARP/EARP deficiency' designates disorders in such individuals., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

T-type calcium channels (Cav3.1 to Cav3.3) regulate low-threshold calcium spikes, burst firing and rhythmic oscillations of neurons and are involved in sensory processing, sleep, and hormone and neurotransmitter release. Here, we examined four heterozygous missense variants in CACNA1I, encoding the Cav3.3 channel, in patients with variable neurodevelopmental phenotypes. The p.(Ile860Met) variant, affecting a residue in the putative channel gate at the cytoplasmic end of the IIS6 segment, was identified in three family members with variable cognitive impairment. The de novo p.(Ile860Asn) variant, changing the same amino acid residue, was detected in a patient with severe developmental delay and seizures. In two additional individuals with global developmental delay, hypotonia, and epilepsy, the variants p.(Ile1306Thr) and p.(Met1425Ile), substituting residues at the cytoplasmic ends of IIIS5 and IIIS6, respectively, were found. Because structure modelling indicated that the amino acid substitutions differentially affect the mobility of the channel gate, we analysed possible effects on Cav3.3 channel function using patch-clamp analysis in HEK293T cells. The mutations resulted in slowed kinetics of current activation, inactivation, and deactivation, and in hyperpolarizing shifts of the voltage-dependence of activation and inactivation, with Cav3.3-I860N showing the strongest and Cav3.3-I860M the weakest effect. Structure modelling suggests that by introducing stabilizing hydrogen bonds the mutations slow the kinetics of the channel gate and cause the gain-of-function effect in Cav3.3 channels. The gating defects left-shifted and increased the window currents, resulting in increased calcium influx during repetitive action potentials and even at resting membrane potentials. Thus, calcium toxicity in neurons expressing the Cav3.3 variants is one likely cause of the neurodevelopmental phenotype. Computer modelling of thalamic reticular nuclei neurons indicated that the altered gating properties of the Cav3.3 disease variants lower the threshold and increase the duration and frequency of action potential firing. Expressing the Cav3.3-I860N/M mutants in mouse chromaffin cells shifted the mode of firing from low-threshold spikes and rebound burst firing with wild-type Cav3.3 to slow oscillations with Cav3.3-I860N and an intermediate firing mode with Cav3.3-I860M, respectively. Such neuronal hyper-excitability could explain seizures in the patient with the p.(Ile860Asn) mutation. Thus, our study implicates CACNA1I gain-of-function mutations in neurodevelopmental disorders, with a phenotypic spectrum ranging from borderline intellectual functioning to a severe neurodevelopmental disorder with epilepsy., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain.)

Marfan syndrome and related disorders are a group of heritable connective tissue disorders and share many clinical features that involve cardiovascular, skeletal, craniofacial, ocular, and cutaneous abnormalities. The majority of affected individuals have aortopathies associated with early mortality and morbidity. Implementation of targeted gene panel next-generation sequencing in these individuals is a powerful tool to obtain a genetic diagnosis. Here, we report on clinical and genetic spectrum of 53 families from India with a total of 83 patients who had a clinical diagnosis suggestive of Marfan syndrome or related disorders. We obtained a molecular diagnosis in 45/53 (85%) index patients, in which 36/53 (68%) had rare variants in FBN1 (Marfan syndrome; 63 patients in total), seven (13.3%) in TGFBR1/TGFBR2 (Loeys-Dietz syndrome; nine patients in total) and two patients (3.7%) in SKI (Shprintzen-Goldberg syndrome). 21 of 41 rare variants (51.2%) were novel. We did not detect a disease-associated variant in 8 (15%) index patients, and none of them met the Ghent Marfan diagnostic criteria. We found the homozygous FBN1 variant p.(Arg954His) in a boy with typical features of Marfan syndrome. Our study is the first reporting on the spectrum of variants in FBN1, TGFBR1, TGFBR2, and SKI in Indian individuals.

Heparan sulfate belongs to the group of glycosaminoglycans (GAGs), highly sulfated linear polysaccharides. Heparan sulfate 2-O-sulfotransferase 1 (HS2ST1) is one of several specialized enzymes required for heparan sulfate synthesis and catalyzes the transfer of the sulfate groups to the sugar moiety of heparan sulfate. We report bi-allelic pathogenic variants in HS2ST1 in four individuals from three unrelated families. Affected individuals showed facial dysmorphism with coarse face, upslanted palpebral fissures, broad nasal tip, and wide mouth, developmental delay and/or intellectual disability, corpus callosum agenesis or hypoplasia, flexion contractures, brachydactyly of hands and feet with broad fingertips and toes, and uni- or bilateral renal agenesis in three individuals. HS2ST1 variants cause a reduction in HS2ST1 mRNA and decreased or absent heparan sulfate 2-O-sulfotransferase 1 in two of three fibroblast cell lines derived from affected individuals. The heparan sulfate synthesized by the individual 1 cell line lacks 2-O-sulfated domains but had an increase in N- and 6-O-sulfated domains demonstrating functional impairment of the HS2ST1. As heparan sulfate modulates FGF-mediated signaling, we found a significantly decreased activation of the MAP kinases ERK1/2 in FGF-2-stimulated cell lines of affected individuals that could be restored by addition of heparin, a GAG similar to heparan sulfate. Focal adhesions in FGF-2-stimulated fibroblasts of affected individuals concentrated at the cell periphery. Our data demonstrate that a heparan sulfate synthesis deficit causes a recognizable syndrome and emphasize a role for 2-O-sulfated heparan sulfate in human neuronal, skeletal, and renal development., (Copyright © 2020 American Society of Human Genetics. All rights reserved.)

In pleiotropic diseases, multiple organ systems are affected causing a variety of clinical manifestations. Here, we report a pleiotropic disorder with a unique constellation of neurological, endocrine, exocrine, and haematological findings that is caused by biallelic MADD variants. MADD, the mitogen-activated protein kinase (MAPK) activating death domain protein, regulates various cellular functions, such as vesicle trafficking, activity of the Rab3 and Rab27 small GTPases, tumour necrosis factor-α (TNF-α)-induced signalling and prevention of cell death. Through national collaboration and GeneMatcher, we collected 23 patients with 21 different pathogenic MADD variants identified by next-generation sequencing. We clinically evaluated the series of patients and categorized the phenotypes in two groups. Group 1 consists of 14 patients with severe developmental delay, endo- and exocrine dysfunction, impairment of the sensory and autonomic nervous system, and haematological anomalies. The clinical course during the first years of life can be potentially fatal. The nine patients in Group 2 have a predominant neurological phenotype comprising mild-to-severe developmental delay, hypotonia, speech impairment, and seizures. Analysis of mRNA revealed multiple aberrant MADD transcripts in two patient-derived fibroblast cell lines. Relative quantification of MADD mRNA and protein in fibroblasts of five affected individuals showed a drastic reduction or loss of MADD. We conducted functional tests to determine the impact of the variants on different pathways. Treatment of patient-derived fibroblasts with TNF-α resulted in reduced phosphorylation of the extracellular signal-regulated kinases 1 and 2, enhanced activation of the pro-apoptotic enzymes caspase-3 and -7 and increased apoptosis compared to control cells. We analysed internalization of epidermal growth factor in patient cells and identified a defect in endocytosis of epidermal growth factor. We conclude that MADD deficiency underlies multiple cellular defects that can be attributed to alterations of TNF-α-dependent signalling pathways and defects in vesicular trafficking. Our data highlight the multifaceted role of MADD as a signalling molecule in different organs and reveal its physiological role in regulating the function of the sensory and autonomic nervous system and endo- and exocrine glands., (© The Author(s) (2020). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

In eukaryotes, the elongation phase of transcription by RNA polymerase II (RNAP II) is regulated by the transcription elongation factor b (P-TEFb), composed of Cyclin-T1 and cyclin-dependent kinase 9. The release of RNAP II is mediated by phosphorylation through P-TEFb that in turn is under control by the inhibitory 7SK small nuclear ribonucleoprotein (snRNP) complex. The 7SK snRNP consists of the 7SK non-coding RNA and the proteins MEPCE, LARP7, and HEXIM1/2. Biallelic LARP7 loss-of-function variants underlie Alazami syndrome characterized by growth retardation and intellectual disability. We report a boy with global developmental delay and seizures carrying the de novo MEPCE nonsense variant c.1552 C > T/p.(Arg518*). mRNA and protein analyses identified nonsense-mediated mRNA decay to underlie the decreased amount of MEPCE in patient fibroblasts followed by LARP7 and 7SK snRNA downregulation and HEXIM1 upregulation. Reduced binding of HEXIM1 to Cyclin-T1, hyperphosphorylation of the RNAP II C-terminal domain, and upregulated expression of ID2, ID3, MRPL11 and snRNAs U1, U2 and U4 in patient cells are suggestive of enhanced activation of P-TEFb. Flavopiridol treatment and ectopic MEPCE protein expression in patient fibroblasts rescued increased expression of six RNAP II-sensitive genes and suggested a possible repressive effect of MEPCE on P-TEFb-dependent transcription of specific genes.

Zimmermann-Laband syndrome (ZLS) is characterized by coarse facial features with gingival enlargement, intellectual disability (ID), hypertrichosis, and hypoplasia or aplasia of nails and terminal phalanges. De novo missense mutations in KCNH1 and KCNK4, encoding K + channels, have been identified in subjects with ZLS and ZLS-like phenotype, respectively. We report de novo missense variants in KCNN3 in three individuals with typical clinical features of ZLS. KCNN3 (SK3/KCa2.3) constitutes one of three members of the small-conductance Ca 2+ -activated K + (SK) channels that are part of a multiprotein complex consisting of the pore-forming channel subunits, the constitutively bound Ca 2+ sensor calmodulin, protein kinase CK2, and protein phosphatase 2A. CK2 modulates Ca 2+ sensitivity of the channels by phosphorylating SK-bound calmodulin. Patch-clamp whole-cell recordings of KCNN3 channel-expressing CHO cells demonstrated that disease-associated mutations result in gain of function of the mutant channels, characterized by increased Ca 2+ sensitivity leading to faster and more complete activation of KCNN3 mutant channels. Pretreatment of cells with the CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole revealed basal inhibition of wild-type and mutant KCNN3 channels by CK2. Analogous experiments with the KCNN3 p.Val450Leu mutant previously identified in a family with portal hypertension indicated basal constitutive channel activity and thus a different gain-of-function mechanism compared to the ZLS-associated mutant channels. With the report on de novo KCNK4 mutations in subjects with facial dysmorphism, hypertrichosis, epilepsy, ID, and gingival overgrowth, we propose to combine the phenotypes caused by mutations in KCNH1, KCNK4, and KCNN3 in a group of neurological potassium channelopathies caused by an increase in K + conductance., (Copyright © 2019 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)