Typhoid Fever and Non-typhoidal Salmonella Outbreaks: A Portrait of Regional Socioeconomic Inequalities in Brazil.

Typhoid fever occurs in an endemic form in Brazil and is a serious public health problem in some regions. In this scenario, further research is urgently needed to identify the associations between socioeconomic factors and typhoid fever, contributing to guiding policy decisions in the country. We aimed to investigate the influence of socioeconomic disparities on the prevalence of typhoid fever and non-typhoidal Salmonella (NTS) in Brazil. A search for data from 2010 to 2019 was carried out with the national health and human development agencies. As milk and derivatives are the fourth food incriminated in food outbreaks in Brazil, analyses for detecting Salmonella spp. in commercial dairy products allowed us to assess whether the outbreaks associated with these foods are due to inadequacies in sanitary control in dairy establishments or whether they are mainly home-based outbreaks. Predictive models validated by the bootstrapping method demonstrate an association of NTS prevalence reduction with improvements in the Sanitation Service Index (Rv ≥ -8 0.686; p ≤ 0.01) and Municipal Human Development Index - MHDI - (Rv = -0.789; p ≤ 0.02). In the North, typhoid fever prevalence had seasonal variability with the rainfall, while sanitation services (Rv ≥-0.684; p ≤ 0.04) and MHDI (Rv ≥-0.949; p ≤ 0.003) directly influenced Northeast and South Brazil. Thus, the unequal distribution of investments in the sanitation sector contributed to disparities in typhoid fever prevalence among Brazilian regions. The absence of Salmonella spp. in commercial samples ratified the collected data that the outbreaks of Salmonella spp. in the Brazilian population occur mainly at residences. These findings show that implementing public health education and increasing investments in sanitation in regions with poor service can control outbreaks of Salmonella spp. in Brazilian endemic areas.

A new case of platelet-type von Willebrand disease supports the recent findings of gain-of-function GP1BA variants outside the C-terminal disulphide loop enhances affinity for von Willebrand factor.

Platelet-type von Willebrand disease (PT-VWD) is a rare autosomal dominant bleeding disorder characterized by an increased ristocetin-induced platelet aggregation (RIPA) and enhanced affinity of platelet glycoprotein Ibα (GPIbα) to von Willebrand factor (VWF). To date, only seven variants have been described with this gain-of-function effect, most of them located in the C-terminal disulphide loop of the VWF-binding domain of GPIbα. We herein describe a patient with moderate bleeding symptoms, mild thrombocytopenia and increased RIPA. By direct sequencing of GP1BA, a novel leucine-rich repeat heterozygous variant was identified (c.580C>T; predictably p.Leu194Phe), strongly suggestive as being the underlying cause for the PT-VWD phenotype of our patient.

Missense MED12 variants in 22 males with intellectual disability: From nonspecific symptoms to complete syndromes.

We describe the phenotype of 22 male patients (20 probands) carrying a hemizygous missense variant in MED12. The phenotypic spectrum is very broad ranging from nonspecific intellectual disability (ID) to the three well-known syndromes: Opitz-Kaveggia syndrome, Lujan-Fryns syndrome, or Ohdo syndrome. The identified variants were randomly distributed throughout the gene (p = 0.993, χ2 test), but mostly outside the functional domains (p = 0.004; χ2 test). Statistical analyses did not show a correlation between the MED12-related phenotypes and the locations of the variants (p = 0.295; Pearson correlation), nor the protein domain involved (p = 0.422; Pearson correlation). In conclusion, establishing a genotype-phenotype correlation in MED12-related diseases remains challenging. Therefore, we think that patients with a causative MED12 variant are currently underdiagnosed due to the broad patients' clinical presentations.

Thrombocytopenia-Absent Radius Syndrome: Descriptions of Three New Cases and a Novel Splicing Variant in RBM8A That Expands the Spectrum of Null Alleles.

Thrombocytopenia-absent radius (TAR) syndrome is a rare congenital disorder characterized by the bilateral absence of the radius and thrombocytopenia, and sometimes by other skeletal, gastrointestinal, cardiac, and renal abnormalities. The underlying genetic defect is usually the compound inheritance of a microdeletion in 1q21.1 (null allele) and a low-frequency, non-coding single nucleotide variant (SNV) in the RBM8A gene (hypomorphic allele). We report three new cases from two unrelated families. The two siblings presented the common genotype, namely the compound heterozygosity for a 1q21.1 microdeletion and the hypomorphic SNV c.-21G>A in RBM8A, whereas the third, unrelated patient presented a rare genotype comprised by two RBM8A variants: c.-21G>A (hypomorphic allele) and a novel pathogenic variant, c.343-2A>G (null allele). Of the eight documented RBM8A variants identified in TAR syndrome patients, four have hypomorphic expression and four behave as null alleles. The present report expands the RBM8A null allele spectrum and corroborates the particularities of RBM8A involvement in TAR syndrome pathogenesis.

Subclinical bovine mastitis associated with Staphylococcus spp. in eleven Uruguayan dairy farms.

INTRODUCTION: Bovine mastitis is the most common disease affecting the dairy industry, with staphylococci being considered as one of the most significant and prevalent causes. This study aimed to assess the presence of staphylococcal subclinical mastitis (SCM) in Uruguayan dairy farms and to identify Staphylococcus aureus (SA) and non-aureus staphylococci (NAS) in milking cows. In addition, the antibiotic susceptibility of isolated staphylococci was evaluated. METHODOLOGY: We tested 546 apparently healthy milking cows from 11 farms for detecting SCM using the California Mastitis Test (CMT). The cows were not treated with antibiotics. CMT-positive samples were cultured, and colonies compatible with Staphylococcus spp. were further identified through molecular techniques. The susceptibility of the Staphylococcus spp. isolates against thirteen antibiotics was determined using the disk diffusion method. RESULTS: Subclinical staphylococcal mastitis was present in almost all (82%) farms. SA (n = 39) was more common than NAS (n = 9) in the 48 samples tested. Isolates exhibited resistance to one, two, and even three different antibiotics. Resistance to penicillin was the most frequent among SA (23/39) and NAS (4/9). No staphylococci isolates exhibited resistance to cefoxitin, vancomycin, trimethoprim-sulfamethoxazole, erythromycin, or clindamycin. CONCLUSIONS: Staphylococcal SCM is one of the most common diseases in Uruguayan dairy farms. SA was the prevalent pathogen, however SA and NAS mastitis coexisted in many farms. NAS were identified and its distribution was similar to other countries. Penicillin had the highest and most frequent percentage of resistance.

Use of the FMR1 Gene Methylation Status to Assess the X-Chromosome Inactivation Pattern: A Stepwise Analysis.

X-chromosome inactivation (XCI) is a developmental process to compensate the imbalance in the dosage of X-chromosomal genes in females. A skewing of the XCI pattern may suggest a carrier status for an X-linked disease or explain the presence of a severe phenotype. In these cases, it is important to determine the XCI pattern, conventionally using the gold standard Human Androgen-Receptor Assay (HUMARA), based on the analysis of the methylation status at a polymorphic CAG region in the first exon of the human androgen receptor gene (AR). The aim of this study was to evaluate whether the methylation status of the fragile mental retardation protein translational regulator gene (FMR1) can provide an XCI pattern similar to that obtained by HUMARA. A set of 48 female carriers of FMR1 gene normal-sized alleles was examined using two assays: HUMARA and a FMR1 methylation PCR (mPCR). Ranges were defined to establish the XCI pattern using the methylation pattern of the FMR1 gene by mPCR. Overall, a 77% concordance of the XCI patterns was obtained between the two assays, which led us to propose a set of key points and a stepwise analysis towards obtaining an accurate result for the XCI pattern and to minimize the underlying pitfalls.

CalDAG-GEFI Deficiency in a Family with Symptomatic Heterozygous and Homozygous Carriers of a Likely Pathogenic Variant in RASGRP2.

RASGRP2 encodes the calcium and diacylglycerol (DAG)-regulated guanine nucleotide exchange factor I (CalDAG-GEFI) identified as a Rap1-activating molecule. Pathogenic variants previously identified in RASGRP2 allowed the characterization of CalDAG-GEFI deficiency as a non-syndromic, autosomal recessive platelet function disease. We report on the clinical manifestations and laboratory features of a Portuguese family with a likely pathogenic variant in RASGRP2 (c.999G>C leading to a p.Lys333Asn change in the CDC25 catalytic domain of CalDAG-GEFI) and discuss the contribution of this variant to the disease manifestations. Based on the study of this family with one homozygous patient and five heterozygous carriers and on a critical analysis of the literature, we challenge previous knowledge that CalDAG-GEFI deficiency only manifests in homozygous patients. Our data suggest that at least for the RASGRP2 variant reported herein, there is a phenotypic expression, albeit milder, in heterozygous carriers.

Impact of juçara (Euterpe edulis) fruit waste extracts on the quality of conventional and antibiotic-free broiler meat.

Juçara (Euterpe edulis) is a native Brazilian palm tree from the Atlantic Forest, whose fruit-processing waste can present high concentration of antioxidant compounds. This research was assessed to determine the antioxidant potential of juçara waste extracts aiming to reduce the lipid and protein oxidation processes on conventional and antibiotic-free broiler meat throughout 9 d during refrigerated storage. The juçara waste extracts were obtained by microwave-assisted extraction. Two different extracts were tested based on the optimum point obtained when checking total phenolic (TPC) contents (Extract P) and antioxidant activity (Extract A) based on a previous study. The treatments using conventional and antibiotic-free broiler meat included: chicken patties without antioxidant addition (AFBNC and CBNC), with synthetic antioxidant (BHT) (AFBPC and CBPC), with Extract P (AFBEP and CBEP) and with Extract A (AFBEA and CBEA), totaling 8 treatments. Antioxidant activity of extracts along with TPC, flavonoid, anthocyanin, and tannin contents of extracts and patties were assessed. Proximate composition, fatty acid profile, lipid and protein oxidation process, and instrumental color were analyzed in patty treatments. Although both extracts had similar content of TPC and tannin, extract A presented the highest anthocyanin, while extract P exhibited the highest flavonoid. While extract A exhibited the highest antioxidant activity, extract P was highly influential in the stability of lipid oxidative degradation in both types of broiler meat (AFBEP and CBEP), and as successful as BHT (AFBPC and CBPC). In addition, extract P was also able to stabilize protein oxidation in conventional broiler meat (CBEP) from the third day, until the end of the storage period. Therefore, the fruit waste extract P of juçara can be a promising source of natural antioxidants to prevent the oxidative process in conventional and antibiotic-free broiler meat.

Development and validation in 500 female samples of a TP-PCR assay to identify AFF2 GCC expansions.

Over 100 X-linked intellectual disability genes have been identified, with triplet repeat expansions at the FMR1 (FRAXA) and AFF2 (FRAXE) genes being the causative agent in two of them. The absence of FRAXE pathognomonic features hampers early recognition, delaying testing and molecular confirmation. Hence, our laboratory uses a multiplex PCR-based strategy to genotype both FRAXA and FRAXE. However, AFF2 expansions are missed giving rise to an uninformative result in around 20% of female samples. To rule out undetected expansions and confirm homozygosity Southern blot analysis is performed being labour- and resource-intensive. The aim of this study is to develop a timely and economic triplet-primed amplification (TP-PCR) screening strategy to size the AFF2 GCC repeat and accurately assess homozygosity as well as pinpoint multiplex-PCR false negatives in female samples. In order to achieve this, validation was performed in a cohort of 500 females with a previous uninformative FRAXE PCR result. Interestingly, the presence of a T > C SNP (rs868949662), contiguous to the GCC repetitive tract, allows triplet primer binding in two additional repeats, increasing the discrimination power of the TP-PCR assay in heterozygous and homozygous samples. Twelve alleles outside the normal range were recognized: eight intermediate and four premutated, which seems relevant considering the rarity of the AFF2 expansions. All genotypes are concordant with that obtained by Southern blotting, confirming this as a strict, reproducible and low-cost homozygosity screening strategy that enables the identification of small expanded alleles missed by the routine multiplex-PCR due to allele dropout. Overall, this assay is capable of spotting multiplex-PCR false negatives besides identifying alleles up to > 80 GCC repeats. Furthermore, the occurrence of intermediate repeat sizes with unexpected frequency, introduces new areas of clinical research in this cohort in understanding these less explored AFF2 repeat sizes and newly associated phenotypes.

Everybody loves cheese: crosslink between persistence and virulence of Shiga-toxin Escherichia coli.

General cheese manufacturing involves high temperatures, fermentation and ripening steps that function as hurdles to microbial growth. On the other hand, the application of several different formulations and manufacturing techniques may create a bacterial protective environment. In cheese, the persistent behavior of Shiga toxin-producing Escherichia coli (STEC) relies on complex mechanisms that enable bacteria to respond to stressful conditions found in cheese matrix. In this review, we discuss how STEC manages to survive to high and low temperatures, hyperosmotic conditions, exposure to weak organic acids, and pH decreasing related to cheese manufacturing, the cheese matrix itself and storage. Moreover, we discuss how these stress responses interact with each other by enhancing adaptation and consequently, the persistence of STEC in cheese. Further, we show how virulence genes eae and tir are affected by stress response mechanisms, increasing either cell adherence or virulence factors production, which leads to a selection of more resistant and virulent pathogens in the cheese industry, leading to a public health issue.

Can the Synergic Contribution of Multigenic Variants Explain the Clinical and Cellular Phenotypes of a Neurodevelopmental Disorder?

We describe an infant female with a syndromic neurodevelopmental clinical phenotype and increased chromosome instability as cellular phenotype. Genotype characterization revealed heterozygous variants in genes directly or indirectly linked to DNA repair: a de novo X-linked HDAC8 pathogenic variant, a paternally inherited FANCG pathogenic variant and a maternally inherited BRCA2 variant of uncertain significance. The full spectrum of the phenotype cannot be explained by any of the heterozygous variants on their own; thus, a synergic contribution is proposed. Complementation studies showed that the FANCG gene from the Fanconi Anaemia/BRCA (FA/BRCA) DNA repair pathway was impaired, indicating that the variant in FANCG contributes to the cellular phenotype. The patient's chromosome instability represents the first report where heterozygous variant(s) in the FA/BRCA pathway are implicated in the cellular phenotype. We propose that a multigenic contribution of heterozygous variants in HDAC8 and the FA/BRCA pathway might have a role in the phenotype of this neurodevelopmental disorder. The importance of these findings may have repercussion in the clinical management of other cases with a similar synergic contribution of heterozygous variants, allowing the establishment of new genotype-phenotype correlations and motivating the biochemical study of the underlying mechanisms.

Integrating Whole-Genome Sequencing in Clinical Genetics: A Novel Disruptive Structural Rearrangement Identified in the Dystrophin Gene (DMD).

While in most patients the identification of genetic alterations causing dystrophinopathies is a relatively straightforward task, a significant number require genomic and transcriptomic approaches that go beyond a routine diagnostic set-up. In this work, we present a Becker Muscular Dystrophy patient with elevated creatinine kinase levels, progressive muscle weakness, mild intellectual disability and a muscle biopsy showing dystrophic features and irregular dystrophin labelling. Routine molecular techniques (Southern-blot analysis, multiplex PCR, MLPA and genomic DNA sequencing) failed to detect a defect in the DMD gene. Muscle DMD transcript analysis (RT-PCR and cDNA-MLPA) showed the absence of exons 75 to 79, seen to be present at the genomic level. These results prompted the application of low-coverage linked-read whole-genome sequencing (WGS), revealing a possible rearrangement involving DMD intron 74 and a region located upstream of the PRDX4 gene. Breakpoint PCR and Sanger sequencing confirmed the presence of a ~8 Mb genomic inversion. Aberrant DMD transcripts were subsequently identified, some of which contained segments from the region upstream of PRDX4. Besides expanding the mutational spectrum of the disorder, this study reinforces the importance of transcript analysis in the diagnosis of dystrophinopathies and shows how WGS has a legitimate role in clinical laboratory genetics.

Usher syndrome and Nebulin-associated myopathy in a single patient due to variants in MYO7A and NEB.

In a patient with Usher syndrome and atypical muscle complaints, we have identified two separate variants in MYO7A andNEB genes by exome sequencing. The homozygous variants in these two recessive genes could explain the full phenotype of our patient.

Development and Validation of a Mathematical Model to Predict the Complexity of FMR1 Allele Combinations.

The polymorphic trinucleotide repetitive region in the FMR1 gene 5'UTR contains AGG interspersions, particularly in normal-sized alleles (CGG < 45). In this range repetitive stretches are typically interrupted once or twice, although alleles without or with three or more AGG interspersions can also be observed. AGG interspersions together with the total length of the repetitive region confer stability and hinder expansion to pathogenic ranges: either premutation (55 < CGG < 200) or full mutation (CGG > 200). The AGG interspersions have long been identified as one of the most important features of FMR1 repeat stability, being particularly important to determine expansion risk estimates in female premutation carriers. We sought to compute the combined AGG interspersion numbers and patterns, aiming to define FMR1 repetitive tract complexity combinations. A mathematical model, the first to compute this cumulative effect, was developed and validated using data from 131 young and healthy females. Plotting of their allelic complexity enabled the identification of two statistically distinct groups - equivalent and dissimilar allelic combinations. The outcome, a numerical parameter designated allelic score, depicts the repeat substructure of each allele, measuring the allelic complexity of the FMR1 gene including the AGGs burden, thus allowing new behavioral scrutiny of normal-sized alleles in females.

αIIbß3 variants in ten families with autosomal dominant macrothrombocytopenia: Expanding the mutational and clinical spectrum.

BACKGROUND: Rare pathogenic variants in either the ITGA2B or ITGB3 genes have been linked to autosomal dominant macrothrombocytopenia associated with abnormal platelet production and function, deserving the designation of Glanzmann Thrombasthenia-Like Syndrome (GTLS) or ITGA2B/ITGB3-related thrombocytopenia. OBJECTIVES: To describe a series of patients with familial macrothrombocytopenia and decreased expression of αIIbß3 integrin due to defects in the ITGA2B or ITGB3 genes. METHODS: We reviewed the clinical and laboratory records of 10 Portuguese families with GTLS (33 patients and 11 unaffected relatives), including the functional and genetic defects. RESULTS: Patients had absent to moderate bleeding, macrothrombocytopenia, low αIIbß3 expression, impaired platelet aggregation/ATP release to physiological agonists and low expression of activation-induced binding sites on αIIbß3 (PAC-1) and receptor-induced binding sites on its ligand (bound fibrinogen), upon stimulation with TRAP-6 and ADP. Evidence for constitutive αIIbß3 activation, occurred in 2 out of 9 patients from 8 families studied, but also in 2 out of 12 healthy controls. We identified 7 missense variants: 3 in ITGA2B (5 families), and 4 in ITGB3 (5 families). Three variants (αIIb: p.Arg1026Trp and p.Arg1026Gln and ß3: p.Asp749His) were previously reported. The remaining (αIIb: p.Gly1007Val and ß3: p.Thr746Pro, p.His748Pro and p.Arg760Cys) are new, expanding the αIIbß3 defects associated with GTLS. The integration of the clinical and laboratory data allowed the identification of two GTLS subgroups, with distinct disease severity. CONCLUSIONS: Previously reported ITGA2B and ITGB3 variants related to thrombocytopenia were clustered in a confined region of the membrane-proximal cytoplasmic domains, the inner membrane clasp. For the first time, variants are reported at the outer membrane clasp, at the transmembrane domain of αIIb, and at the membrane distal cytoplasmic domains of ß3. This is the largest single-center series of inherited macrothrombocytopenia associated with αIIbß3 variants published to date.

Two Compound Heterozygous Variants in SNX14 Cause Stereotypies and Dystonia in Autosomal Recessive Spinocerebellar Ataxia 20.

Autosomal Recessive Spinocerebellar Ataxia 20, SCAR20, is a rare condition characterized by intellectual disability, lack of speech, ataxia, coarse facies and macrocephaly, caused by SNX14 variants. While all cases described are due to homozygous variants that generally result in loss of protein, so far there are no other cases of reported compound heterozygous variants. Here we describe the first non-consanguineous SCAR20 family, the second Portuguese, with two siblings presenting similar clinical features caused by compound heterozygous SNX14 variants: NM_001350532.1:c.1195C>T, p.(Arg399*) combined with a novel complex genomic rearrangement. Quantitative PCR (Q-PCR), long-range PCR and sequencing was used to elucidate the region and mechanisms involved in the latter: two deletions, an inversion and an AG insertion: NM_001350532.1:c.[612+3028_698-2759del;698-2758_698-516inv;698-515_1171+1366delinsAG]. In silico analyses of these variants are in agreement with causality, enabling a genotype-phenotype correlation in both patients. Clinical phenotype includes dystonia and stereotypies never associated with SCAR20. Overall, this study allowed to extend the knowledge of the phenotypic and mutational spectrum of SCAR20, and to validate the role of Sorting nexin-14 in a well-defined neurodevelopmental syndrome, which can lead to cognitive impairment. We also highlight the value of an accurate clinical evaluation and deep phenotyping to disclose the molecular defect underlying highly heterogeneous condition such as intellectual disability.

EMQN best practice guidelines for genetic testing in dystrophinopathies.

Dystrophinopathies are X-linked diseases, including Duchenne muscular dystrophy and Becker muscular dystrophy, due to DMD gene variants. In recent years, the application of new genetic technologies and the availability of new personalised drugs have influenced diagnostic genetic testing for dystrophinopathies. Therefore, these European best practice guidelines for genetic testing in dystrophinopathies have been produced to update previous guidelines published in 2010.These guidelines summarise current recommended technologies and methodologies for analysis of the DMD gene, including testing for deletions and duplications of one or more exons, small variant detection and RNA analysis. Genetic testing strategies for diagnosis, carrier testing and prenatal diagnosis (including non-invasive prenatal diagnosis) are then outlined. Guidelines for sequence variant annotation and interpretation are provided, followed by recommendations for reporting results of all categories of testing. Finally, atypical findings (such as non-contiguous deletions and dual DMD variants), implications for personalised medicine and clinical trials and incidental findings (identification of DMD gene variants in patients where a clinical diagnosis of dystrophinopathy has not been considered or suspected) are discussed.

Unveiling the genetic etiology of primary ciliary dyskinesia: When standard genetic approach is not enough.

PURPOSE: Primary ciliary dyskinesia (PCD) is a ciliopathy caused by dysfunction of motile cilia. As there is still no standard PCD diagnostics, the final diagnosis requires a combination of several tests. The genetic screening is a hallmark for the final diagnosis and requires high-throughput techniques, such as whole-exome sequencing (WES). Nevertheless, WES has limitations that may prevent a definitive genetic diagnosis. Here we present a case that demonstrates how the PCD genetic diagnosis may not be trivial. MATERIALS/METHODS: A child with PCD and situs inversus totalis (designated as Kartagener syndrome (KS)) was subjected to clinical assessments, ultrastructural analysis of motile cilia, extensive genetic evaluation by WES and chromosomal array analysis, bioinformatic analysis, gene expression analysis and immunofluorescence to identify the genetic etiology. His parents and sister, as well as healthy controls were also evaluated. RESULTS: Here we show that a disease-causing variant in the USP11 gene and copy number variations in CRHR1 and KRT34 genes may be involved in the patient PCD phenotype. None of these genes were previously reported in PCD patients and here we firstly show its presence and immunolocalization in respiratory cells. CONCLUSIONS: This work highlights how the genetic diagnosis can turn to be rather complex and that combining several approaches may be needed. Overall, our results contribute to increase the understanding of the genetic factors involved in the pathophysiology of PCD/KS, which is of paramount importance to assist the current diagnosis and future development of newer therapies.

Clinical and Genetic Analysis of Children with Kartagener Syndrome.

Primary ciliary dyskinesia (PCD) is a rare autosomal recessive disorder characterized by dysfunction of motile cilia causing ineffective mucus clearance and organ laterality defects. In this study, two unrelated Portuguese children with strong PCD suspicion underwent extensive clinical and genetic assessments by whole-exome sequencing (WES), as well as ultrastructural analysis of cilia by transmission electron microscopy (TEM) to identify their genetic etiology. These analyses confirmed the diagnostic of Kartagener syndrome (KS) (PCD with situs inversus). Patient-1 showed a predominance of the absence of the inner dynein arms with two disease-causing variants in the CCDC40 gene. Patient-2 showed the absence of both dynein arms and WES disclosed two novel high impact variants in the DNAH5 gene and two missense variants in the DNAH7 gene, all possibly deleterious. Moreover, in Patient-2, functional data revealed a reduction of gene expression and protein mislocalization in both genes' products. Our work calls the researcher's attention to the complexity of the PCD and to the possibility of gene interactions modelling the PCD phenotype. Further, it is demonstrated that even for well-known PCD genes, novel pathogenic variants could have importance for a PCD/KS diagnosis, reinforcing the difficulty of providing genetic counselling and prenatal diagnosis to families.