Hypomorphic RAG deficiency: impact of disease burden on survival and thymic recovery argues for early diagnosis and HSCT.

Patients with hypomorphic mutations in the RAG1 or RAG2 gene present with either Omenn syndrome or atypical combined immunodeficiency with a wide phenotypic range. Hematopoietic stem cell transplantation (HSCT) is potentially curative, but data are scarce. We report on a worldwide cohort of 60 patients with hypomorphic RAG variants who underwent HSCT, 78% of whom experienced infections (29% active at HSCT), 72% had autoimmunity, and 18% had granulomas pretransplant. These complications are frequently associated with organ damage. Eight individuals (13%) were diagnosed by newborn screening or family history. HSCT was performed at a median of 3.4 years (range 0.3-42.9 years) from matched unrelated donors, matched sibling or matched family donors, or mismatched donors in 48%, 22%, and 30% of the patients, respectively. Grafts were T-cell depleted in 15 cases (25%). Overall survival at 1 and 4 years was 77.5% and 67.5% (median follow-up of 39 months). Infection was the main cause of death. In univariable analysis, active infection, organ damage pre-HSCT, T-cell depletion of the graft, and transplant from a mismatched family donor were predictive of worse outcome, whereas organ damage and T-cell depletion remained significant in multivariable analysis (hazard ratio [HR] = 6.01, HR = 8.46, respectively). All patients diagnosed by newborn screening or family history survived. Cumulative incidences of acute and chronic graft-versus-host disease were 35% and 22%, respectively. Cumulative incidences of new-onset autoimmunity was 15%. Immune reconstitution, particularly recovery of naïve CD4+ T cells, was faster and more robust in patients transplanted before 3.5 years of age, and without organ damage. These findings support the indication for early transplantation.

Assessment of the cost-effectiveness of alternative bovine tuberculosis surveillance protocols in French cattle farms using the mixed interferon gamma test.

Periodic screening in farms, using intradermal cervical comparative tuberculin test (ICCT), is a component of the French ante mortem surveillance of bovine tuberculosis (bTB). Previous studies have estimated the cost-effectiveness of the French mandatory bTB screening protocols. In these protocols, a second ICCT (ICCT2) is performed 42 days after the first one (ICCT1), either on the entire herd (strict protocol) or in series on animals with non-negative results (reactors) to ICCT1 (compliant protocol). The 42-days interval reduced protocols' cost-effectiveness. To minimize this interval, we suggested two alternative protocols, in which a mixed interferon gamma test (IFNMIX), with better sensitivity than ICCT2 and comparable specificity, replaces the ICCT2, and is carried out directly after the ICCT1. In the strict alternative protocol, reactors to ICCT1 are culled to perform laboratory analyses (PCR, bacteriology). Negative results to these analyses imply the IFNMIX testing of the entire herd. In the compliant alternative protocol, only reactor(s) to ICCT1 are tested with IFNMIX, and animals with positive results to IFNMIX are culled for laboratory analyses. We evaluated these protocols through scenario tree modelling. The estimated cost-effectiveness indexes showed that the compliant alternative protocol was the most efficient. The strict protocols (mandatory and alternative) were never the most efficient, but were the most effective. Therefore, using IFNMIX instead of ICCT2 may be useful in reducing the costs of the compliant protocol used when the probability of infection is considered low. The strict alternative protocol may become more attractive would IFNMIX's price decreased.

Assessment of bovine tuberculosis surveillance effectiveness in French wildlife: An operational approach.

Bovine tuberculosis (bTB) is a chronic, zoonotic, bacterial disease mostly caused by Mycobacterium bovis, which can affect both domestic and wild species. France was officially declared bTB-free in 2001 but faced since 2004 an increase of the prevalence in cattle. Since 2001, bTB has been detected in several wild species: red deer (Cervus elaphus), roe deer (Capreolus capreolus), wild boar (Sus scrofa) and badger (Meles meles). Infected wild species constitute a major threat, because they may contribute to the maintenance of the infection in cattle and prevent eradication. In 2011, a surveillance system, Sylvatub, was implemented nationwide, to monitor the epidemiological status of bTB in mainland France. Our objective in this study was to assess the effectiveness of one of Sylvatub's passive surveillance system components (SSCs), which is based on the visual inspection of hunted animals (wild boars, red and roe deer) throughout mainland France. The following effectiveness criteria were evaluated: individual-level and component group-level positive and negative predictive values, and individual-level and component group-level probabilities of type I error ("false-positive" error) and type II error ("false-negative" error). These criteria were estimated quantitatively, at the scale of the département (a French administrative area of similar size to a county), with a stochastic scenario tree model. Individual negative predictive values were high, whereas individual positive predictive values were poor, whatever the species considered, and the training of hunters did not improve these effectiveness criteria sufficiently. The individual-level probability of type I error was relatively low, but the individual-level probability of type II error was generally high and was therefore an issue. However, increasing the proportion of trained hunters decreased this probability effectively. At group level, the size of the population surveyed had a marked impact on the effectiveness criteria: both the component group-level negative predictive value and the component group-level probability of type II error decreased rapidly with increasing population size. The conclusions drawn from such rationale may directly benefit stakeholders and actors in the field in their day to day practice of the surveillance processes. Thus, the assessment method used in this article presents the advantage of being operational, as well as being applicable to any surveillance system.

The IL-2RG R328X nonsense mutation allows partial STAT-5 phosphorylation and defines a critical region involved in the leaky-SCID phenotype.

In addition to their detection in typical X-linked severe combined immunodeficiency, hypomorphic mutations in the interleukin (IL)-2 receptor common gamma chain gene (IL2RG) have been described in patients with atypical clinical and immunological phenotypes. In this leaky clinical phenotype the diagnosis is often delayed, limiting prompt therapy in these patients. Here, we report the biochemical and functional characterization of a nonsense mutation in exon 8 (p.R328X) of IL2RG in two siblings: a 4-year-old boy with lethal Epstein-Barr virus-related lymphoma and his asymptomatic 8-month-old brother with a Tlow B+ natural killer (NK)+ immunophenotype, dysgammaglobulinemia, abnormal lymphocyte proliferation and reduced levels of T cell receptor excision circles. After confirming normal IL-2RG expression (CD132) on T lymphocytes, signal transducer and activator of transcription-1 (STAT-5) phosphorylation was examined to evaluate the functionality of the common gamma chain (γc ), which showed partially preserved function. Co-immunoprecipitation experiments were performed to assess the interaction capacity of the R328X mutant with Janus kinase (JAK)3, concluding that R328X impairs JAK3 binding to γc . Here, we describe how the R328X mutation in IL-2RG may allow partial phosphorylation of STAT-5 through a JAK3-independent pathway. We identified a region of three amino acids in the γc intracellular domain that may be critical for receptor stabilization and allow this alternative signaling. Identification of the functional consequences of pathogenic IL2RG variants at the cellular level is important to enable clearer understanding of partial defects leading to leaky phenotypes.

Integrating data of veterinarians' practices in assessing the cost effectiveness of three components of the bovine tuberculosis surveillance system by intradermal tuberculin testing in French cattle farms through a scenario-tree approach.

Disease surveillance systems' effectiveness relies on participants following prescribed practices. We developed a general method to improve a previous cost-effectiveness evaluation of three French screening program protocols for bovine tuberculosis (bTB) to account for the practices of participants by scenario tree modelling. This method relies on: 1) semi-directive interviews of participants to identify the variability of practices and potentially influential factors, and to understand the sociological context; 2) a quantitative survey, based on multiple-choice questions, to quantify various practices and identify significantly influential factors by multivariable regression analyses; 3) addition of the scenario-tree nodes corresponding to the practices and their influential factors and configuration of the new limbs according to the data of the quantitative survey. We used this approach to integrate data concerning veterinary practices and identify some failures to conform to regulatory guidelines regarding intradermal cervical comparative tuberculin test (SICCT) (testing and notification of non-negative results). Such nonconformities appeared to be mainly caused by cattle restraint issues and the perception of veterinarians of the bTB control program. Indeed, their perception of that program significantly influenced veterinarians' practices. We modelled the influence of the SICCT practices on the SICCT results. The incorporation of these data led to a major decrease of the herd sensitivity estimations relative to the previous assessments that did not incorporate data of practices (15% to 42% decrease). This result shows the important impact of veterinarians' practices and their influencing factors (such as perception of the bTB control program) on the effectiveness of the surveillance system.

Cost-effectiveness of two different protocols for animal tracing investigations of bovine tuberculosis outbreaks in France.

In the French bovine tuberculosis (bTB) surveillance program, tracing-on and back investigations have a major importance as, in 2016, they represented about 21 % of the detected outbreaks. Building on our previous work on the other surveillance system components (Poirier et al., 2019), we evaluated for the first time the sensitivity and the cost of the two existing protocols of bTB's tracing-on investigations trough scenario tree modelling with a stochastic approach. We used French databases (national database for bovine identification and database recording all bTB surveillance and control results) and direct and indirect costs collected in a previous study. These assessments allowed us to calculate the cost-effectiveness index (cost/sensitivity) of each tracing-on protocol. In the first protocol (trace-and-cull protocol), the animal(s) linking the farm to an outbreak are systematically culled for bacteriology, PCR and histology testing. In the second protocol (trace-and-test protocol), the traced animal is culled only if it had non-negative result to an intradermal cervical comparative tuberculin test (ICCT). We estimated herd sensitivity of the two tracing-on protocols for 12 herd types defined by their production type, size and herd turnover. For the trace-and-cull protocol, mean herd sensitivity was estimated between 67.3 % [66.8-67.7]CI95 % and 89.2 % [88.7-89.7]CI95 % and between 51.2 % [50.8-51.5]CI95 % and 73.1 % [72.6-73.6]CI95 % for the trace-and-test protocol, depending on herd type. The trace-and-cull protocol was between 278 €/herd and 717 €/herd more expensive than the trace-and-test protocol, depending on herd type. Regardless of herd type, the trace-and-cull protocol had the smaller cost/sensitivity ratio and was therefore the most cost-effective protocol. That work showed that systematically culling traced animals to perform bacteriology and PCR on them (trace-and-cull protocol) is associated with a better herd sensitivity and is more cost-effective for all herd types. That is consistent with French veterinary authorities' recommendations but does not account for sociological aspects such as the bond between the farmer and his animals. Yet, cost-effectiveness difference was minor in small dairy and beef herds with a low turnover, suggesting the protocol could be chosen depending on the epidemiological context in those herds.

Cost-effectiveness assessment of three components of the bovine tuberculosis surveillance system by intradermal tuberculin testing in French cattle farms by a scenario tree approach.

In most officially bovine tuberculosis (bTB)-free countries, bTB has not been fully eradicated. Costly and time-consuming surveillance and control measures are therefore still in place to control this infection. An officially bTB-free status, both at the national and at the herd level, influences whether and when animals can be sold. Thus, this infection is still an economic issue, justifying measures towards its eradication. An evaluation of the cost-effectiveness of such measures would be highly useful, especially to optimise the costs of control measures and their adaptation to a local epidemiological context. We evaluated the cost-effectiveness of three mandatory surveillance protocols currently used in France by herd type (type of production, size, and turnover of the herd) under French field conditions. The first protocol ("strict") implies the direct slaughter and post-mortem analyses of any intradermal cervical tuberculin test (ICT) reactor, and negative results to a second intradermal cervical comparative tuberculin test (ICCT) to regain bTB-free status of the herd. In the second protocol ("compliant quick-path") bTB-free status can be regain if post-mortem analyses of reactors to the first ICT are negative. In the third protocol ("compliant slow-path"), ICCT-reactive animals are tested using the interferon gamma assay; the results of this test influence the path of further investigation. We built scenario trees for each of these protocols at the animal level. They allowed us to estimate herd sensitivity and the total cost of each protocol by herd type. The protocols could be ordered by decreasing herd sensitivity and cost, regardless of the herd type, as follows: strict protocol, compliant quick-path protocol, and compliant slow-path protocol. We calculated a cost-effectiveness index to evaluate the cost-effectiveness of each protocol. The strict protocol was never the most cost-effective, regardless of herd type, due to higher costs relative to the other protocols, despite better herd sensitivity. We found the compliant quick-path to be the most cost-effective protocol for big beef, big dairy, and mixed herds. The compliant slow-path was the most cost-effective for small-scale beef and dairy herds. All differences were significant. This comparison of the cost-effectiveness of the protocols by herd type could help authorities to choose the most suitable protocol in the investigation of suspected cases, depending on the herd type, but could be improved by accounting for important sociological data, such as the acceptability of the protocols.

Mathematical modeling and simulation in animal health. Part III: Using nonlinear mixed-effects to characterize and quantify variability in drug pharmacokinetics.

A common feature of human and veterinary pharmacokinetics is the importance of identifying and quantifying the key determinants of between-patient variability in drug disposition and effects. Some of these attributes are already well known to the field of human pharmacology such as bodyweight, age, or sex, while others are more specific to veterinary medicine, such as species, breed, and social behavior. Identification of these attributes has the potential to allow a better and more tailored use of therapeutic drugs both in companion and food-producing animals. Nonlinear mixed effects (NLME) have been purposely designed to characterize the sources of variability in drug disposition and response. The NLME approach can be used to explore the impact of population-associated variables on the relationship between drug administration, systemic exposure, and the levels of drug residues in tissues. The latter, while different from the method used by the US Food and Drug Administration for setting official withdrawal times (WT) can also be beneficial for estimating WT of approved animal drug products when used in an extralabel manner. Finally, NLME can also prove useful to optimize dosing schedules, or to analyze sparse data collected in situations where intensive blood collection is technically challenging, as in small animal species presenting limited blood volume such as poultry and fish.

Biocorona formation on gold nanoparticles modulates human proximal tubule kidney cell uptake, cytotoxicity and gene expression.

Gold nanoparticles (AuNP) adsorb macromolecules to form a protein corona (PC) after systemic delivery, to which the kidney as the primary excretory organ is constantly exposed. The role of the PC on AuNP cell uptake and toxicity was investigated in vitro in human proximal tubule cells (HPTC) using 40 and 80nm branched polyethylenimine (BPEI), lipoic acid (LA) and polyethylene glycol (PEG) coated AuNP with or without (bare) PCs composed of human plasma (HP) or human serum albumin (HSA) for 0.25 to 24h. Time-dependent intracellular uptake, assessed by ICP-MS showed PC modulated cell uptake and cytotoxicity; with bare 40nm BPEI-AuNP showing the greatest responses. All AuNP showed minimal to no cytokine release. At the nontoxic dose, 40nm bare BPEI-AuNP significantly modified gene expression related to immunotoxicity, steatosis, and mitochondrial metabolism; while at the high dose, pathways of DNA damage and repair, apoptosis, fatty acid metabolism and heat shock response were modulated. HP corona BPEI-AuNP response was comparable to control. These studies clearly showed reduced uptake and cytotoxicity, as well as differentiated gene expression of AuNP with PCs, questioning the utility of in vitro studies using bare NP to assess in vivo effects. Significantly, only cationic bare BPEI-AuNP had HPTC uptake or cytotoxicity suggesting the relative safety of PEG and LA-AuNP as nanomedicine constructs.

Further delineation of a rare recessive encephalomyopathy linked to mutations in GFER thanks to data sharing of whole exome sequencing data.

BACKGROUND: Alterations in GFER gene have been associated with progressive mitochondrial myopathy, congenital cataracts, hearing loss, developmental delay, lactic acidosis and respiratory chain deficiency in 3 siblings born to consanguineous Moroccan parents by homozygosity mapping and candidate gene approach (OMIM#613076). Next generation sequencing recently confirmed this association by the finding of compound heterozygous variants in 19-year-old girl with a strikingly similar phenotype, but this ultra-rare entity remains however unknown from most of the scientific community. MATERIALS AND METHODS: Whole exome sequencing was performed as part of a "diagnostic odyssey" for suspected mitochondrial condition in 2 patients, presenting congenital cataracts, progressive encephalomyopathy and hypotrophy and detected unreported compound heterozygous variants in GFER. RESULTS: Thanks to an international data sharing, we found 2 additional patients carrying compound heterozygous variants in GFER. Reverse phenotyping confirmed the phenotypical similarities between the 4 patients. Together with the first literature reports, the review of these 8 cases from 4 unrelated families enables us to better describe this apparently homogeneous disorder, with the clinical and biological stigmata of mitochondrial disease. CONCLUSION: This report highlights the clinical utility of whole exome sequencing and reverse phenotyping for the diagnosis of ultra-rare diseases and underlines the importance of a broad data sharing for accurate clinical delineation of previously unrecognized entities.

Mosaic-activating FGFR2 mutation in two fetuses with papillomatous pedunculated sebaceous naevus.

Papillomatous pedunculated sebaceous naevus (PPSN) has been described as a subtype of sebaceous naevus (SN), typically affecting the scalp and face. In contrast with Schimmelpenning syndrome, no cerebral, ocular or skeletal anomalies have hitherto been reported. We report two unrelated fetuses with PPSN, one with large pink exophytic tumours, the other with minor features but similar microscopic findings. We performed whole-exome sequencing in affected skin tissue from fetus 1, which identified a postzygotic de novo FGFR2 c.1144T>C (p.Cys382Arg) mutation in 34·6% of reads which was absent in the parents' blood. Targeted deep sequencing of FGFR2 confirmed its mosaic status in additional affected skin from fetus 1, and identified the same substitution in 26% of reads in affected skin from fetus 2. FGFR2 p.Cys382Arg is a known somatic driver mutation in human cancer, previously reported to result in activation of RAS signalling. A similar paralogous missense mutation in the transmembrane domain of FGFR3 (p.Gly380Arg) has been reported in keratinocytic epidermal naevi. Our findings define a distinct clinical and molecular subgroup of SN, beside HRAS or KRAS-related SN, and expand the spectrum of mosaic skin conditions associated with receptor tyrosine kinase mutations.

Autosomal recessive mutations in THOC6 cause intellectual disability: syndrome delineation requiring forward and reverse phenotyping.

THOC6 is a part of the THO complex, which is involved in coordinating mRNA processing with export. The THO complex interacts with additional components to form the larger TREX complex (transcription export complex). Previously, a homozygous missense mutation in THOC6 in the Hutterite population was reported in association with syndromic intellectual disability. Using exome sequencing, we identified three unrelated patients with bi-allelic mutations in THOC6 associated with intellectual disability and additional clinical features. Two of the patients were compound heterozygous for a stop and a missense mutation, and the third was homozygous for a missense mutation; the missense mutations were predicted to be pathogenic by in silico analysis and modeling. Clinical features of the three newly identified patients and those previously reported are reviewed; intellectual disability is moderate to severe, and malformations are variable including renal and heart defects, cleft palate, microcephaly, and corpus callosum dysgenesis. Facial features are variable and include tall forehead, short upslanting palpebral fissures +/- deep set eyes, and a long nose with overhanging columella. These subtle facial features render the diagnosis difficult to make in isolation with certainty. Our results expand the mutational and clinical spectrum of this rare disease, confirm that THOC6 is an intellectual disability causing gene, while providing insight into the importance of the THO complex in neurodevelopment.

Autosomal recessive truncating MAB21L1 mutation associated with a syndromic scrotal agenesis.

We report on a boy with a rare malformative association of scrotum agenesis, ophthalmological anomalies, cerebellar malformation, facial dysmorphism and global development delay. The reported patient was carrying a homozygous frameshift in MAB21L1 detected by whole-exome sequencing, considered as the most likely disease-causing variant. Mab21l1 knockout mice present a strikingly similar malformative association of ophthalmological malformations of the anterior chamber and preputial glands hypoplasia. We hypothesize that MAB21L1 haploinsufficiency cause a previously undescribed syndrome with scrotal agenesis, ophthalmological anomalies, facial dysmorphism and gross psychomotor delay as remarkable hallmarks. Four cases from the literature were reported with features suggestive of a similar and recognizable clinical entity. We hypothesize that MAB21L1 should be the culprit gene in these patients.

Autosomal recessive variations of TBX6, from congenital scoliosis to spondylocostal dysostosis.

Proximal 16p11.2 microdeletions are recurrent microdeletions with an overall prevalence of 0.03%. In patients with segmentation defects of the vertebra (SDV), a burden of this microdeletion was observed with TBX6 as a candidate gene for SDV. In a published cohort of patients with congenital scoliosis (CS), TBX6 haploinsufficiency was compound heterozygous with a common haplotype. Besides, a single three-generation family with spondylocostal dysostosis (SCD) was reported with a heterozygous stop-loss of TBX6. These observations questioned both on the inheritance mode and on the variable expressivity associated with TBX6-associated SDV. Based on a national recruitment of 56 patients with SDV, we describe four patients with variable SDV ranging from CS to SCD associated with biallelic variations of TBX6. Two patients with CS were carrying a proximal 16p11.2 microdeletion associated with the previously reported haplotype. One patient with extensive SDV was carrying a proximal 16p11.2 microdeletion associated with a TBX6 rare missense change. One patient with a clinical diagnosis of SCD was compound heterozygous for two TBX6 rare missense changes. The three rare variants were affecting the chromatin-binding domain. Our data illustrate the variable expressivity of recessive TBX6 ranging from CS to SCD.

Protocol for diversion of confirmed positive bulk raw milk tankers to calf ranches - A review of the Pharmacokinetics of tetracyclines and sulfonamides in veal calves.

The tetracyclines (TTC) and sulfonamides are among the most common residues found in bulk raw milk samples. Detection of drug residues in bulk milk (BM) tankers demonstrates that the product is not suitable for human consumption. Discarding BM with residue-contaminated milk is a waste of a valuable commodity, and a repurposing for consumption at calf ranches is a way to recapture some value. However, if calves consuming milk with drug residues are slaughtered for veal, their meat could contain drug residues. The objective of this review is to provide a residue avoidance strategy for TTC and sulfonamide residues in veal. To determine the pharmacokinetic properties of each drug a structured review of the literature was performed and the study inclusion criteria were that the publication used dairy breed calves, with body weight <330 kg or <6 months of age. The most pertinent parameters were determined to be plasma, tissue elimination half-lives, and systemic bioavailability. The results of this review were integrated with milk and tissue testing levels of quantification and tissue tolerances to formulate a recommended withdrawal interval for calves ingesting this milk. The suggested withdrawal interval of 20 days will ensure that no veal calves will test positive for residues from being fed this milk.