Woodhouse-Sakati syndrome in an Indian patient with a novel pathogenic variant.

Woodhouse-Sakati syndrome consists of hypogonadism, diabetes mellitus, alopecia, ECG abnormalities, and dystonia. This condition is caused by the loss of function of the DCAF17 gene. Most of the patients have been reported from Greater Middle Eastern countries. We report a 38 male from southern India who presented with syncope and massive hemoptysis due to ruptured bronchopulmonary collaterals. He also had alopecia, cataracts, recently diagnosed diabetes and hypogonadism. Whole exome sequencing showed a novel homozygous truncating variant in the DCAF17 gene. Despite embolization of the aortopulmonary collaterals, the patient died of recurrent hemoptysis.

Pathogenic variants in SMARCA1 cause an X-linked neurodevelopmental disorder modulated by NURF complex composition.

Pathogenic variants in ATP-dependent chromatin remodeling proteins are a recurrent cause of neurodevelopmental disorders (NDDs). The NURF complex consists of BPTF and either the SNF2H (SMARCA5) or SNF2L (SMARCA1) ISWI-chromatin remodeling enzyme. Pathogenic variants in BPTF and SMARCA5 were previously implicated in NDDs. Here, we describe 40 individuals from 30 families with de novo or maternally inherited pathogenic variants in SMARCA1. This novel NDD was associated with mild to severe ID/DD, delayed or regressive speech development, and some recurrent facial dysmorphisms. Individuals carrying SMARCA1 loss-of-function variants exhibited a mild genome-wide DNA methylation profile and a high penetrance of macrocephaly. Genetic dissection of the NURF complex using Smarca1, Smarca5, and Bptfsingle and double mouse knockouts revealed the importance of NURF composition and dosage for proper forebrain development. Finally, we propose that genetic alterations affecting different NURF components result in a NDD with a broad clinical spectrum.

The landscape of reported VUS in multi-gene panel and genomic testing: Time for a change.

PURPOSE: Variants of uncertain significance (VUS) are a common result of diagnostic genetic testing and can be difficult to manage with potential misinterpretation and downstream costs, including time investment by clinicians. We investigated the rate of VUS reported on diagnostic testing via multi-gene panels (MGPs) and exome and genome sequencing (ES/GS) to measure the magnitude of uncertain results and explore ways to reduce their potentially detrimental impact. METHODS: Rates of inconclusive results due to VUS were collected from over 1.5 million sequencing test results from 19 clinical laboratories in North America from 2020 to 2021. RESULTS: We found a lower rate of inconclusive test results due to VUSs from ES/GS (22.5%) compared with MGPs (32.6%; P < .0001). For MGPs, the rate of inconclusive results correlated with panel size. The use of trios reduced inconclusive rates (18.9% vs 27.6%; P < .0001), whereas the use of GS compared with ES had no impact (22.2% vs 22.6%; P = ns). CONCLUSION: The high rate of VUS observed in diagnostic MGP testing warrants examining current variant reporting practices. We propose several approaches to reduce reported VUS rates, while directing clinician resources toward important VUS follow-up.

Monoallelic intragenic POU3F2 variants lead to neurodevelopmental delay and hyperphagic obesity, confirming the gene's candidacy in 6q16.1 deletions.

While common obesity accounts for an increasing global health burden, its monogenic forms have taught us underlying mechanisms via more than 20 single-gene disorders. Among these, the most common mechanism is central nervous system dysregulation of food intake and satiety, often accompanied by neurodevelopmental delay (NDD) and autism spectrum disorder. In a family with syndromic obesity, we identified a monoallelic truncating variant in POU3F2 (alias BRN2) encoding a neural transcription factor, which has previously been suggested as a driver of obesity and NDD in individuals with the 6q16.1 deletion. In an international collaboration, we identified ultra-rare truncating and missense variants in another ten individuals sharing autism spectrum disorder, NDD, and adolescent-onset obesity. Affected individuals presented with low-to-normal birth weight and infantile feeding difficulties but developed insulin resistance and hyperphagia during childhood. Except for a variant leading to early truncation of the protein, identified variants showed adequate nuclear translocation but overall disturbed DNA-binding ability and promotor activation. In a cohort with common non-syndromic obesity, we independently observed a negative correlation of POU3F2 gene expression with BMI, suggesting a role beyond monogenic obesity. In summary, we propose deleterious intragenic variants of POU3F2 to cause transcriptional dysregulation associated with hyperphagic obesity of adolescent onset with variable NDD.

Biallelic pathogenic variants in roundabout guidance receptor 1 associate with syndromic congenital anomalies of the kidney and urinary tract.

Congenital anomalies of the kidney and urinary tract (CAKUT) represent the most common cause of chronic kidney failure in children. Despite growing knowledge of the genetic causes of CAKUT, the majority of cases remain etiologically unsolved. Genetic alterations in roundabout guidance receptor 1 (ROBO1) have been associated with neuronal and cardiac developmental defects in living individuals. Although Slit-Robo signaling is pivotal for kidney development, diagnostic ROBO1 variants have not been reported in viable CAKUT to date. By next-generation-sequencing methods, we identified six unrelated individuals and two non-viable fetuses with biallelic truncating or combined missense and truncating variants in ROBO1. Kidney and genitourinary manifestation included unilateral or bilateral kidney agenesis, vesicoureteral junction obstruction, vesicoureteral reflux, posterior urethral valve, genital malformation, and increased kidney echogenicity. Further clinical characteristics were remarkably heterogeneous, including neurodevelopmental defects, intellectual impairment, cerebral malformations, eye anomalies, and cardiac defects. By in silico analysis, we determined the functional significance of identified missense variants and observed absence of kidney ROBO1 expression in both human and murine mutant tissues. While its expression in multiple tissues may explain heterogeneous organ involvement, variability of the kidney disease suggests gene dosage effects due to a combination of null alleles with mild hypomorphic alleles. Thus, comprehensive genetic analysis in CAKUT should include ROBO1 as a new cause of recessively inherited disease. Hence, in patients with already established ROBO1-associated cardiac or neuronal disorders, screening for kidney involvement is indicated.

Characterization of responses initiated by different Toll-like receptor 2 ligands in chicken spleen cells.

Toll-like receptors (TLRs) are pattern recognition receptors that mediate host responses to pathogens by promoting cellular activation and the production of cytokines. Ligands for TLRs are conserved structural motifs of pathogens termed pathogen-associated molecular patterns. In the case of TLR2, these ligands include peptidoglycan, lipomannan and lipopeptides. In mammals, it has been shown that different TLR2 ligands induce distinct cytokine responses. However, whether a similar phenomenon occurs in chickens remains to be determined. To this end, chicken splenocytes were stimulated with three different TLR2 ligands: Pam3CSK4, FSL-1 and lipomannan, and the relative gene expression of several cytokines was quantified at 2, 6 and 18h post-stimulation. The results suggest that Pam3 and FSL-1 modulate the kinetics of the pro-inflammatory cytokine response differently, as Pam3 induced a robust interleukin (IL)-1ß response, while FSL-1 induced an early and prolonged up-regulation of IL-8. Furthermore, it appears that all three TLR2 ligands induce a mixed T-helper (TH) 1 and 2-like response, as characterized by the up-regulation of IFN-γ, IL-12, IL-4 and IL-13. In conclusion, we have demonstrated that different TLR2 ligands may induce different cytokine responses in chicken splenocytes. Future studies may be aimed at examining the immunomodulating effects of these ligands in vivo.

Chicken erythrocytes respond to Toll-like receptor ligands by up-regulating cytokine transcripts.

Avian erythrocytes are nucleated cells of myeloid origin that are able to actively transcribe and translate proteins. Although their role in gas exchange and transportation has been well described, it has recently been shown that chicken erythrocytes produce cytokines in response to Toll-like receptor (TLR) ligands, which raises the possibility that they also contribute to host immunity. To this end, the objective of the study was to gain some further insight into the immunological role of erythrocytes by identifying the repertoire of TLRs that they express and to elucidate their responses to the TLR3 and TLR21 ligands poly I:C and CpG oligodeoxynucleotide (ODN), respectively. The results suggest that erythrocytes constitutively express transcripts for TLRs 2, 3, 4, 5, and 21, as well as for many immunological genes including type I interferons (IFN) and interleukin (IL)-8. Moreover, it was found that treatment with both poly I:C and CpG ODN up-regulated transcripts for type I IFNs, while only poly I:C up-regulated IL-8 transcripts and enhanced the production of nitrite. Future studies may be aimed at further characterizing the immunological role of erythrocytes.

Toll-like receptor ligands induce the expression of interferon-gamma and interleukin-17 in chicken CD4+ T cells.

BACKGROUND: Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors that mediate host responses to pathogens. To date, at least 10 different TLRs have been identified in chickens including TLR2, which binds lipopeptides and other similar ligands such as Pam3CSK4, TLR3, which binds double stranded RNA as well as synthetic molecules such as poly I:C, TLR4, which binds lipopolysaccharide (LPS), and TLR21, which binds CpG DNA motifs. In mammals, TLRs have been detected on CD4+ T cells where they mediate cellular survival, proliferation and the production of cytokines. However, the TLR-mediated responses in chicken CD4+ T cells remain to be determined. As such, the objective of the present study was to elucidate the kinetics of cytokine response to several different TLR ligands in chicken CD4+ T cells. RESULTS: The results suggest that these cells express TLRs 2, 3, 4 and 21 at the transcript level, and treatment with ligands for these TLRs significantly influenced the expression of the cytokines interferon (IFN)-γ and interleukin (IL)-17, but not IL-4, IL-10 and IL-13. Specifically, treatment with Pam3CSK4, poly I:C and LPS up-regulated IFN-γ transcripts, while CpG ODN significantly down-regulated them. In contrast, at least one dose of each of the TLR ligands, except for Pam3CSK4, significantly down-regulated IL-17 transcripts. CONCLUSION: Chicken CD4+ T cells respond to ligands for TLRs 2, 3, 4 and 21 by up-regulating or down-regulating cytokine transcripts. Future studies may consider exploring how these TLR ligands may modulate other effector functions in chicken CD4+ T cells, as well as in other T cell subsets such as CD8+ T cells.

Treatment with ligands for toll-like receptors 2 and 5 induces a mixed T-helper 1- and 2-like response in chicken splenocytes.

Toll-like receptors (TLRs) play an important role in the induction of host responses to pathogens. Interactions between TLRs and their ligands result in the production of cytokines that modulate the adaptive immune response through polarizing CD4+ T cells into either T-helper (T(H))1 or T(H)2 phenotypes. In this regard, TLR2 and TLR5 ligands have been shown to induce responses in mammals that are biased toward T(H)1 or T(H)2 phenotypes. However, whether a similar phenomenon occurs in chickens remains to be elucidated. To this end, chicken splenocytes were stimulated with the TLR2 ligand Pam3CSK4 and the TLR5 ligand flagellin, and the relative expression of several cytokines and transcription factors was quantified at 1, 3, 8, and 18 h poststimulation. The results suggest that both TLR ligands induce a mixed T(H)1- and T(H)2-like response, as characterized by the upregulation of both the T(H)1-associated cytokine interferon-γ and the T(H)1-inducing cytokine interleukin (IL)-12, in addition to the T(H)2-associated cytokine IL-4, and in the case of flagellin, IL-13 as well. Future studies may be aimed at assessing the adjuvant potential of these ligands.

Characterization of responses elicited by Toll-like receptor agonists in cells of the bursa of Fabricius in chickens.

Toll-like receptors (TLRs) are an evolutionarily conserved group of pattern recognition receptors that play an important role in mediating host-responses to pathogens. Several TLRs have been identified in chickens and their expression has been detected in many immune cell subsets including in B cells. However, the mechanisms through which TLRs modulate B cell responses have not been well characterized in chickens. The aim of the present study was to elucidate the responses mounted by cells of the bursa of Fabricius to treatment with the TLR 3, 4 and 21 ligands, poly I:C, lipopolysaccharide (LPS) and CpG oligodeoxynucleotides (ODN), respectively. The relative expression of several immune system genes was quantified at 1, 3, 8 and 18 h post-treatment. The results show that all three ligands induced the up-regulation of interferon (IFN)-γ and interleukin (IL)-10 transcripts and promoted the up-regulation of transcripts associated with antigen presentation, namely CD80 and major histocompatibility complex (MHC) class II. Furthermore, the results indicated that LPS and poly I:C induced the greatest IFN-γ and IL-10 responses, respectively, while CpG ODN was the most efficacious inducer of CD80 and MHC-II expression. Future studies may be aimed at elucidating the mechanisms of TLR-mediated activation of chicken B cells. These mechanisms may be then exploited for the development of adjuvants with enhanced ability to stimulate B cell responses.

Characterization of chicken thrombocyte responses to Toll-like receptor ligands.

Thrombocytes are the avian equivalent to mammalian platelets. In addition to their hemostatic effects, mammalian platelets rely in part on pattern recognition receptors, such as the Toll-like receptors (TLR), to detect the presence of pathogens and signal the release of certain cytokines. Ligands for TLRs include lipopolysaccharide (LPS), which is bound by TLR4, as well as unmethylated CpG DNA motifs, which are bound by TLR9 in mammals and TLR21 in chickens. Similar to mammalian platelets, avian thrombocytes have been shown to express TLR4 and secrete some pro-inflammatory cytokines in response to LPS treatment. However, the full extent of the contributions made by thrombocytes to host immunity has yet to be elucidated. Importantly, the mechanisms by which TLR stimulation may modulate thrombocyte effector functions have not been well characterized. As such, the objective of the present study was to gain further insight into the immunological role of thrombocytes by analyzing their responses to treatment with ligands for TLR4 and TLR21. To this end, we quantified the relative expression of several immune system genes at 1, 3, 8 and 18 hours post-treatment using real-time RT-PCR. Furthermore, production of nitric oxide and phagocytic activity of thrombocytes was measured after their activation with TLR ligands. We found that thrombocytes constitutively express transcripts for both pro- and anti-inflammatory cytokines, in addition to those associated with anti-viral responses and antigen presentation. Moreover, we found that both LPS and CpG oligodeoxynucleotides (ODN) induced robust pro-inflammatory responses in thrombocytes, as characterized by more than 100 fold increase in interleukin (IL)-1ß, IL-6 and IL-8 transcripts, while only LPS enhanced nitric oxide production and phagocytic capabilities. Future studies may be aimed at examining the responses of thrombocytes to other TLR ligands.