Prevalence of kidney health genetic variants in adults with sickle cell nephropathy.

The pathophysiology and genetic risk for sickle cell disease (SCD)-related chronic kidney disease (CKD) are not well understood. In 70 adults with SCD-related CKD and without APOL1 inherited in a high-risk pattern, 24 (34%) had pathogenic variants in candidate genes using KidneySeq™. A moderate impact INF2 variant was observed in 20 (29%) patients and those with 3 versus 0-2 pathogenic or moderate impact glomerular genetic variants had higher albuminuria and lower estimated glomerular filtration rate (adjusted p ≤ 0.015). Using a panel of preselected genes implicated in kidney health, we observed several variants in people with sickle cell nephropathy.

Targeted broad-based genetic testing by next-generation sequencing informs diagnosis and facilitates management in patients with kidney diseases.

BACKGROUND: The clinical diagnosis of genetic renal diseases may be limited by the overlapping spectrum of manifestations between diseases or by the advancement of disease where clues to the original process are absent. The objective of this study was to determine whether genetic testing informs diagnosis and facilitates management of kidney disease patients. METHODS: We developed a comprehensive genetic testing panel (KidneySeq) to evaluate patients with various phenotypes including cystic diseases, congenital anomalies of the kidney and urinary tract (CAKUT), tubulointerstitial diseases, transport disorders and glomerular diseases. We evaluated this panel in 127 consecutive patients ranging in age from newborns to 81 years who had samples sent in for genetic testing. RESULTS: The performance of the sequencing pipeline for single-nucleotide variants was validated using CEPH (Centre de'Etude du Polymorphism) controls and for indels using Genome-in-a-Bottle. To test the reliability of the copy number variant (CNV) analysis, positive samples were re-sequenced and analyzed. For patient samples, a multidisciplinary review board interpreted genetic results in the context of clinical data. A genetic diagnosis was made in 54 (43%) patients and ranged from 54% for CAKUT, 53% for ciliopathies/tubulointerstitial diseases, 45% for transport disorders to 33% for glomerulopathies. Pathogenic and likely pathogenic variants included 46% missense, 11% nonsense, 6% splice site variants, 23% insertion-deletions and 14% CNVs. In 13 cases, the genetic result changed the clinical diagnosis. CONCLUSION: Broad genetic testing should be considered in the evaluation of renal patients as it complements other tests and provides insight into the underlying disease and its management.

Screening of Living Kidney Donors for Genetic Diseases Using a Comprehensive Genetic Testing Strategy.

Related living kidney donors (LKDs) are at higher risk of end-stage renal disease (ESRD) compared with unrelated LKDs. A genetic panel was developed to screen 115 genes associated with renal diseases. We used this panel to screen six negative controls, four transplant candidates with presumed genetic renal disease and six related LKDs. After removing common variants, pathogenicity was predicted using six algorithms to score genetic variants based on conservation and function. All variants were evaluated in the context of patient phenotype and clinical data. We identified causal variants in three of the four transplant candidates. Two patients with a family history of autosomal dominant polycystic kidney disease segregated variants in PKD1. These findings excluded genetic risk in three of four relatives accepted as potential LKDs. A third patient with an atypical history for Alport syndrome had a splice site mutation in COL4A5. This pathogenic variant was excluded in a sibling accepted as an LKD. In another patient with a strong family history of ESRD, a negative genetic screen combined with negative comparative genomic hybridization in the recipient facilitated counseling of the related donor. This genetic renal disease panel will allow rapid, efficient and cost-effective evaluation of related LKDs.

High temperature is essential for preserved human sperm function during the devitrification process.

Sperm vitrification is a cryopreservation method based on high-speed freezing by direct exposure of cells in liquid nitrogen (N2L), thereby avoiding the traditional cooling curves of freezing. The objective of this work was to determine the optimal warming temperature for vitrified human spermatozoa in order to maintain their fertilisation potential. Spermatozoa were cryopreserved by direct plunging into N2L and warmed at different temperatures for 5 and 10 s at 38, 40 and 42 °C. Sperm motility was evaluated by the CASA system and the sperm membrane function by HOST test. It was detected that progressive motility of sperm warmed at 38, 40 and 42 °C was 26.4 ± 8.4%; 56.6 ± 16.3% and 65.4 ± 15%, respectively, with statistically significant differences between the temperatures of 38 and 40 °C and 38 and 42 °C (P < 0.05). The plasma membrane function evaluated by HOST test was better preserved at 42 °C (76.3 ± 2.0%) compared to 40 °C (43 ± 2%) and 38 °C (65.6 ± 1.5%). The temperature in the thawing process can affect the motility and plasma membrane integrity and function. The warming at 42 °C for thawed vitrified sperm is the optimum temperature to preserve the sperm physiological parameters.

Thymidine phosphorylase is both a therapeutic and a suicide gene in a murine model of mitochondrial neurogastrointestinal encephalomyopathy.

Suicide gene therapy (SGT) is a promising strategy for treating cancer. In this work, we show that thymidine phosphorylase (TP) deficiency, the underlying genetic defect in mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), presents an opportunity to apply SGT using capecitabine, a commonly used prodrug that is converted into 5-fluorouracil by TP. Using an immortalised B-lymphoblastoid cell line from a patient with MNGIE, the tumourigenic EL-4 cell line, lentiviral vectors encoding TP and a double knockout (Tymp(-/-)Upp1(-/-)) murine model, we found that EL-4 cell-derived TP(+) tumours were exquisitely sensitive to capecitabine and generated a significant local bystander effect. In addition, we detected a spontaneous cytolytic immune response in a significant fraction of the animals surviving more than 20 days after termination of the therapy. These data indicate that, in individuals lacking TP expression, TP is a highly specific suicide gene, which can be used to treat tumours that could hypothetically arise in MNGIE patients undergoing gene therapy, as these tumours will likely originate from the gene-modified cells and will be selectively targeted by capecitabine. These observations have important implications for gene therapy for MNGIE.

Anti-myelin antibodies play an important role in the susceptibility to develop proteolipid protein-induced experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is the most common demyelinating disease of the central nervous system. It is an autoimmune disorder in which activated T cells cross the blood-brain barrier (BBB) to initiate an inflammatory response that leads to demyelination and axonal damage. The key mechanisms responsible for disease initiation are still unknown. We addressed this issue in experimental autoimmune encephalomyelitis (EAE), the animal model of MS. It is widely known that EAE manifests only in certain strains when immunized with myelin proteins or peptides. We studied the differential immune responses induced in two mouse strains that are susceptible or resistant to EAE induction when they are immunized with the 139-151 peptide of proteolipid protein, an encephalitogenic peptide capable of inducing EAE in the susceptible strain. The adequate combination of major histocompatibility complex alleles and myelin peptides triggered in susceptible mice a T helper type 17 (Th17) response capable of inducing the production of high-affinity anti-myelin immunoglobulin (Ig)G antibodies. These were not detected in resistant mice, despite immunization with the encephalitogenic peptide in junction with complete Freund's adjuvant and pertussis toxin, which mediate BBB disruption. These data show the pivotal role of Th17 responses and of high-affinity anti-myelin antibodies in EAE induction and that mechanisms that prevent their appearance can contribute to resistance to EAE.

Suspected Autosomal Recessive Polycystic Kidney Disease but Cerebellar Vermis Hypoplasia, Oligophrenia Ataxia, Coloboma, and Hepatic Fibrosis (COACH) Syndrome in Retrospect, A Delayed Diagnosis Aided by Genotyping and Reverse Phenotyping: A Case Report and A Review of the Literature.

The clinical features of cerebellar vermis hypoplasia, oligophrenia, ataxia, coloboma, and hepatic fibrosis (COACH) characterize the rare autosomal recessive multisystem disorder called COACH syndrome. COACH syndrome belongs to the spectrum of Joubert syndrome and related disorders (JSRDs) and liver involvement distinguishes COACH syndrome from the rest of the JSRD spectrum. Developmental delay and oculomotor apraxia occur early but with time, these can improve and may not be readily apparent or no longer need active medical management. Congenital hepatic fibrosis and renal disease, on the other hand, may develop late, and the temporal incongruity in organ system involvement may delay the recognition of COACH syndrome. We present a case of a young adult presenting late to a Renal Genetics Clinic for evaluation of renal cystic disease with congenital hepatic fibrosis, clinically suspected to have autosomal recessive polycystic kidney disease. Following genetic testing, a reevaluation of his medical records from infancy, together with reverse phenotyping and genetic phasing, led to a diagnosis of COACH syndrome.

Confirming genes influencing risk to cleft lip with/without cleft palate in a case-parent trio study.

A collection of 1,108 case-parent trios ascertained through an isolated, nonsyndromic cleft lip with or without cleft palate (CL/P) was used to replicate the findings from a genome-wide association study (GWAS) conducted by Beaty et al. (Nat Genet 42:525-529, 2010), where four different genes/regions were identified as influencing risk to CL/P. Tagging SNPs for 33 different genes were genotyped (1,269 SNPs). All four of the genes originally identified as showing genome-wide significance (IRF6, ABCA4 and MAF, plus the 8q24 region) were confirmed in this independent sample of trios (who were primarily of European and Southeast Asian ancestry). In addition, eight genes classified as 'second tier' hits in the original study (PAX7, THADA, COL8A1/FILIP1L, DCAF4L2, GADD45G, NTN1, RBFOX3 and FOXE1) showed evidence of linkage and association in this replication sample. Meta-analysis between the original GWAS trios and these replication trios showed PAX7, COL8A1/FILIP1L and NTN1 achieved genome-wide significance. Tests for gene-environment interaction between these 33 genes and maternal smoking found evidence for interaction with two additional genes: GRID2 and ELAVL2 among European mothers (who had a higher rate of smoking than Asian mothers). Formal tests for gene-gene interaction (epistasis) failed to show evidence of statistical interaction in any simple fashion. This study confirms that many different genes influence risk to CL/P.

Expression and mutation analyses implicate ARHGAP29 as the etiologic gene for the cleft lip with or without cleft palate locus identified by genome-wide association on chromosome 1p22.

BACKGROUND: Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a common birth defect with complex etiology reflecting the action of multiple genetic and environmental factors. Genome-wide association studies have successfully identified five novel loci associated with NSCL/P, including a locus on 1p22.1 near the ABCA4 gene. Because neither expression analysis nor mutation screening support a role for ABCA4 in NSCL/P, we investigated the adjacent gene ARHGAP29. METHODS: Mutation screening for ARHGAP29 protein coding exons was conducted in 180 individuals with NSCL/P and controls from the United States and the Philippines. Nine exons with variants in ARHGAP29 were then screened in an independent set of 872 cases and 802 controls. Arhgap29 expression was evaluated using in situ hybridization in murine embryos. RESULTS: Sequencing of ARHGAP29 revealed eight potentially deleterious variants in cases including a frameshift and a nonsense variant. Arhgap29 showed craniofacial expression and was reduced in a mouse deficient for Irf6, a gene previously shown to have a critical role in craniofacial development. CONCLUSION: The combination of genome-wide association, rare coding sequence variants, craniofacial specific expression, and interactions with IRF6 support a role for ARHGAP29 in NSCL/P and as the etiologic gene at the 1p22 genome-wide association study locus for NSCL/P. This work suggests a novel pathway in which the IRF6 gene regulatory network interacts with the Rho pathway via ARHGAP29. Birth Defects Research (Part A) 2012. © 2012 Wiley Periodicals, Inc.

CRISPLD2 variants including a C471T silent mutation may contribute to nonsyndromic cleft lip with or without cleft palate.

OBJECTIVE: To assess the association between nonsyndromic (NS) cleft lip with or without cleft palate (CL(P)) and single-nucleotide polymorphisms (SNPs) within the CRISPLD2 gene (cysteine-rich secretory protein LCCL domain containing 2). DESIGN: Four SNPs within the CRISPLD2 gene domain (rs1546124, rs8061351, rs2326398, rs4783099) were genotyped to test for association via family-based association methods. PARTICIPANTS: A total of 5826 individuals from 1331 families in which one or more family member is affected with CL(P). RESULTS: Evidence of association was seen for SNP rs1546124 in U.S. (p  =  .02) and Brazilian (p  =  .04) Caucasian cohorts. We also found association of SNP rs1546124 with cleft palate alone (CP) in South Americans (Guatemala and ECLAMC) and combined Hispanics (Guatemala, ECLAMC, and Texas Hispanics; p  =  .03 for both comparisons) and with both cleft lip with cleft palate (CLP; p  =  .04) and CL(P) (p  =  .02) in North Americans. Strong evidence of association was found for SNP rs2326398 with CP in Asian populations (p  =  .003) and with CL(P) in Hispanics (p  =  .03) and also with bilateral CL(P) in Brazilians (p  =  .004). In Brazilians, SNP rs8061351 showed association with cleft subgroups incomplete CL(P) (p  =  .004) and unilateral incomplete CL(P) (p  =  .003). Prediction of SNP functionality revealed that the C allele in the C471T silent mutation (overrepresented in cases with CL(P) presents two putative exonic splicing enhancer motifs and creates a binding site AP-2 alpha, a transcription factor involved in craniofacial development. CONCLUSIONS: Our results support the hypothesis that variants in the CRISPLD2 gene may be involved in the etiology of NS CL(P).

Genetic studies in the Nigerian population implicate an MSX1 mutation in complex oral facial clefting disorders.

BACKGROUND: Orofacial clefts are the most common malformations of the head and neck, with a worldwide prevalence of 1 in 700 births. They are commonly divided into CL(P) and CP based on anatomic, genetic, and embryologic findings. A Nigerian craniofacial anomalies study (NigeriaCRAN) was set up in 2006 to investigate the role of gene-environment interaction in the origin of orofacial clefts in Nigeria. SUBJECTS AND METHODS: DNA isolated from saliva from Nigerian probands was used for genotype association studies and direct sequencing of cleft candidate genes: MSX1 , IRF6 , FOXE1, FGFR1 , FGFR2 , BMP4 , MAFB, ABCA4 , PAX7, and VAX1 , and the chromosome 8q region. RESULTS: A missense mutation A34G in MSX1 was observed in nine cases and four HapMap controls. No other apparent causative variations were identified. Deviation from Hardy Weinberg equilibrium (HWE) was observed in these cases (p = .00002). A significant difference was noted between the affected side for unilateral CL (p = .03) and bilateral clefts and between clefts on either side (p = .02). A significant gender difference was also observed for CP (p = .008). CONCLUSIONS: Replication of a mutation previously implicated in other populations suggests a role for the MSX1 A34G variant in the development of CL(P).

Paving the way towards an effective treatment for multiple sclerosis: advances in cell therapy.

Multiple sclerosis (MS) is a leading cause of chronic neurological disability in young to middle-aged adults, affecting ~2.5 million people worldwide. Currently, most therapeutics for MS are systemic immunosuppressive or immunomodulatory drugs, but these drugs are unable to halt or reverse the disease and have the potential to cause serious adverse events. Hence, there is an urgent need for the development of next-generation treatments that, alone or in combination, stop the undesired autoimmune response and contribute to the restoration of homeostasis. This review analyzes current MS treatments as well as different cell-based therapies that have been proposed to restore homeostasis in MS patients (tolerogenic dendritic cells, regulatory T cells, mesenchymal stem cells, and vaccination with T cells). Data collected from preclinical studies performed in the experimental autoimmune encephalomyelitis (EAE) model of MS in animals, in vitro cultures of cells from MS patients and the initial results of phase I/II clinical trials are analyzed to better understand which parameters are relevant for obtaining an efficient cell-based therapy for MS.

Thermal hysteresis of Morin transition in hematite particles.

Rhombohedral shaped, single crystal hematite particles with narrow size distribution (D(TEM) = 93 +/- 2 nm) were prepared by hydrolysis of iron chloride and polymerisation in water. The results of field dependent magnetization measurements at different warming-cooling rates and ac susceptibility measurements at varying frequencies are reported and discussed. Thermal hysteresis (DeltaT(M)) associated with the Morin transition and field dependence of the Morin temperature (T(M)) are observed in warming-cooling cycles (DeltaT(M) = 25 and 13 K for H = 0.1 and 3 T, respectively) due to the first order phase transition. A frequency dependence of ac susceptibility is observed above T(M), as a result of the relaxation of the magnetic moment of hematite particles in the weak-ferromagnetic phase.

Genome scan, fine-mapping, and candidate gene analysis of non-syndromic cleft lip with or without cleft palate reveals phenotype-specific differences in linkage and association results.

OBJECTIVES: Non-syndromic orofacial clefts, i.e. cleft lip (CL) and cleft palate (CP), are among the most common birth defects. The goal of this study was to identify genomic regions and genes for CL with or without CP (CL/P). METHODS: We performed linkage analyses of a 10 cM genome scan in 820 multiplex CL/P families (6,565 individuals). Significant linkage results were followed by association analyses of 1,476 SNPs in candidate genes and regions, utilizing a weighted false discovery rate (wFDR) approach to control for multiple testing and incorporate the genome scan results. RESULTS: Significant (multipoint HLOD >or=3.2) or genome-wide-significant (HLOD >or=4.02) linkage results were found for regions 1q32, 2p13, 3q27-28, 9q21, 12p11, 14q21-24 and 16q24. SNPs in IRF6 (1q32) and in or near FOXE1 (9q21) reached formal genome-wide wFDR-adjusted significance. Further, results were phenotype dependent in that the IRF6 region results were most significant for families in which affected individuals have CL alone, and the FOXE1 region results were most significant in families in which some or all of the affected individuals have CL with CP. CONCLUSIONS: These results highlight the importance of careful phenotypic delineation in large samples of families for genetic analyses of complex, heterogeneous traits such as CL/P.

Minimal Change Disease With Nephrotic Syndrome Associated With Coronavirus Disease 2019 After Apolipoprotein L1 Risk Variant Kidney Transplant: A Case Report.

Kidney injury is a well-known complication in people with coronavirus disease 2019 (COVID-19). In kidney transplant recipients with COVID-19, presentation with nephrotic syndrome has not been well described. We report on a 49-year-old black female kidney transplant recipient who presented 25 years after transplant with clinical features of nephrotic syndrome following a diagnosis of COVID-19. Histologic examination showed acute tubular injury with unremarkable glomeruli on light microscopy and diffuse foot process effacement of podocytes on electron microscopy, consistent with minimal change-like podocyte injury. Apolipoprotein L1 (APOL1) genetic testing confirmed 2 high-risk APOL1 alleles in the kidney donor. We speculate that COVID-19-induced systemic or local cytokine release could serve as a second hit in the presence of APOL1 risk alleles and mediate a podocytopathy manifesting as nephrotic syndrome. The presented case with minimal change-like disease, occurring in the context of the donor high-risk APOL1 genotype, extends the spectrum of clinical manifestations in COVID-19-associated nephropathy.

Identification of novel candidate genes associated with cleft lip and palate using array comparative genomic hybridisation.

AIM AND METHOD: We analysed DNA samples isolated from individuals born with cleft lip and cleft palate to identify deletions and duplications of candidate gene loci using array comparative genomic hybridisation (array-CGH). RESULTS: Of 83 syndromic cases analysed we identified one subject with a previously unknown 2.7 Mb deletion at 22q11.21 coinciding with the DiGeorge syndrome region. Eighteen of the syndromic cases had clinical features of Van der Woude syndrome and deletions were identified in five of these, all of which encompassed the interferon regulatory factor 6 (IRF6) gene. In a series of 104 non-syndromic cases we found one subject with a 3.2 Mb deletion at chromosome 6q25.1-25.2 and another with a 2.2 Mb deletion at 10q26.11-26.13. Analyses of parental DNA demonstrated that the two deletion cases at 22q11.21 and 6q25.1-25.2 were de novo, while the deletion of 10q26.11-26.13 was inherited from the mother, who also has a cleft lip. These deletions appear likely to be causally associated with the phenotypes of the subjects. Estrogen receptor 1 (ESR1) and fibroblast growth factor receptor 2 (FGFR2) genes from the 6q25.1-25.2 and 10q26.11-26.13, respectively, were identified as likely causative genes using a gene prioritization software. CONCLUSION: We have shown that array-CGH analysis of DNA samples derived from cleft lip and palate subjects is an efficient and productive method for identifying candidate chromosomal loci and genes, complementing traditional genetic mapping strategies.

Comparison of dendritic cells obtained from autoimmunty-prone and resistant rats.

Dendritic cells (DC) are responsible for the initiation and shaping of the adaptive immune response and are in the focus of autoimmunity research. We were interested in comparison of DC obtained from autoimmunity-prone Dark Agouti (DA) rats and autoimmunity-resistant Albino Oxford (AO) rats. DC were generated from bone marrow precursors and matured (mDC) by lipopolysaccharide. Tolerogenic DC (tolDC) obtained by vitamin D3 treatment were studied in parallel. Profile of cytokine production was different in AO and DA mDC and tolDC. Expression of MHC class II molecules and CD86 were higher in DA DC, while vitamin D3 reduced their expression in dendritic cells of both strains. Allogeneic proliferation of CD4+ T cells was reduced by AO tolDC, but not with DA tolDC in comparison to respective mDC. Finally, expression of various genes identified as differentially expressed in human mDC and tolDC was also analyzed in AO and DA DC. Again, AO and DA DC differed in the expression of the analyzed genes. To conclude, AO and DA DC differ in production of cytokines, expression of antigen presentation-related molecules and in regulation of CD4+ T proliferation. The difference is valuable for understanding the divergence of the strains in their susceptibility to autoimmunity.

Tolerogenic dendritic cell-based treatment for multiple sclerosis (MS): a harmonised study protocol for two phase I clinical trials comparing intradermal and intranodal cell administration.

INTRODUCTION: Based on the advances in the treatment of multiple sclerosis (MS), currently available disease-modifying treatments (DMT) have positively influenced the disease course of MS. However, the efficacy of DMT is highly variable and increasing treatment efficacy comes with a more severe risk profile. Hence, the unmet need for safer and more selective treatments remains. Specifically restoring immune tolerance towards myelin antigens may provide an attractive alternative. In this respect, antigen-specific tolerisation with autologous tolerogenic dendritic cells (tolDC) is a promising approach. METHODS AND ANALYSIS: Here, we will evaluate the clinical use of tolDC in a well-defined population of MS patients in two phase I clinical trials. In doing so, we aim to compare two ways of tolDC administration, namely intradermal and intranodal. The cells will be injected at consecutive intervals in three cohorts receiving incremental doses of tolDC, according to a best-of-five design. The primary objective is to assess the safety and feasibility of tolDC administration. For safety, the number of adverse events including MRI and clinical outcomes will be assessed. For feasibility, successful production of tolDC will be determined. Secondary endpoints include clinical and MRI outcome measures. The patients' immune profile will be assessed to find presumptive evidence for a tolerogenic effect in vivo. ETHICS AND DISSEMINATION: Ethics approval was obtained for the two phase I clinical trials. The results of the trials will be disseminated in a peer-reviewed journal, at scientific conferences and to patient associations. TRIAL REGISTRATION NUMBERS: NCT02618902 and NCT02903537; EudraCT numbers: 2015-002975-16 and 2015-003541-26.

Dual promoters control expression of the Bacillus anthracis virulence factor AtxA.

The AtxA virulence regulator of Bacillus anthracis is required for toxin and capsule gene expression. AtxA is a phosphotransferase system regulatory domain-containing protein whose activity is regulated by phosphorylation/dephosphorylation of conserved histidine residues. Here we report that transcription of the atxA gene occurs from two independent promoters, P1 (previously described by Dai et al. [Z. Dai, J. C. Sirard, M. Mock, and T. M. Koehler, Mol. Microbiol. 16:1171-1181, 1995]) and P2, whose transcription start sites are separated by 650 bp. Both promoters have -10 and -35 consensus sequences compatible with recognition by sigma(A)-containing RNA polymerase, and neither promoter depends on the sporulation sigma factor SigH. The dual promoter activity and the extended untranslated mRNA suggest that as-yet-unknown regulatory mechanisms may act on this region to influence the level of AtxA in the cell.