Expanding the allelic spectrum of ELOVL4-related autosomal recessive neuro-ichthyosis.

BACKGROUND: Very long-chain fatty acids (VLCFAs) composed of more than 20 carbon atoms are essential in the biosynthesis of cell membranes in the brain, skin, and retina. VLCFAs are elongated beyond 28 carbon atoms by ELOVL4 enzyme. Variants in ELOVL4 are associated with three Mendelian disorders: autosomal dominant (AD) Stargardt-like macular dystrophy type 3, AD spinocerebellar ataxia, and autosomal recessive disorder congenital ichthyosis, spastic quadriplegia and impaired intellectual development (ISQMR). Only seven subjects from five unrelated families with ISQMR have been described, all of which have biallelic single-nucleotide variants. METHODS: We performed clinical exome sequencing on probands from four unrelated families with neuro-ichthyosis. RESULTS: We identified three novel homozygous ELOVL4 variants. Two of the families originated from the same Saudi tribe and had the exact homozygous exonic deletion in ELOVL4, while the third and fourth probands had two different novel homozygous missense variants. Seven out of the eight affected subjects had profound developmental delay, epilepsy, axial hypotonia, peripheral hypertonia, and ichthyosis. Delayed myelination and corpus callosum hypoplasia were seen in two of five subjects with brain magnetic rosonance imaging and cerebral atrophy in three. CONCLUSION: Our study expands the allelic spectrum of ELOVL4-related ISQMR. The detection of the same exonic deletion in two unrelated Saudi family from same tribe suggests a tribal founder mutation.

T cells of colorectal cancer patients' stimulated by neoantigenic and cryptic peptides better recognize autologous tumor cells.

BACKGROUND: Patients with cancers that exhibit extraordinarily high somatic mutation numbers are ideal candidates for immunotherapy and enable identifying tumor-specific peptides through stimulation of tumor-reactive T cells (Tc). METHODS: Colorectal cancers (CRC) HROC113 and HROC285 were selected based on high TMB, microsatellite instability and HLA class I expression. Their HLA ligandome was characterized using mass spectrometry, compared with the HLA ligand atlas and HLA class I-binding affinity was predicted. Cryptic peptides were identified using Peptide-PRISM. Patients' Tc were isolated from either peripheral blood (pTc) or tumor material (tumor-infiltrating Tc, TiTc) and expanded. In addition, B-lymphoblastoid cells (B-LCL) were generated and used as antigen-presenting cells. pTc and TiTc were stimulated twice for 7 days using peptide pool-loaded B-LCL. Subsequently, interferon gamma (IFNγ) release was quantified by ELISpot. Finally, cytotoxicity against autologous tumor cells was assessed in a degranulation assay. RESULTS: 100 tumor-specific candidate peptides-97 cryptic peptides and 3 classically mutated neoantigens-were selected. The neoantigens originated from single nucleotide substitutions in the genes IQGAP1, CTNNB1, and TRIT1. Cryptic and neoantigenic peptides inducing IFNγ secretion of Tc were further investigated. Stimulation of pTc and TiTc with neoantigens and selected cryptic peptides resulted in increased release of cytotoxic granules in the presence of autologous tumor cells, substantiating their improved tumor cell recognition. Tetramer staining showed an enhanced number of pTc and TiTc specific for the IQGAP1 neoantigen. Subpopulation analysis prior to peptide stimulation revealed that pTc mainly consisted of memory Tc, whereas TiTc constituted primarily of effector and effector memory Tc. This allows to infer that TiTc reacting to neoantigens and cryptic peptides must be present within the tumor microenvironment. CONCLUSION: These results prove that the analyzed CRC present both mutated neoantigenic and cryptic peptides on their HLA class I molecules. Moreover, stimulation with these peptides significantly strengthened tumor cell recognition by Tc. Since the overall number of neoantigenic peptides identifiable by HLA ligandome analysis hitherto is small, our data emphasize the relevance of increasing the target scope for cancer vaccines by the cryptic peptide category.

Suleiman-El-Hattab syndrome: a histone modification disorder caused by TASP1 deficiency.

BACKGROUND: TASP1 encodes an endopeptidase activating histone methyltransferases of the KMT2 family. Homozygous loss-of-function variants in TASP1 have recently been associated with Suleiman-El-Hattab syndrome. We report six individuals with Suleiman-El-Hattab syndrome and provide functional characterization of this novel histone modification disorder in a multi-omics approach. METHODS: Chromosomal microarray/exome sequencing in all individuals. Western blotting from fibroblasts in two individuals. RNA sequencing and proteomics from fibroblasts in one individual. Methylome analysis from blood in two individuals. Knock-out of tasp1 orthologue in zebrafish and phenotyping. RESULTS: All individuals had biallelic TASP1 loss-of-function variants and a phenotype including developmental delay, multiple congenital anomalies (including cardiovascular and posterior fossa malformations), a distinct facial appearance and happy demeanor. Western blot revealed absence of TASP1. RNA sequencing/proteomics showed HOX gene downregulation (HOXA4, HOXA7, HOXA1 and HOXB2) and dysregulation of transcription factor TFIIA. A distinct methylation profile intermediate between control and Kabuki syndrome (KMT2D) profiles could be produced. Zebrafish tasp1 knock-out revealed smaller head size and abnormal cranial cartilage formation in tasp1 crispants. CONCLUSION: This work further delineates Suleiman-El-Hattab syndrome, a recognizable neurodevelopmental syndrome. Possible downstream mechanisms of TASP1 deficiency include perturbed HOX gene expression and dysregulated TFIIA complex. Methylation pattern suggests that Suleiman-El-Hattab syndrome can be categorized into the group of histone modification disorders including Wiedemann-Steiner and Kabuki syndrome.

Novel Autosomal Recessive Splice-Altering Variant in PRKD1 Is Associated with Congenital Heart Disease.

Congenital heart defects (CHDs) are the most common types of birth defects, and global incidence of CHDs is on the rise. Despite the prevalence of CHDs, the genetic determinants of the defects are still in the process of being identified. Herein, we report a consanguineous Saudi family with three CHD affected daughters. We used whole exome sequencing (WES) to investigate the genetic cause of CHDs in the affected daughters. We found that all affected individuals were homozygous for a novel splice-altering variant (NM_001330069.1: c.265-1G>T) of PRKD1, which encodes a calcium/calmodulin-dependent protein kinase in the heart. The homozygous variant was found in the affected patients with Pulmonary Stenosis (PS), Truncus Arteriosis (TA), and Atrial Septal Defect (ASD). Based on the family's pedigree, the variant acts in an autosomal recessive manner, which makes it the second autosomal recessive variant of PRKD1 to be identified with a link to CHDs, while all other previously described variants act dominantly. Interestingly, the father of the affected daughters was also homozygous for the variant, though he was asymptomatic of CHDs himself. Since both of his sisters had CHDs as well, this raises the possibility that the novel PRKD1 variant may undergo autosomal recessive inheritance mode with gender limitation. This finding confirms that CHD can be associated with both dominant and recessive mutations of the PRKD1 gene, and it provides a new insight to genotype-phenotype association between PRKD1 and CHDs. To our knowledge, this is the first report of this specific PRKD1 mutation associated with CHDs.

Successful application of genome sequencing in a diagnostic setting: 1007 index cases from a clinically heterogeneous cohort.

Despite clear technical superiority of genome sequencing (GS) over other diagnostic methods such as exome sequencing (ES), few studies are available regarding the advantages of its clinical application. We analyzed 1007 consecutive index cases for whom GS was performed in a diagnostic setting over a 2-year period. We reported pathogenic and likely pathogenic (P/LP) variants that explain the patients' phenotype in 212 of the 1007 cases (21.1%). In 245 additional cases (24.3%), a variant of unknown significance (VUS) related to the phenotype was reported. We especially investigated patients which had had ES with no genetic diagnosis (n = 358). For this group, GS diagnostic yield was 14.5% (52 patients with P/LP out of 358). GS should be especially indicated for ES-negative cases since up to 29.6% of them  could benefit from GS testing (14.5% with P/LP, n = 52 and 15.1% with VUS, n = 54). Genetic diagnoses in most of the ES-negative/GS-positive cases were determined by technical superiority of GS, i.e., access to noncoding regions and more uniform coverage. Importantly, we reported 79 noncoding variants, of which, 41 variants were classified as P/LP. Interpretation of noncoding variants remains challenging, and in many cases, complementary methods based on direct enzyme assessment, biomarker testing and RNA analysis are needed for variant classification and diagnosis. We present the largest cohort of patients with GS performed in a clinical setting to date. The results of this study should direct the decision for GS as standard second-line, or even first-line stand-alone test.

Fetuin-A aggravates lipotoxicity in podocytes via interleukin-1 signaling.

Sterile inflammation is considered critical in the pathogenesis of diabetic nephropathy (DN). Here we show that Fetuin-A (FetA) or lipopolysaccharide (LPS) exacerbate palmitic acid-induced podocyte death, which is associated with a strong induction of monocyte chemoattractant protein-1 (MCP-1) and keratinocyte chemoattractant (KC). Moreover, blockage of TLR4 prevents MCP-1 and KC secretion and attenuates podocyte death induced by palmitic acid alone or combined with FetA. In addition, inhibition of interleukin-1 (IL-1) signaling by anakinra, a recombinant human IL-1Ra, or a murinized anti-IL-1ß antibody attenuates the inflammatory and ultimate cell death response elicited by FetA alone or combined with palmitic acid. In vivo short-term therapy of diabetic DBA/2J mice with an anti-IL1-ß antibody for 4 weeks prevented an increase in serum FetA and considerably decreased urinary tumor necrosis alpha (TNF-α), a known risk factor for DN progression. In summary, our results suggest that FetA similarly to LPS leads to an inflammatory response in podocytes, which exacerbates palmitic acid-induced podocyte death and our data imply a critical role for IL-1ß signaling in this process. The study offers the rational for prolonged in vivo studies aimed at testing anti-IL-1ß therapy for prevention and treatment of DN.

Angiotensin II induces interleukin-1ß-mediated islet inflammation and ß-cell dysfunction independently of vasoconstrictive effects.

Pathological activation of the renin-angiotensin system (RAS) is associated with the metabolic syndrome, and the new onset of type 2 diabetes can be delayed by RAS inhibition. In animal models of type 2 diabetes, inhibition of the RAS improves insulin secretion. However, the direct effects of angiotensin II on islet function and underlying mechanisms independent of changes in blood pressure remain unclear. Here we show that exposure of human and mouse islets to angiotensin II induces interleukin (IL)-1-dependent expression of IL-6 and MCP-1, enhances ß-cell apoptosis, and impairs mitochondrial function and insulin secretion. In vivo, mice fed a high-fat diet and treated with angiotensin II and the vasodilator hydralazine to prevent hypertension showed defective glucose-stimulated insulin secretion and deteriorated glucose tolerance. Application of an anti-IL-1ß antibody reduced the deleterious effects of angiotensin II on islet inflammation, restored insulin secretion, and improved glycemia. We conclude that angiotensin II leads to islet dysfunction via induction of inflammation and independent of vasoconstriction. Our findings reveal a novel role for the RAS and an additional rationale for the treatment of type 2 diabetic patients with an IL-1ß antagonist.

Susceptibility of podocytes to palmitic acid is regulated by fatty acid oxidation and inversely depends on acetyl-CoA carboxylases 1 and 2.

Type 2 diabetes is characterized by dyslipidemia with elevated free fatty acids (FFAs). Loss of podocytes is a hallmark of diabetic nephropathy, and podocytes are susceptible to saturated FFAs, which induce endoplasmic reticulum (ER) stress and podocyte death. Genome-wide association studies indicate that expression of acetyl-CoA carboxylase (ACC) 2, a key enzyme of fatty acid oxidation (FAO), is associated with proteinuria in type 2 diabetes. Here, we show that stimulation of FAO by aminoimidazole-4-carboxamide-1ß-D-ribofuranoside (AICAR) or by adiponectin, activators of the low-energy sensor AMP-activated protein kinase (AMPK), protects from palmitic acid-induced podocyte death. Conversely, inhibition of carnitine palmitoyltransferase (CPT-1), the rate-limiting enzyme of FAO and downstream target of AMPK, augments palmitic acid toxicity and impedes the protective AICAR effect. Etomoxir blocked the AICAR-induced FAO measured with tritium-labeled palmitic acid. The beneficial effect of AICAR was associated with a reduction of ER stress, and it was markedly reduced in ACC-1/-2 double-silenced podocytes. In conclusion, the stimulation of FAO by modulating the AMPK-ACC-CPT-1 pathway may be part of a protective mechanism against saturated FFAs that drive podocyte death. Further studies are needed to investigate the potentially novel therapeutic implications of these findings.

Susceptibility of podocytes to palmitic acid is regulated by stearoyl-CoA desaturases 1 and 2.

Type 2 diabetes mellitus is characterized by dyslipidemia with elevated free fatty acids (FFAs). Loss of podocytes is a hallmark of diabetic nephropathy, and podocytes are highly susceptible to saturated FFAs but not to protective, monounsaturated FFAs. We report that patients with diabetic nephropathy develop alterations in glomerular gene expression of enzymes involved in fatty acid metabolism, including induction of stearoyl-CoA desaturase (SCD)-1, which converts saturated to monounsaturated FFAs. By IHC of human renal biopsy specimens, glomerular SCD-1 induction was observed in podocytes of patients with diabetic nephropathy. Functionally, the liver X receptor agonists TO901317 and GW3965, two known inducers of SCD, increased Scd-1 and Scd-2 expression in cultured podocytes and reduced palmitic acid-induced cell death. Similarly, overexpression of Scd-1 attenuated palmitic acid-induced cell death. The protective effect of TO901317 was associated with a reduction of endoplasmic reticulum stress. It was lost after gene silencing of Scd-1/-2, thereby confirming that the protective effect of TO901317 is mediated by Scd-1/-2. TO901317 also shifted palmitic acid-derived FFAs into biologically inactive triglycerides. In summary, SCD-1 up-regulation in diabetic nephropathy may be part of a protective mechanism against saturated FFA-derived toxic metabolites that drive endoplasmic reticulum stress and podocyte death.

Regulation of podocyte survival and endoplasmic reticulum stress by fatty acids.

Apoptosis of podocytes is considered critical in the pathogenesis of diabetic nephropathy (DN). Free fatty acids (FFAs) are critically involved in the pathogenesis of diabetes mellitus type 2, in particular the regulation of pancreatic ß cell survival. The objectives of this study were to elucidate the role of palmitic acid, palmitoleic, and oleic acid in the regulation of podocyte cell death and endoplasmic reticulum (ER) stress. We show that palmitic acid increases podocyte cell death, both apoptosis and necrosis of podocytes, in a dose and time-dependent fashion. Palmitic acid induces podocyte ER stress, leading to an unfolded protein response as reflected by the induction of the ER chaperone immunoglobulin heavy chain binding protein (BiP) and proapoptotic C/EBP homologous protein (CHOP) transcription factor. Of note, the monounsaturated palmitoleic and oleic acid can attenuate the palmitic acid-induced upregulation of CHOP, thereby preventing cell death. Similarly, gene silencing of CHOP protects against palmitic acid-induced podocyte apoptosis. Our results offer a rationale for interventional studies aimed at testing whether dietary shifting of the FFA balance toward unsaturated FFAs can delay the progression of DN.