Heterozygous loss-of-function DHX9 variants are associated with neurodevelopmental disorders: Human genetic and experimental evidences

DExH-box helicases are involved in unwinding of RNA and DNA. Among the 16 DExH-box genes, monoallelic variants of DHX16, DHX30, DHX34, and DHX37 are known to be associated with neurodevelopmental disorders. In particular, DHX30 is well established as a causative gene for neurodevelopmental disorders. Germline variants of DHX9, the closest homolog of DHX30, have not been reported until now as being associated with congenital disorders in humans, except that one de novo heterozygous variant, p.(Arg1052Gln) of the gene was identified during comprehensive screening in a patient with autism; unfortunately, the phenotypic details of this individual are unknown. Herein, we report a patients with a heterozygous de novo missense variant, p.(Gly414Arg) of DHX9 who presented with a short stature, intellectual disability, and ventricular non-compaction cardiomyopathy. The variant was located in the glycine codon of the ATP-binding site, G-C-G-K-T. To assess the pathogenicity of these variants, we generated transgenic Drosophila lines expressing human wild-type and mutant DHX9 proteins: 1) the mutant proteins showed aberrant localization both in the nucleus and the cytoplasm; 2) ectopic expression of wild-type protein in the visual system led to the rough eye phenotype, whereas expression of the mutant proteins had minimal effect; 3) overexpression of the wild-type protein in the retina led to a reduction in axonal numbers, whereas expression of the mutant proteins had a less pronounced effect. Furthermore, in a gene-editing experiment of Dhx9 G416 to R416, corresponding to p.(Gly414Arg) in humans, heterozygous mice showed a reduced body size, reduced emotionality, and cardiac conduction abnormality. In conclusion, we established that heterozygosity for a loss-of-function variant of DHX9 can lead to a new neurodevelopmental disorder.     

Various phenotypes of disease associated with mutated DGKE gene

Atypical haemolytic uraemic syndrome and steroid-resistant nephrotic syndrome are highly rare kidney diseases that can occur in childhood. In some cases, genetic variants may trigger these conditions, although in atypical haemolytic uraemic syndrome they mostly confer only a predisposition to the disease. Most variants causing atypical haemolytic uraemic syndrome were identified in genes encoding proteins regulating the complement pathway; on the other hand, there are approximately 58 genes encoding distinct proteins primarily causing steroid-resistant nephrotic syndrome. We present a child with steroid-resistant nephrotic syndrome and a confirmed homozygous c.966G > A, p.Trp322Ter pathogenic variant in DGKE. This variant was also found in compound with a novel DGKE heterozygous deletion c.171delG, p.Ser58Alafs*111 in a patient from our paediatric cohort with atypical haemolytic uraemic syndrome. Both cases presented with hypertension, nephrotic proteinuria and severe acute kidney injury followed by renal recovery; however, their renal histology was different. In this paper, we deal with the clinical course of children with disrupted DGKE, including the steroid-resistant nephrotic syndrome and atypical haemolytic uraemic syndrome overlap.     

ATP7B variant spectrum in a French pediatric Wilson disease cohort

Background/aimThe spectrum of ATP7B variants varies significantly according to geographic distribution, and there is insufficient data on the variants observed in the French population.MethodsClinical data of 113 children included in the French WD national registry were gathered from March 01, 1995 to July 01, 2020. Data included epidemiological, clinical, laboratory, genetics.ResultsDiagnosis was made at a mean age of 11.0 ± 4.1 years (range 1–18 years). At diagnosis, 91 patients (79.8 %) had hepatic manifestations, 18 (15.8 %) presented neurological manifestations, and 4 patients (3.5 %) were asymptomatic. Only 29 patients (25 %) were homozygous for a variant. We have found a total of 102 different variants including 14 novel variants. Recurrent variant p.His1069Gln was the most prevalent, n = 31 alleles (14,2%), with only seven homozygous; in contrast 55% of variants are identified in only one family. 45% were truncating variants. In respect of mutated exon, the three most prevalent were exon 14 (16.5%), exon 8 (13.8%), and exon 3 (11.5%). When considering patients with two Nonsense / Frameshift variants as a group and those with two Missense variants, we found significantly lower ceruloplasmin for the former: 2.8 ± 0.7 mg/dl vs 8.4 ± 5mg/dl (p<0.05).Conclusionp.His1069Gln is the most frequent variant (14,2%) and exons 14, 8, and 2 of the ATP7B gene account for 41.7% of total variants. However, there is significant heterogeneity in the French population concerning the other ATP7B variants. Nonsense / Frameshift variants were associated with lower ceruloplasmin levels.     

Phenotypic overlap between cardioacrofacial dysplasia-2 and oral-facial-digital syndrome

Oral-facial digital (OFD) syndrome is characterized by abnormalities of the face (hypertelorism and low set-ears), oral cavity (multiple frenula, lingual hamartoma, or lobulated tongue) and extremities (postaxial polydactyly). At least 19 genes have been implicated in the development of OFD syndrome. Herein, we report the case a 13-year-old patient with atrioventricular septal defect, moderate intellectual disability, epilepsy, and features of OFD, including multiple oral frenula, and postaxial polydactyly of the hands and feet. The patient had a de novo heterozygous variant in PRKACB: chr1(GRCh37):g.84700915T > C, c.1124T > C (NM_182948.4), p.(Phe375Ser). To date, four patients with pathogenic monoallelic variants in PRKACB have been reported, and the condition associated with these variants is referred to as Cardioacrofacial dysplasia-2 (CAFD2, MIM619143). Previously reported features of this condition include congenital heart disease (e.g., atrioventricular septal defect) and postaxial polydactyly, and two of the patients had multiple oral frenula. We suggest that a significant phenotypic overlap exists between CAFD2 and OFD syndrome, in that these patients especially share the features of postaxial polydactyly and multiple oral frenula. The phenotypic similarity between patients with CAFD2 and classic OFD syndrome with an OFD1 variant might be explained by the recent in vitro experimental finding that a protein kinase A subunit encoded by PRKACB directly phosphorylates the OFD1 protein. From the standpoint of genetic counseling, OFD syndrome type1, the prototypic form of OFD, exhibits an X-linked dominant inheritance pattern, whereas other forms of OFD syndrome exhibit an autosomal recessive inheritance pattern. Recognition of CAFD2 as a differential diagnosis or forme fruste of OFD syndrome suggests that an autosomal dominant pattern of inheritance should also be considered during genetic counseling.     

The outcome of targeted NGS screening in patients with syndromic forms of sagittal and pansynostosis - IL11RA is an emerging core-gene for pansynostosis

Here, we have studied the prevalence and spectrum of genetic alterations in syndromic forms of sagittal and pansynostosis. Eighteen patients with sagittal synostosis (isolated or combined with other synostoses, except coronal) or pansynostosis were phenotypically assessed by retrospective analysis of medical records, three-dimensional computed tomography skull reconstructions, and registered photos. Patient DNAs were analyzed using a targeted next-generation sequencing (NGS) panel including 63 craniosynostosis (CS) related genes. Pathogenic and likely pathogenic variants were found in 72% of the cases, mainly affecting FGFR2, TWIST1, IL11RA, and SKI. Two patients that were negative at NGS screening – one with a supernumerary marker chromosome with duplication of 15q25.2q26.3 and one with a pathogenic PHEX variant – were identified using microarray and single gene analysis, respectively. The overall diagnostic rate in the cohort was thus 83%. We identified two novel likely pathogenic variants in FGFR2 (NM_022970.3: c.811_812delGGinsCC, p.Gly271Pro) and TWIST1 (NM_000474.3: c.476T > A, p.Leu159His), and a novel variant of unclear phenotypic significance in RUNX2 (NM_001024630.3: c.340G > A, p.Val114Ile) which could suggest a modulatory effect. Notably, we also identified three new patients with pansynostosis and a Crouzon-like phenotype with IL11RA mutation. Targeted NGS using a broad panel of CS-related genes is a simple and powerful tool for detecting pathogenic mutations in patients with syndromic forms of CS and multiple suture involvement, in particular pansynostosis. Our results provide additional evidence of an association between pansynostosis and IL11RA, an emerging core gene for autosomal recessive CS.     

Interrogation of selected genes influencing serum LDL-Cholesterol levels in patients with well characterized NAFLD

BackgroundThe clinical significance of rare mutations in LDL metabolism genes on nonalcoholic fatty liver disease (NAFLD) severity is not well understood.ObjectiveTo examine the significance of mutations in LDL metabolism genes including apolipoprotein B (APOB), proprotein convertase subtilisin kexin 9 (PCSK9) and LDL receptor (LDLR) in patients with NAFLD.MethodsPatients with biopsy-confirmed NAFLD from the NASH Clinical Research Network studies were stratified into 3 groups of LDL-C (≤50 mg/dL, 130–150 mg/dL, ≥ 190 mg/dL) and then 120 (40 per group) were randomly selected from the strata. We examined the presence of mutations on LDL genes and analyzed its association with selected NAFLD-related features. Multivariable analyses were adjusted for age, race, gender and use of statins.ResultsAmong 40 patients with LDL-C ≤ 50 mg/dL, 7 (18%) patients had heterozygous variants in APOB and 2 had heterozygous variants in PCSK9 (5%). We also found heterozygous mutations in 3 (8%) patients with LDL-C ≥ 190 mg/dL; 2 and 1 located in LDLR and APOE genes, respectively. Compared to wild-type controls with LDL-C ≤ 50, APOB carriers displayed higher levels of alanine aminotransferase (85.86 ± 35.14 U/L vs 45.61 ± 20.84 U/L, Adj. P = 0.002) and steatosis >66% (57% vs 24%, Adj. P = 0.050). These associations remained statistically significant after excluding statin users. Other histological features of NAFLD severity were not different between wild-type controls and APOB mutation carriers.ConclusionMutations in the APOB gene are common among NAFLD patients with very low LDL-C and may be associated with increased aminotransferase levels and steatosis severity.     

A splice site and copy number variant responsible for TTC25-related primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder of motile cilia. With few exceptions, PCD is an autosomal recessive condition, and there are over 40 genes associated with the condition. We present a case of a newborn female with clinical features of PCD, specifically the Kartagener syndrome phenotype, due to variants in TTC25. This gene has been previously associated with PCD in three families. Two multi-gene panels performed as a neonate and at two years of age were uninformative. Exome sequencing was performed by the Care4Rare Canada Consortium on a research basis, and an apparent homozygous intronic variant (TTC25:c.1145+1G > A) was identified that was predicted to abolish the canonical splice donor activity of exon 8. The child's mother was a heterozygous carrier of the variant. The paternal sample did not show the splice variant, and homozygosity was observed across the paternal locus. Microarray analysis showed a 50 kb heterozygous deletion spanning the genes TTC25 and CNP. This is the first example of a pathogenic gross deletion in trans with a splice variant, resulting in TTC25-related PCD.     

Defining the clinical validity of genes reported to cause pulmonary arterial hypertension

PurposePulmonary arterial hypertension (PAH) is a rare, progressive vasculopathy with significant cardiopulmonary morbidity and mortality. Genetic testing is currently recommended for adults diagnosed with heritable, idiopathic, anorexigen-, hereditary hemorrhagic telangiectasia–, and congenital heart disease–associated PAH, PAH with overt features of venous/capillary involvement, and all children diagnosed with PAH. Variants in at least 27 genes have putative evidence for PAH causality. Rigorous assessment of the evidence is needed to inform genetic testing.MethodsAn international panel of experts in PAH applied a semi-quantitative scoring system developed by the NIH Clinical Genome Resource to classify the relative strength of evidence supporting PAH gene-disease relationships based on genetic and experimental evidence.ResultsTwelve genes (BMPR2, ACVRL1, ATP13A3, CAV1, EIF2AK4, ENG, GDF2, KCNK3, KDR, SMAD9, SOX17, and TBX4) were classified as having definitive evidence and 3 genes (ABCC8, GGCX, and TET2) with moderate evidence. Six genes (AQP1, BMP10, FBLN2, KLF2, KLK1, and PDGFD) were classified as having limited evidence for causal effects of variants. TOPBP1 was classified as having no known PAH relationship. Five genes (BMPR1A, BMPR1B, NOTCH3, SMAD1, and SMAD4) were disputed because of a paucity of genetic evidence over time.ConclusionWe recommend that genetic testing includes all genes with definitive evidence and that caution be taken in the interpretation of variants identified in genes with moderate or limited evidence. Genes with no known evidence for PAH or disputed genes should not be included in genetic testing.     

Long-term follow-up and novel genotype-phenotype analysis of monozygotic twins with ATP1A3 mutation in Alternating Hemiplegia of Childhood-2

Alternating Hemiplegia of Childhood (AHC) is a rare disorder characterized by frequent, transient attacks of hemiplegia involving either side of the body or both in association to several other disturbances including dystonic spells, abnormal ocular movements, autonomic manifestations, epileptic seizures and cognitive impairment. The clinical manifestations usually start before the age of 18 months. Two forms of the disorder known as AHC-1 (MIM#104290) and AHC-2 (MIM#614820) depends on mutations in ATP1A2 and ATP1A3 genes respectively, with over 75% of AHC caused by a mutation in the ATP1A3 gene. Herewith, we report serial clinical follow-up data of monozygotic (MZ) twin sisters, who presented in early life bath-induced dystonia, signs of acute encephalopathy at the age of 2 years, hemiplegic spells, and motor dysfunction after the age of 3 years, and in young/adult frequent episodes of headache with drastic reduction of paroxysmal motor attacks. The molecular analysis revealed a known pathogenic variant p.Asn773Ser (rs606231437) in ATP1A3 gene associated with an unusual and moderate AHC-2 phenotype, with mild cognitive impairment and lack of epilepsy. The aim of this study is to analyze the clinical phases of the MZ twins, and to investigate the novel genotype–phenotype correlation.     

Heart transplantation in Danon disease: Long term single centre experience and review of the literature

Danon disease is characterized by hypertrophic cardiomyopathy, skeletal myopathy, and intellectual disability due to deficiency of the lysosome-associated membrane protein-2 (LAMP-2). Although heart transplantation is considered an option for end stage Danon cardiomyopathy, scarce information is available about long term follow up. We report on long term follow up (14.7 years, IQ range 9–21 years) of 4 patients, transplanted for Danon disease cardiomyopathy, showing two LAMP-2 gene variants, the novel c.815T > C and the previously reported c.294G > A. We have also analysed previous published paper on this topic comparing available data from different follow up. Being a skeletal and cardiac muscle disease, with systemic effects, long term results about HTx are indispensable to justify any treatments in this subset of patients.     

Novel BRCA2 pathogenic genotype and breast cancer phenotype discordance in monozygotic triplets

BRCA1/2 genes with high-penetrance are tumor suppressor and tumor susceptibility genes that play important roles in the homologous recombination mechanism in DNA repair and increase breast cancer risk. Variants in BRCA1 or BRCA2 are the main causes of familial and early-onset breast cancer. This study investigated pathogenic variant belonging to the BRCA2 gene splice region in monozygotic triplets. A 44-year-old woman was diagnosed with breast cancer when she was 32 years old. Her monozygotic sister had a history of breast cancer. No malignancy was detected in the third one of the monozygotic triplets. Sanger sequencing was used to evaluate the BRCA1/2 gene status of the patient and family members. It was figured out that they had the same genetic variant, a heterozygous germ-line splice region variant (c.7008-1G > C) in the BRCA2 gene. This novel splice region variant may be a new pathogenic variant of the BRCA2 gene. Its association with breast cancers needs to be further verified in more patient cases.     

Expanding the clinical spectrum of SOX18-related Hypotrichosis-lymphedema-telangiectasia-renal defect syndrome

Pathogenic variants in SOX18 are associated with hypotrichosis-lymphedema-telangiectasia-renal defects syndrome (HLTRS) and hypotrichosis-lymphedema-telangiectasia syndrome (HLTS). Eleven patients with SOX18 related HLTRS/HLTS have been previously described. Cardinal features include varying degrees of hypotrichosis, lymphedema and telangiectasias. We report a 15-year-old female patient with a likely de novo SOX18 pathogenic variant identified on duo exome sequencing. In addition to the classic features, the currently reported patient presented with novel clinical features including musculoskeletal abnormalities and strikingly poor wound healing. Chronic skin ulcers have been a major cause of morbidity for the patient and have led to significant functional limitation. Further, our experience with wound management has been detailed. We hope to improve understanding of the clinical spectrum of this ultra-rare disorder by reviewing the phenotypic features in all reported patients including our patient.     

Role of the Klebsiella pneumoniae TolC porin in antibiotic efflux

The major Gram-negative gated efflux channel TolC has been extensively characterized in Escherichia coli but there is minimal information about Klebsiella pneumoniae TolC. Using an arabinose-inducible plasmid-based expression system, we show that the K. pneumoniae TolC complements the efflux defect in an E. coli K-12 ΔtolC strain, restoring wild-type levels of resistance towards most antibiotics suggesting that it can interact with the E. coli AcrB efflux pump. We characterize the efflux properties of K. pneumoniae TolC using an orthogonal whole cell-based assay and quantify the extrusion of environment-sensitive fluorescent probes and contrast the findings with the E. coli ortholog.     

Characterization of the cagA-gene in Helicobacter pylori in Mongolia and detection of two EPIYA-A enriched CagA types

Helicobacter pylori infection is strongly associated with gastritis, gastroduodenal ulcer disease and gastric carcinoma. The virulence of H. pylori strains increases with the presence of the pathogenicity island PAI, which encodes a Type 4 Secretion System and the oncoprotein CagA. Two major CagA types can be distinguished by differences in the repetitive EPIYA region in the C-terminal sequence; the more virulent East Asian CagA type with EPIYA-A, -B, and -D motifs and the Western CagA type with EPIYA-A, -B, and C motifs, the virulence of which is associated with the multitude of EPIYA-C motifs.In this study, the cagA gene was characterized in H. pylori strains isolated from Mongolians suffering from gastritis (80%) or ulcer (20%). The EPIYA region of 53 isolates was determined by PCR-amplification of overlapping cagA regions and subsequent Sanger sequencing. Only one H. pylori isolate carried the East Asian type (ABD) and 52 isolates the Western type of CagA, thereof 30 the EPIYA type ABC, 19 the ABCC type and one each of type ABCCCC, AAABC and AAAAB. An amino acid exchange from EPIYA-B to EPIYT-B was predominantly found in CagA proteins in strains with < 2 EPIYA-C copies (n = 25/32; p = 0.015) including the two EPIYA-A enriched CagA proteins, which have not been described to date. Due to the amino acid triplet preceding the EPIYA motif and strength of predicted phosphorylation, the multiple EPIYA-A motifs A2, A3 and A4 were shown to cluster with EPIYA-B and EPIYT-B with the unique feature of amino acid E in position ? 4 to Y of EPIYA. It has been described that tyrosine-phosphorylated EPIYA-A and -B motifs counteract the EPIYA-C-driven signaling towards host cell transformation and malignancy. Thus, Mongolian H. pylori strains carrying CagA proteins not only with a few EPIYA-C segments but also with multiplied EPIYA-A segments are probably less virulent; a thesis that needs further investigation at the protein level.     

Atg8 and Ire1 in combination regulate the autophagy-related endoplasmic reticulum stress response in Candida albicans

The unfolded protein response (UPR) is an important pathway to prevent endoplasmic reticulum (ER) stress in eukaryotic cells. In Saccharomyces cerevisiae, Ire1 is a key regulatory factor required for HAC1 gene splicing for further production of functional Hac1 and activation of UPR gene expression. Autophagy is another mechanism involved in the attenuation of ER stress by ER-phagy, and Atg8 is a core protein in autophagy. Both autophagy and UPR are critical for ER stress response, but whether they act individually or in combination in Candida albicans is unknown. In this study, we explored the interaction between Ire1 and the autophagy protein Atg8 for the ER stress response by constructing the atg8Δ/Δire1Δ/Δ double mutant in the pathogenic fungus C. albicans. Compared to the single mutants atg8Δ/Δ or ire1Δ/Δ, atg8Δ/Δire1Δ/Δ exhibited much higher sensitivity to various ER stress-inducing agents and more severe attenuation of UPR gene expression under ER stress. Further investigations showed that the double mutant had a defect in ER-phagy, which was associated with attenuated vacuolar fusion under ER stress. This study revealed that Ire1 and Atg8 in combination function in the activation of the UPR and ER-phagy to maintain ER homeostasis under ER stress in C. albicans.     

Outcomes of corticosteroid treatment in critical Ill adult patients with respiratory viruses-related community acquired pneumonia – a propensity-matched case control study

ObjectivesTo assess the outcomes of corticosteroid treatment in critically ill patients with respiratory virus–related community-acquired pneumonia (CAP).Materials/methodsAdult patients who were admitted to the intensive care unit and had a polymerase chain reaction–confirmed diagnosis of respiratory virus–related CAP were included. Patients with and without corticosteroid treatment during the hospital course were retrospectively compared using a propensity score–matched case–control analysis.ResultsFrom January 2018 to December 2020, 194 adult patients were enrolled with 1:1 matching. The 14-day and 28-day mortality rates did not differ significantly between patients treated with and without corticosteroids (14-day mortality: 7% versus 14%, P = 0.11; 28-day mortality: 15% versus 20%, P = 0.35). However, multivariate analysis by using a Cox regression model revealed that corticosteroid treatment was an independent factor predicting decreased mortality (adjusted odds ratio, 0.46; 95% confidence interval, 0.22–0.97, P = 0.04). Subgroup analysis revealed lower 14-day and 28-day mortality rates in patients younger than 70 years treated with corticosteroids than in those not treated with corticosteroids (14-day mortality: 6% versus 23%; P = 0.01 and 28-day mortality: 12% versus 27%; P = 0.04).ConclusionsNon-elderly patients with severe respiratory virus–related CAP are more likely to benefit from corticosteroid treatment than elderly patients.     

Cardiomyopathy prevalence exceeds 30% in individuals with TTN variants and early atrial fibrillation

PurposeTTN truncating variants (TTNtvs) represent the largest known genetic cause of dilated cardiomyopathies (DCMs), however their penetrance for DCM in general populations is low. More broadly, patients with cardiomyopathies (CMs) often exhibit other cardiac conditions, such as atrial fibrillation (Afib), which has also been linked to TTNtvs. This retrospective analysis aims to characterize the relationship between different cardiac conditions in those with TTNtvs and identify individuals with the highest risk of DCM.MethodsIn this work we leverage longitudinal electronic health record and exome sequencing data from approximately 450,000 individuals in 2 health systems to statistically confirm and pinpoint the genetic footprint of TTNtv-related diagnoses aside from CM, such as Afib, and determine whether vetting additional significantly associated phenotypes better stratifies CM risk across those with TTNtvs. We focused on TTNtvs in exons with a percentage spliced in >90% (hiPSI TTNtvs), a representation of constitutive cardiac expression.ResultsWhen controlling for CM and Afib, other cardiac conditions retained only nominal association with TTNtvs. A sliding window analysis of TTNtvs across the locus confirms that the association is specific to hiPSI exons for both CM and Afib, with no meaningful associations in percent spliced in ≤90% exons (loPSI TTNtvs). The combination of hiPSI TTNtv status and early Afib diagnosis (before age 60) found a subset of TTNtv individuals at high risk for CM. The prevalence of CM in this subset was 33%, a rate that was 3.5 fold higher than that in individuals with hiPSI TTNtvs (9% prevalence), 5-fold higher than that in individuals without TTNtvs with early Afib (6% prevalence), and 80-fold higher than that in the general population.ConclusionOur retrospective analyses revealed that those with hiPSI TTNtvs and early Afib (～1/2900) have a high prevalence of CM (33%), far exceeding that in other individuals with TTNtvs and in those without TTNtvs with an early Afib diagnosis. These results show that combining phenotypic information along with genomic population screening can identify patients at higher risk for progressing to symptomatic heart failure.