Novel Heterozygous Missense Variants in Diacylglycerol Kinase Epsilon and Complement Factor I: Potential Pathogenic Association With Atypical Hemolytic Uremic Syndrome.

Hemolytic uremic syndrome (HUS) is a thrombotic microangiopathy (TMA), which copresents with microangiopathic hemolytic anemia, thrombocytopenia, and kidney injury. While typical HUS is normally preceded by infections such as Shiga-toxin-producing Escherichia coli, atypical HUS (aHUS) has a genetic component that leads to dysregulation of the alternative complement pathway. We report a case of a 69-year-old female who developed aHUS after undergoing an elective knee surgery. Genetic testing revealed novel mutations affecting diacylglycerol kinase epsilon (DGKE) protein and complement factor I (CFI) that were not reported before as pathogenic. The patient was treated with eculizumab, leading to the complete resolution of TMA with no lasting organ damage.

Clinical features and management of atypical hemolytic uremic syndrome patient with DGKE gene variants: a case report.

Background: Atypical hemolytic uremic syndrome (aHUS) with diacylglycerol kinase epsilon (DGKE) gene variant is a rare variant of thrombotic microangiopathy (TMA). The information on the clinical features, management and long-term outcomes of DGKE-aHUS patients have not yet been fully elucidated. The aim of this study was to report a novel variant of the DGKE gene in a Chinese population with aHUS. Case presentation: The present work reports a 7-month-old boy with aHUS, possibly triggered by gastrointestinal infection, without complement activation, with little response to plasma therapy and nephroprotective measures. The patient died during the 8th week of his hospital stay. The causes of death were intracranial hemorrhage and multiorgan dysfunction. Comprehensive WES of peripheral blood-derived DNA revealed two heterozygous variations in the DGKE exon region: NM_003647.2, c.610dup, p.Thr204Asnfs*4 and deletion of exons 4-6. Conclusions: This case suggest that atypical HUS with DGKE gene variant has a poor prognosis with a high mortality rate, which typically manifests in the first year of life and presents as a systemic disease with early-onset HUS with rapidly worsening renal function and chronic proteinuria. There is no specific treatment for DGKE-aHUS. There have an uncertain benefit of plasma therapy for DGKE-aHUS patients. The literature demonstrated that anti-complement therapy showed benefits for DGKE-aHUS with complement activation and autoantibodies during the overt TMA presentation but did not prevent TMA relapses. Early diagnosis and treatment may prevent complications and improve prognosis.

Thrombotic microangiopathy in children.

The syndrome of thrombotic microangiopathy (TMA) is a clinical-pathological entity characterized by microangiopathic hemolytic anemia, thrombocytopenia, and end organ involvement. It comprises a spectrum of underlying etiologies that may differ in children and adults. In children, apart from ruling out shigatoxin-associated hemolytic uremic syndrome (HUS) and other infection-associated TMA like Streptococcus pneumoniae-HUS, rare inherited causes including complement-associated HUS, cobalamin defects, and mutations in diacylglycerol kinase epsilon gene must be investigated. TMA should also be considered in the setting of solid organ or hematopoietic stem cell transplantation. In this review, acquired and inherited causes of TMA are described with a focus on particularities of the main causes of TMA in children. A pragmatic approach that may help the clinician tailor evaluation and management is provided. The described approach will allow for early initiation of treatment while waiting for the definitive diagnosis of the underlying TMA.

Novel Mutations in the DGKE Gene in Two Indian Patients with Early-onset Atypical Haemolytic Uraemic Syndrome.

Atypical haemolytic uremic syndrome (aHUS) is a clinically and genetically heterogeneous condition caused by a complex interplay between genomic susceptibility factors and environmental influences. Pathogenic variants in the DGKE gene are recently identified in cases with infantile-onset autosomal recessive aHUS. The presence of low serum C3 levels, however, has rarely been described in cases of DGKE-associated aHUS. Molecular genetic testing was performed by a commercial next-generation sequencing (NGS) panel as well and by an in-house developed targeted NGS for DGKE gene. Copy number variations (CNVs) were computed from NGS data by calculating a normalised copy number ratio of aligned number of reads at targeted genomic regions against multiple reference regions of the same sample and multiple controls. We report here two such novel clinically relevant variants (c.727_730delTTGT and c.251_259delGCGCCTTC) in the DGKE gene, in two families of infantile aHUS with low serum C3 levels.

A patient with a homozygous diacylglycerol kinase epsilon (DGKE) gene mutation with atypical haemolytic uraemic syndrome and low C3 responded well to eculizumab: a case report.

BACKGROUND: Atypical haemolytic uraemic syndrome (aHUS) is a rare systemic syndrome characterized by non-immune haemolytic anaemia, thrombocytopenia, and kidney injury. In most cases, alternative complement pathway dysregulation is the identifying cause. Recently, other genetic causes have been identified, including a mutation in the diacylglycerol kinase epsilon (DGKE) gene, which theoretically affect the coagulation pathway and does not affect the complement pathway. Data about the management of these patients are limited. Ideal management and definitive treatment protocols have not yet been established. CASE PRESENTATION: A three-year-old boy presented with features of atypical haemolytic uraemic syndrome (aHUS) and low complement C3. He was presumed to have complement-mediated aHUS and was managed empirically with eculizumab. Two weeks after starting eculizumab, his haemoglobin levels, platelet count, and complement C3 level normalized but he continued to have non-nephrotic range proteinuria. His genetic testing revealed a homozygous DGKE mutation, with no other mutation detected. Six months after presentation, the patient was still in remission with no features of aHUS, a trial of weaning eculizumab by increasing dose interval was followed by nephrotic range proteinuria and severe oedema. His proteinuria improved and his oedema resolved after resuming his recommended eculizumab dose. CONCLUSIONS: DGKE gene mutation can lead to aHUS with theoretically no complement dysregulation. However, some patients with this mutation show alternative complement pathway activation. This case report describes a patient with aHUS due to a DGKE gene mutation and low C3 levels who responded to eculizumab, adding to the previously reported cases of patients with DGKE gene mutations who had complete remission with no relapse with C5 blockers and/or plasma exchange. A randomized controlled study on patients with DGKE mutations might be beneficial in understanding the disease and generating a management protocol.

Cyclosporine A relieved proteinuria and hypoproteinemia in DGKE nephropathy.

BACKGROUND: The DGKE gene encodes the diacylglycerol kinase epsilon (DGKε). Loss-of-function mutations of DGKE caused a group of rare renal diseases, which are called DGKE nephropathy. We report the clinical manifestations and therapeutic effects of a patient diagnosed with DGKE nephropathy. CASE REPORT: The patient's initial symptoms were fever, diarrhea, eyelid edema, acute anemia, acute thrombocytopenia, an elevation of plasm D-dimer, proteinuria, microscopic hematuria, without oliguria or renal insufficiency at the age of 7.6 months. Hemolytic uremic syndrome was diagnosed. His proteinuria and hematuria turned out negative 2 months later. Proteinuria was noticed again at the age of 5.5-year old when he was brought to the hospital because of failure to thrive. Since then, he had been noticed with persistent proteinuria. RESULTS: Genetic analysis revealed 2 novel heterozygous mutations on DGKE of the patient. Renal pathology mimicked membrane proliferative glomerulonephritis (MPGN). CONCLUSIONS: After a 5-month treatment of cyclosporine A (CsA), proteinuria and hypoproteinemia have relieved apparently. We also observed an improvement of his growth.

Whole exome sequencing revealed a novel homozygous variant in the DGKE catalytic domain: a case report of familial hemolytic uremic syndrome.

BACKGROUND: Atypical hemolytic uremic syndrome (aHUS) is a rare disease characterized by microangiopathic hemolytic anemia caused by small vessel thrombosis, thrombocytopenia, and renal failure. The common cause of aHUS is a dysregulation in the alternative complement pathway. Mutations in none complement genes such as diacylglycerol kinase epsilon (DGKE) can also result in this syndrome. CASE PRESENTATION: Here, we report on a 19-year-old female with the clinical diagnosis of aHUS, who has unaffected consanguineous parents and an older sibling who was deceased from aHUS when she was seven months old. We performed whole exome sequencing (WES) followed by evaluation of detected variants for functional significance, using several online prediction tools. Next, in order to confirm the detected pathogenic variant in proband and segregation analysis in her family, Sanger sequencing was done. The novel variant was analyzed in terms of its impact on the protein 3-dimensional structure by computational structural modeling. The results revealed that the proband carried a novel homozygous missense variant in DGKE located in exon 6 of the gene (NM_003647.3, c.942C > G [p.Asn314Lys]), and in silico analysis anticipated it as damaging. Protein computational study confirmed the influence of potential pathogenic variant on structural stability and protein function. CONCLUSION: We suggest that some variations in the catalytic domain of DGKE like p.Asn314Lys which can cause alterations in secondary and 3-D structure of protein, might lead to aHUS.

Molecular basis and outcomes of atypical haemolytic uraemic syndrome in Czech children.

Atypical haemolytic uraemic syndrome is an ultra-rare, life-threatening disease. Causative variants in genes that encode complement factors can be identified in 40-70% of cases. We performed genetic analysis of 21 Czech children with atypical haemolytic uraemic syndrome. Genetic or acquired predisposition to the disease was identified in the majority of our patients: CFHR1 and CFHR3 deletions in 14/21 (67%; 13 of them were positive for anti-complement factor H antibodies), variants in complement genes or DGKE in 13/21 (62%). Multiple genetic findings were identified in eight patients (38%). The incidence of atypical haemolytic uraemic syndrome in the Czech paediatric population was estimated to be 0.092 (CI 0.053-0.131) cases per million inhabitants and 0.92 (CI 0.53-1.32) cases per 100,000 births for the entire reporting period. Ten patients were initially treated with plasma exchange and eight with eculizumab or with a combination of eculizumab and plasma exchange. At the last follow-up, 20 patients were alive and one patient had end-stage renal disease.Conclusion: The incidence of atypical haemolytic uraemic syndrome in the Czech paediatric population corresponds to the reported incidence in Europe. We detected the unusually high rate of CFHR1/CFHR3 deletions associated with anti-complement factor H antibodies in Czech paediatric patients. Treatment by eculizumab led to superior outcomes and prevention of the disease relapses compared with plasma exchange therapy. Our results may help to understand the polygenic nature of atypical haemolytic uraemic syndrome as a disease that results from a combination of various risk factors. What is Known: • Atypical haemolytic uraemic syndrome (aHUS) is considered a polygenic and multifactorial disease. Genetic predisposition to aHUS is identified in 40-70% of children. • Anti-complement factor H antibodies are usually found in 6-25% of affected children. What is New: • Potentially causative genetic or acquired factors were confirmed in the majority of patients. The prevailing finding was the unusually high rate of CFHR1/CFHR3 deletions associated with anti-complement factor H antibodies (62% of patients). • The incidence of aHUS in Czech children is 0.092 (CI 0.053-0.131) cases per million inhabitants and 0.92 (CI 0.53-1.32) cases per 100,000 births for the entire reporting period.

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.

A novel compound heterozygous mutation in DGKE in a Chinese patient causes atypical hemolytic uremic syndrome.

Objectives: DGKE mutations can lead to hemolysis and thrombus in patients with atypical hemolytic uremic syndrome (aHUS). However, the sequence variants of DGKE in Chinese patients with aHUS have not been reported, and the protein function and crystal structure of DGKE remain unresolved.Methods: Targeted exome sequencing was accomplished in one affected patient from each family using the Illumina NextSeq 500 platform. Protein modeling and functional analysis in DGKE were also performed to understand the impact of identified variants on the phenotype.Results: We report a novel compound heterozygous mutation in the DGKE gene in a Chinese consanguineous family in which a child was diagnosed with aHUS, which includes a c.231C>G missense mutation and a c.790_791delTG frameshift mutation derived from his father and mother, respectively. Our bioinformatic analysis suggested that the allelic mutations at different sites in DGKE yield abnormal crystal structures and conformations, leading to dysregulation of its downstream signaling.Conclusions: Our study further expands the spectrum of the sequence variants reported in the DGKE gene and also indicates that different races may have different DGKE variants. Moreover, the altered structures and conformations, caused by DGKE mutations, disrupt the binding of DGKE with its partners, and leading to the occurrence of aHUS.

Diacylglycerol kinase epsilon nephropathy: late diagnosis and therapeutic implications.

A 17-year-old male presented thrombotic microangiopathy (TMA) at 6 months of age with arterial hypertension, anemia, thrombocytopenia and kidney injury improving with plasma infusions. Fourteen years later, he was diagnosed with severe arterial hypertension, increase in serum creatinine and chronic TMA on kidney biopsy. Eculizumab was started and after 18 months of treatment, he persisted with hypertension, decline in renal function and proteinuria. Genetic analysis demonstrated mutation in diacylglycerol kinase epsilon (DGKe). Complement blockade was stopped. This case of late diagnosis of DGKe nephropathy highlights the importance of genetic testing in patients presenting TMA during the first year of life.

The Phenotypic Spectrum of Nephropathies Associated with Mutations in Diacylglycerol Kinase ε.

The recent discovery of mutations in the gene encoding diacylglycerol kinase ε (DGKE) identified a novel pathophysiologic mechanism leading to HUS and/or MPGN. We report ten new patients from eight unrelated kindreds with DGKE nephropathy. We combined these cases with all previously published cases to characterize the phenotypic spectrum and outcomes of this new disease entity. Most patients presented with HUS accompanied by proteinuria, whereas a subset of patients exhibited clinical and histologic patterns of MPGN without TMA. We also report the first two patients with clinical and histologic HUS/MPGN overlap. DGKE-HUS typically manifested in the first year of life but was not exclusively limited to infancy, and viral triggers frequently preceded HUS episodes. We observed signs of complement activation in some patients with DGKE-HUS, but the role of complement activation remains unclear. Most patients developed a slowly progressive proteinuric nephropathy: 80% of patients did not have ESRD within 10 years of diagnosis. Many patients experienced HUS remission without specific treatment, and a few patients experienced HUS recurrence despite complete suppression of the complement pathway. Five patients received renal allografts, with no post-transplant recurrence reported. In conclusion, we did not observe a clear genotype-phenotype correlation in patients with DGKE nephropathy, suggesting additional factors mediating phenotypic heterogeneity. Furthermore, the benefits of anti-complement therapy are questionable but renal transplant may be a feasible option in the treatment of patients with this condition.

Loss of diacylglycerol kinase epsilon in mice causes endothelial distress and impairs glomerular Cox-2 and PGE2 production.

Thrombotic microangiopathy (TMA) is a disorder characterized by microvascular occlusion that can lead to thrombocytopenia, hemolytic anemia, and glomerular damage. Complement activation is the central event in most cases of TMA. Primary forms of TMA are caused by mutations in genes encoding components of the complement or regulators of the complement cascade. Recently, we and others have described a genetic form of TMA caused by mutations in the gene diacylglycerol kinase-ε (DGKE) that encodes the lipid kinase DGKε (Lemaire M, Fremeaux-Bacchi V, Schaefer F, Choi MR, Tang WH, Le Quintrec M, Fakhouri F, Taque S, Nobili F, Martinez F, Ji WZ, Overton JD, Mane SM, Nurnberg G, Altmuller J, Thiele H, Morin D, Deschenes G, Baudouin V, Llanas B, Collard L, Majid MA, Simkova E, Nurnberg P, Rioux-Leclerc N, Moeckel GW, Gubler MC, Hwa J, Loirat C, Lifton RP. Nat Genet 45: 531-536, 2013; Ozaltin F, Li BH, Rauhauser A, An SW, Soylemezoglu O, Gonul II, Taskiran EZ, Ibsirlioglu T, Korkmaz E, Bilginer Y, Duzova A, Ozen S, Topaloglu R, Besbas N, Ashraf S, Du Y, Liang CY, Chen P, Lu DM, Vadnagara K, Arbuckle S, Lewis D, Wakeland B, Quigg RJ, Ransom RF, Wakeland EK, Topham MK, Bazan NG, Mohan C, Hildebrandt F, Bakkaloglu A, Huang CL, Attanasio M. J Am Soc Nephrol 24: 377-384, 2013). DGKε is unrelated to the complement pathway, which suggests that unidentified pathogenic mechanisms independent of complement dysregulation may result in TMA. Studying Dgke knockout mice may help to understand the pathogenesis of this disease, but no glomerular phenotype has been described in these animals so far. Here we report that Dgke null mice present subclinical microscopic anomalies of the glomerular endothelium and basal membrane that worsen with age and develop glomerular capillary occlusion when exposed to nephrotoxic serum. We found that induction of cyclooxygenase-2 and of the proangiogenic prostaglandin E2 are impaired in Dgke null kidneys and are associated with reduced expression of the antithrombotic cell adhesion molecule platelet endothelial cell adhesion molecule-1/CD31 in the glomerular endothelium. Notably, prostaglandin E2 supplementation was able to rescue motility defects of Dgke knockdown cells in vitro and to restore angiogenesis in a test in vivo. Our results unveil an unexpected role of Dgke in the induction of cyclooxygenase-2 and in the regulation of glomerular prostanoids synthesis under stress.

Complement activation in diseases presenting with thrombotic microangiopathy.

The complement system contains a great deal of biological "energy". This is demonstrated by the atypical hemolytic uremic syndrome (aHUS), which is a thrombotic microangiopathy (TMA) characterized by endothelial and blood cell damage and thrombotic vascular occlusions. Kidneys and often also other organs (brain, lungs and gastrointestinal tract) are affected. A principal pathophysiological feature in aHUS is a complement attack against endothelial cells and blood cells. This leads to platelet activation and aggregation, hemolysis, prothrombotic and inflammatory changes. The attacks can be triggered by infections, pregnancy, drugs or trauma. Complement-mediated aHUS is distinct from bacterial shiga-toxin (produced e.g. by E. coli O:157 or O:104 serotypes) induced "typical" HUS, thrombotic thrombocytopenic purpura (TTP) associated with ADAMTS13 (an adamalysin enzyme) dysfunction and from a recently described disease related to mutations in intracellular diacylglycerol kinase ε (DGKE). Mutations in proteins that regulate complement (factor H, factor I, MCP/CD46, thrombomodulin) or promote (C3, factor B) amplification of its alternative pathway or anti-factor H antibodies predispose to aHUS. The fundamental defect in aHUS is an excessive complement attack against cellular surfaces. This can be due to 1) an inability to regulate complement on self cell surfaces, 2) hyperactive C3 convertases or 3) complement activation and coagulation promoting changes on cell surfaces. The most common genetic cause is in factor H, where aHUS mutations disrupt its ability to recognize protective polyanions on surfaces where C3b has become attached. Most TMAs are thus characterized by misdirected complement activation affecting endothelial cell and platelet integrity.