Advertisment

Indian Journal of Nephrology About us |  Subscription |  e-Alerts  | Feedback | Login   
  Print this page Email this page   Small font sizeDefault font sizeIncrease font size
 Home | Current Issue | Archives| Ahead of print | Search |Instructions |  Editorial Board  

Users Online:1506

Official publication of the Indian Society of Nephrology
  Search
 
  
 ~  Similar in PUBMED
 ~  Search Pubmed for
 ~  Search in Google Scholar for
 ~Related articles
 ~  Article in PDF (1,029 KB)
 ~  Citation Manager
 ~  Access Statistics
 ~  Reader Comments
 ~  Email Alert *
 ~  Add to My List *
* Registration required (free)  

 
   Abstract
  Introduction
  Case Details
  Discussion
   References
   Article Figures
   Article Tables

 Article Access Statistics
    Viewed659    
    Printed15    
    Emailed0    
    PDF Downloaded33    
    Comments [Add]    

Recommend this journal

 


 
  Table of Contents  
CASE REPORT
Year : 2021  |  Volume : 31  |  Issue : 2  |  Page : 182-186
 

Novel mutations in the DGKE gene in two indian patients with early-onset atypical haemolytic uraemic syndrome


1 Nephrology Unit, King Edward Memorial Hospital, India
2 GenePath Diagnostics India Pvt. Ltd., 1260/B, J M Road, Pune, India
3 Paeditrics Department, King Edward Memorial Hospital, Pune, India
4 GenePath Diagnostics Inc. Ann Arbor, Michigan, USA
5 GenePath Diagnostics India Pvt. Ltd., 1260/B, J M Road, Pune; I-SHARE Foundation, 1260/B, J M Road, Pune, India

Date of Submission01-Oct-2019
Date of Acceptance16-Feb-2020
Date of Web Publication02-Apr-2021

Correspondence Address:
Dr. Meenal Agarwal
GenePath Diagnostics India Private limited, Pune, and I-SHARE Foundation, 1260/B, J M Road, Pune,
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijn.IJN_336_19

Rights and Permissions

  Abstract 


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.


Keywords: Atypical haemolytic uremic syndrome, DGKE, next-generation sequencing, novel mutations


How to cite this article:
Sharma J, Lobo V, Singhal J, Anand S, Kadam S, Ranade S, Gangodkar P, Ganesan K, Phadke N, Agarwal M. Novel mutations in the DGKE gene in two indian patients with early-onset atypical haemolytic uraemic syndrome. Indian J Nephrol 2021;31:182-6

How to cite this URL:
Sharma J, Lobo V, Singhal J, Anand S, Kadam S, Ranade S, Gangodkar P, Ganesan K, Phadke N, Agarwal M. Novel mutations in the DGKE gene in two indian patients with early-onset atypical haemolytic uraemic syndrome. Indian J Nephrol [serial online] 2021 [cited 2021 Sep 22];31:182-6. Available from: https://www.indianjnephrol.org/text.asp?2021/31/2/182/313020



  Introduction Top


Haemolytic uraemic syndrome (HUS) falls under thrombotic microangiopathies (TMA) characterised by a triad of thrombocytopenia, microangiopathic anaemia and acute kidney injury. Atypical HUS (aHUS) is a subset of HUS without coexisting/preceding infections or coexisting diseases. Compared to typical HUS, aHUS are associated with a poorer clinical outcome, have higher chances of end-stage renal disease (ESRD) and exhibit increased episodes of relapses. Most aHUS cases have a dysregulated complement pathway thought to result from a complex interaction of genetic and acquired factors.[1] Other non-complement-associated aHUS pathologies have also been identified; hence, newer terminologies like complement-HUS, diacylglycerol kinase ε (DGKE) mutation–HUS and cobalamin C (cblC) defect-HUS might be more appropriate.[2] Biallelic loss-of-function mutations (or pathogenic variants) in the DGKE gene was established as a cause of infantile-onset aHUS (in 27% cases) and young-onset nephrotic syndrome in 2013.[3] Here, we report two novel genomic variants in the DGKE gene in two unrelated Indian families. Both cases also exhibited low serum C3 levels; additionally, one patient exhibited high anti-factor H antibody levels.


  Case Details Top


Case 1 was an 11-month-old boy, born in a third-degree consanguineous family presenting with anasarca and hypertension. The investigation results were suggestive of microangiopathic haemolytic anaemia, deranged renal function, high anti-factor H antibody titres (1210 AU/mL) and a C3 level of 40 mg/dL (Normal: 90–180 mg/dL). Daily plasma infusions (PIs) and immunosuppression with prednisolone and azathioprine were initiated. The patient underwent two sessions of acute peritoneal dialysis (PD), each lasting 72 h. After 2 weeks of treatment, his haematological parameters improved; however, his eGFR remained between 25 and 30 mL/min/1.73 m2 (Normal eGFR = 48–117.2 mL/min/1.73 m2).[4] The frequency of PIs was decreased to alternate days. Two weeks later, he became oliguric again and his renal function deteriorated. PD and daily PIs were re-initiated; however, the patient's parents decided to withdraw the patient from further management.

Case 2 was a 5-month old male, born in a nonconsanguineous family and was referred with a diagnosis of HUS and eGFR of 16 mL/min/1.73 m2 (normal eGFR = 43.9–99.5 mL/min/1.73 m2).[4] Anti-CFH antibody levels were normal (8.85 AU/mL) and had a C3 level of 46 mg/dL. Acute PD was initiated, and daily PIs were administered during the 1st week, followed by PIs on alternate days. Haematological and renal parameters improved (no features of haemolysis, eGFR = 56 mL/min/1.73 m2) following 3 weeks of PIs. Four weeks later, his urine output decreased, and renal parameters worsened (eGFR = 34 mL/min/1.73 m2). The parents withdrew the patient from further management.

Anti-complement factor H antibody testing

To check the level of the anti-CFH antibodies, ELISA was performed with the Viditest anti-complement factor H kit (Vidia; ODZ-166) and standardised against the reference method at National Renal Complement Therapeutics Centre, Newcastle University. The normal range for our population was determined from healthy blood donors, school children and patients admitted with diseases other than HUS.

Molecular genetic testing

DNA was extracted from the peripheral blood sample using the DNeasy Blood and Tissue Kit (Qiagen, Germany) according to the manufacturer's protocol. For case 1, next-generation sequencing (NGS) was performed using the Trusight One Panel (Illumina; Cat. # 15046895) followed by analysis using an in-house developed bioinformatics pipeline. The DGKE gene is not included in this NGS panel. Therefore, a targeted amplicon-based NGS assay was developed to analyse the DGKE gene. Briefly, the assay involved an initial PCR in which the DGKE gene was amplified in three segments—exon 1, exons 2–6 and exons 7–11 using primers previously published in the literature.[5] Exon one, which is 916 bp in length, was amplified using a ramp-up PCR and Quantitect qPCR mastermix (Qiagen; Cat # 204543). Long-range PCRs were used to amplify exons 2–6 and exons 7–11 in separate tubes using Q5 Hotstart HiFi DNA polymerase (New England Biolabs (NEB); cat # M0493L), which are 5.4 kb and 6.7 kb in length, respectively. Furthermore, the PCR products were purified with ExoSap [Exonuclease I (NEB; M0293) and shrimp alkaline phosphatase (NEB; M0371)] to digest unused, single-stranded oligonucleotide primers and unincorporated nucleotides and quantified using a Qubit fluorometric assay (Life Technologies, CA, USA). These purified PCR products were pooled/normalised and then subjected to transposon-mediated adapter ligation (tagmentation) using the NexteraXT library preparation kit (Illumina; cat. # 15032355), followed by barcoding and adaptor ligation of the pooled amplicons with custom-synthesised oligonucleotides. The adaptor-ligated products were analysed for their quality on an Agilent Bioanalyzer and quantified accurately with an in-house qPCR and a Qubit fluorometric assay. The library was sequenced on an Illumina MiSeq sequencer using paired-end sequencing-by-synthesis chemistry. Sequencing for case 2 was performed using a 6640-gene (including the coding region of DGKE gene) clinical exome-capture assay at an external laboratory.

Bioinformatic analysis was performed using an in-house developed pipeline on both datasets including alignment of the FASTQ reads to a GRCh37/hg19 human reference genome assembly. For copy number variations (CNVs) determination, we developed an in-house pipeline 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 control samples. A ratio <0.65 was suggestive of a heterozygous deletion whereas a ratio >1.3 was suggestive of duplication. An absence of aligned reads was highly suggestive of a homozygous deletion.

In case 1, we found a novel 4-bp deletion [c. 727_730delTTGT] in DGKE exon 4, predicted to shift the protein reading frame and was reported as 'likely pathogenic' [Figure 1]a. Findings were confirmed by targeted capillary sequencing [Figure 1]b in the patient (in a homozygous state). In case 2, a 9-bp in-frame homozygous deletion [c. 251_259delGCGCCTTCT] was detected in DGKE exon 2 [Figure 1]c. This variant has not been previously reported in the literature. Bioinformatically, this variant is predicted to cause a 3-amino acid (A-F-C) deletion [Figure 1]c. In silico pathogenicity prediction algorithms including MutationTaster and LoFTool predicted this variant as disease-causing and possibly damaging, respectively. Based on the available evidence and clinical correlation, this variant was classified as a variant of unknown significance, possibly pathogenic.
Figure 1: (a) Visualisation of the next generation sequencing (NGS) data for case 1 aligned to the reference genome shows a homozygous deletion of four bases (in pink) in DGKE exon 4 resulting in a frameshift that is predicted to be pathogenic. (b) Snapshot of Mutation Surveyer software (SoftGenetics. Pennsylvania, USA) highlighting the homozygous deletion in case 1. (c) NGS data for case 2 aligned to the reference genome shows homozygous deletion of nine bases (pink) in DGKE exon 2 resulting in a deletion of three amino acids (A-F-C), which was predicted to be variant of unknown significance possibly pathogenic

Click here to view


No clinically relevant sequence variants were detected in the C3, CFB, CFH, CFI, CD46 (MCP) and THBD genes. In both these cases, complete coding regions of these genes along with exon-intron boundaries were covered with >20X coverage. Additionally, clinically relevant CNVs were not detected in the CFHR1/CFHR3 genes.


  Discussion Top


We report two novel clinically relevant variants, in the coding region of the DGKE gene in two infantile aHUS cases with low serum C3 levels. This report expands the spectrum of DGKE gene variants and associated clinical phenotype. We also describe the development and validation of a custom amplicon-based targeted NGS assay for the DGKE gene.

The presence of biallelic pathogenic/likely-pathogenic variants in the DGKE was first reported by Lemaire et al. in two unrelated families, each with two affected children with infantile-onset aHUS and unaffected parents.[3] None of them had evidence of complement system activation and did not harbour mutations in any of the complement genes, suggesting that mutations in the DGKE gene cause a thrombotic phenotype independent of complement activation. The same group sequenced the DGKE gene in 47 unrelated paediatric aHUS without the presence of anti-factor H antibodies and found mutations in the DGKE gene in nine children (19.1%).[5] These children had a relapsing course of aHUS before 5 years of age and progressed to chronic kidney disease (CKD) by the second decade of life. Two of them were on anti-complement therapy (eculizumab and fresh frozen plasma infusions respectively). Ozaltin et al. identified mutations in the DGKE gene in three unrelated Turkish families as the cause of glomerular microangiopathy with histologic signs of membranoproliferative glomerulonephritis (MPGN) and endothelial dysfunction.[6] No complement data were reported in this study. The clinical phenotype of DGKE-associated diseases was expanded to hypocomplementemic aHUS by Westland et al.[7] They described two siblings with infantile HUS, one with low serum C3 at presentation and the other at 5 months after onset. Both siblings were managed with PIs at gradually increasing intervals. Capillary sequencing confirmed the presence of homozygous p.K101X mutation in the DGKE gene in both cases. These children did not exhibit the presence of anti-factor H antibodies or mutations in the complement genes. In the literature, only nine cases of infancy/early childhood aHUS with DGKE gene mutations have been reported who did not have anti-factor H antibodies but had evidence of complement activation on a presentation or on follow-up [Table 1]. These children were managed with various combinations of PIs, plasma exchange and eculizumab along with other supportive therapy. In the reported cases, the patients were treated with plasma infusions rather than exchanges. This was done keeping in mind view of the very small size of the infants, which increased the risk of adverse events and the technical difficulty in performing the procedure. Chinchilla et al.[8] reported two children with DGKE gene mutations and low C3 levels; one child harboured an additional mutation in the C3 gene and a risk haplotype in the MCP gene in a homozygous state and the other child harboured a heterozygous THBD gene mutation and a high-risk haplotype in the CFH gene, thereby explaining the low C3 levels in both of them. None of the patients in our study harboured any clinically relevant variants in complement pathway genes. It remains unclear whether aHUS-associated DGKE deficiency directly initiates complement activation or whether these patients harbour other genetic abnormalities in modifier genes that affect the complement cascade.
Table 1: Patients with DGKE mutation and evidence of complement activation

Click here to view


Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Noris M, Caprioli J, Bresin E, Mossali C, Pianetti G, Gamba S, et al. Relative role of genetic complement abnormalities in sporadic and familial aHUS and their impact on clinical phenotype. Clin J Am Soc Nephrol 2010;5:1844-59.  Back to cited text no. 1
    
2.
Fakhouri F, Zuber J, Frémeaux-Bacchi V, Loriat C. Haemolytic uraemic syndrome. Lancet 2017;390:681-96.  Back to cited text no. 2
    
3.
Fremeaux-Bacchi V, Fakhouri F, Garnier A, Bienaimé F, Dragon-Durey MA, Ngo S, et al. Genetics and outcome of atypical hemolytic uremic syndrome: A nationwide French series comparing children and adults. Clin J Am Soc Nephrol 2013;8:554-62.  Back to cited text no. 3
    
4.
Piepsz A, Tondeur M, Ham H. Revisiting normal(51) Cr-ethylenediaminetetraacetic acid clearance values in children. Eur J Nucl Med Mol Imaging 2006;33:1477-82.  Back to cited text no. 4
    
5.
Lemaire M, Frémeaux-Bacchi V, Schaefer F, Choi M, Tang WH, Le Quintrec M, et al. Recessive mutations in DGKE cause atypical haemolytic-uremic syndrome. Nat Genet 2013;45:531-6.  Back to cited text no. 5
    
6.
Ozaltin F, Li B, Rauhauser A, An S, Soylemezoglu O, Gonul I, et al. DGKE variants cause a glomerular microangiopathy that mimics membranoproliferative GN. J Am Soc Nephrol 2013;24:377-84.  Back to cited text no. 6
    
7.
Westland R, Bodria M, Carrea A, Lata S, Scolari F, Fremeaux-Bacchi V, et al. Phenotypic expansion of DGKE-associated diseases J Am Soc Nephrol 2014;25:1408-14.  Back to cited text no. 7
    
8.
Chinchilla D, Pinto S, Hoppe B, Adragna M, Lopez L, Roldan M, et al. Complement mutations in DGKE-associated aHUS. Clin J Am Soc Nephrol 2014;9:1-9.  Back to cited text no. 8
    
9.
Azukaitis K, Simkova E, Majid M, Galiano M, Benz K, Amann K, et al. The phenotypic spectrum of nephropathies associated with mutations in diacylglycerol kinaseε. J Am Soc Nephrol 2017;28:3066-75.  Back to cited text no. 9
    
10.
Mele C, Lemaire M, Latropoulos P, Piras R, Bresin E, Bettoni S, et al. Characterization of a new DGKE intronic mutation in genetically unsolved cases of familial atypical haemolytic uremic syndrome. Clin J Am Soc Nephrol 2015;10:1011-9.  Back to cited text no. 10
    
11.
Miyata T, Uchida Y, Ohta T, Urayama K, Yoshida Y, Fujimura Y. Atypical haemolytic uraemic syndrome in a Japanese patient with DGKE genetic mutations. Thromb Haemost 2015;114:862-3.  Back to cited text no. 11
    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1]



 

Top
Print this article  Email this article
 

    

Indian Journal of Nephrology
Published by Wolters Kluwer - Medknow
Online since 20th Sept '07