POS-028 Rare genetic alterations in patients with C3 glomerulopathy-something to ponder upon

Field: Medicine (Health Sciences)

IntroductionAbout 15-20% patients of C3 Glomerulopathy (C3G) have genetic defects described in complement factor H (CFH), factor I (CFI), factor B (CFB) or C3 genes. Larger gene deletion/duplications are also identified in CFH-related (CFHR) genes. In our Indian cohort we documented previously the common variations using screening of limited genes. However, expanding our diagnostic panels and selecting cases with family history or death in family with renal disease, progression to ESRD, post-transplant recurrence and disease associated conditions, we found some rare variants in alternate complement pathway (ACP) related genes which are documented in present study.MethodsParticipants Biopsy proven 19 cases of C3G (DDD-10, C3GN-9) were selected where there was family history or progression to ESRD, post-transplant recurrence. Genetic variants of C3G were tested by whole exome, or targeted panel or individual gene. Clinical, serological details with latest follow-up was used to compare final outcome.Targeted NGS panel DesigningTargeted NGS panel for ACP related genes was designed using high specificity primers for C3, CFH, CFB, CFI, CFB, CFD, properdin, MCP, CFHR1, CFHR2, CFHR3, CFHR4, CFHR5, DGKE, THBD, ADAMT13, ADAMT19 and CR1 genes using https://ampliseq.com.Library preparation and SequencingDNA was isolated and library was prepared using Ion AmpliSeq library kit 2.0. All 100pM libraries were pooled and template positive Ion Sphere Particles (ISPs) were prepared using Ion 520 OT2 kit. After enrichment of these ISPs, sequencing was done on Ion Torrent-S5 system (Thermo Fisher Scientific).Data analysis The data generated (*.bam files) was loaded into Ion Reporter™ Software 5.12. Alignment of the sequencing reads was done with reference GRCh38 and variant calling was performed. All variants having functional effect (frameshift insertion, stop loss, frameshift deletion, missense, and nonsense) and coverage ≥30 reads were filtered for analysis.MLPAMLPA was done in 16 cases of C3G and 8-healthy controls in cases where deletions was suspected on NGS data. This was done using SALSA MLPA probemix P236-B1 CFH region kit (MRC-Holland). Samples were run on 3500dx genetic analyzer (Applied Biosystems), and data was analyzed using Coffalyser.Net software.ResultsIn these patients of C3G, we found 85 exonic, 11 UTR and 144 intronic variants in present C3G cohort. Of 85 exonic, 40 variants were synonymous and 45 were non-synonymous of which 12 were described in ClinVar (03 pathogenic, 08 benign, 01-other) and 33 (01 stop codon, 05 insertion/deletion, 27 missense) in dbSNP (Figure-1). There was no difference between variants found in DDD and C3GN except for rs11749126 (ADAMTS19) which was present only in C3GN. We found rare exonic variants of different genes in C3G (DDD-11, C3GN-13; Table-1). MLPA highlighted deletion in exon 01, 09, 17 and 22 of CFH gene. CFHR1 Intr-1 was duplicated in 09 and deleted in 02 patients. This duplication was not seen in controls (Figure-2). CFHR3-6 deletion was also found in 05 C3G patients.ConclusionsIn present study, we documented 24 rare exonic variants in all cases of C3G which had some clinical clues to indicate worse outcome. ADAMTS19 gene variants was exclusively present only in C3GN. Duplications of CFHR1 Intr.1 was also rare variation present in both DDD and C3GN, however, present findings need further validation in bigger cohort.Conflict of interest Corporate sponsored research or other substantive relationships:Prof. Ritambhra Nada received 3 years research grant from Science and Engineering Research Board (SERB) vide letter number DST No: EMR/2017/004124. Dr. Ashwani Kumar received a postdoctoral Research Associate Fellowship from ICMR during the period of this work. IntroductionAbout 15-20% patients of C3 Glomerulopathy (C3G) have genetic defects described in complement factor H (CFH), factor I (CFI), factor B (CFB) or C3 genes. Larger gene deletion/duplications are also identified in CFH-related (CFHR) genes. In our Indian cohort we documented previously the common variations using screening of limited genes. However, expanding our diagnostic panels and selecting cases with family history or death in family with renal disease, progression to ESRD, post-transplant recurrence and disease associated conditions, we found some rare variants in alternate complement pathway (ACP) related genes which are documented in present study.