Renal transplantation provides the best long-term treatment for chronic renal failure. Single-nucleotide polymorphisms (SNPs) play a major role in the understanding of the genetic basis of many complex human diseases. Also, the genetics of human phenotype variation could be understood by knowing the functions of these SNPs. It is still a major challenge to identify the functional SNPs in a disease-related gene. This work explored how SNPs mutations in HLA-DRB1 gene could affect renal transplantation rejection. This study was carried out in Ahmed Gasim Hospital, Renal Dialysis Center during the period, from September 2012 to November 2013. Blood samples from five Sudanese patients (different families) with known renal transplantation rejection were collected before hemodialysis, furthermore one blood sample for control. DNA sequences results and detected SNPs were analyzed using bioinformatics tools (BLAST, SIFT, nsSNP Analyzer, PolyPhen, I-mutant, BioEdit, CPH, Chimera, Box shade and Project Hope). In addition, international databases were used for datasets [NCBI, Uniprot]. Results showed that, three SNPs were detected; two of three SNPs were predicted as tolerant or benign (rs1059575, novel) and one was deleterious (rs17885437). This study concluded that the identification of pathological SNPs could be an answer to unknown causes for a lot of organ transplantation rejection cases.
Around the world in 2002 there were over 1.1 million patients estimated to have end stage renal disease (ESRD) with addition of 7% annually [
Human leukocyte antigen (HLA) system is the name of human major histocompatibilty complex (MHC). A group of cell-surface antigen-presenting proteins are encoded by a region on the short arm of chromosome: 6 in the distal portion of the 21.3 band; several different types of gene are arranged in the form of three regions: class I, II and III. Most of these genes are polymorphic, arranged close together and are generally inherited as a haplotype [
HLA-DRB1 belongs to the HLA class II beta chain paralogs. The class II molecule is a heterodimer consisting of an alpha (DRA) and a beta chain (DRB), both anchored in the membrane. It plays a central role in the immune system by presenting peptides derived from extracellular proteins. Class II molecules are expressed in antigen presenting cells (APC: B lymphocytes, dendritic cells, macrophages). The beta chain is approximately 26 - 28 kilo Dalton (kDa). It is encoded by 6 exons. Exon one encodes the commander peptide; exons two and three encode the two extracellular domains; exon four encodes the transmembrane domain; and exon five encodes the cytoplasmic tail. Within the DR molecule the beta chain contains all the polymorphisms specifying the peptide binding specificities. DRB1 is expressed at a level five times higher than its paralogs DRB3, DRB4 and DRB5; also DRB1 is present in all individuals and the Allelic variants of DRB1 are linked with either none or one of the genes DRB3, DRB4 and DRB5. In addition, there are five related pseudogenes: DRB2, DRB6, DRB7, DRB8 and DRB9 [
Single nucleotide polymorphisms (SNPs) are variations of a single base, either between two homologous chromosomes within a single individual, or between two individuals [
This study was a hospital-based case control study, in Ahmed Gasim hospital in the period from September 2012 to November 2013. Five individuals from different families, had undergone renal transplantation whether they develop (acute/hyper acute/chronic) rejection. Sample size include five patients and one control, convenience sampling technique was choose which is based on elements selected from a population on the basis of what elements are easy to obtain. Sometimes a convenience sample is called a grab sample as we essentially grab members from the population for our sample. This is a type of sampling technique that does not rely upon a random process, such as we see in a simple random sample, to generate a sample. Committee of ministry of health, Khartoum state approved the protocol and a written informed consent was obtained from all participants prior to study participation.
Genomic DNA (gDNA) were extracted from the patient’s peripheral blood leukocytes using CinnaPure DNA extractions kits. Sequencing done for the area within HLA-DRB1 gene (301 base pairs), region location of chromosome 6: 32584100, 32584400. Using Roche/454 Genome Sequencer FLX + Titanium, Deep sequencing up to 1000 bp read length and up to 1.1 Gb/run.
Firstly; DNA sequences were compared with the NCBI human reference genome (www.ncbi.nlm.nih.gov) to check DNA sequencing quality and specificity by using BLAST (basic local alignment search tool), it’s a fast sequence similarity searching (http://blast.ncbi.nlm.nih.gov/Blast.cgi) (
http://www.ch.embnet.org/software/BOX_form.html. Thirdly; three identified SNPs were submitted to NCBI human reference genome for the second time to check if the identified SNPs are known or novel, then protein sequence of SNPs were get from UniProt database (
Sample code No. | Gene | SNP ID | Chromosome location | New nucleotide base pair (bp) | Protein ID | Amino acid change |
---|---|---|---|---|---|---|
Pt 0.0014 | HLA-DRB1 Gene bank ID: 3123 | rs1059575 | Chr6: 32584308 | G | NP_002115 | D57E |
Novel | Chr6: 32584307 | C | - | S56S | ||
Pt 0.0016 | rs17885437 | chr6: 32584259 | G | NP_002115 | G74R |
SNP to facilitate the interpretation of results, e.g., structural environment and multiple sequence alignment [
(http://gpcr2.biocomp.unibo.it/cgi/predictors/I-Mutant3.0/I-Mutant3.0.cgi), Project Hope
(http://www.cmbi.ru.nl/hope/home). Fourthly; protein sequence of deleterious SNP placed to get protein secondary structure using GOR IV tool (secondary structure prediction tool) and result shown in
SNP ID | Amino acid variant | Phenotype prediction | Environment | Area buried | Frac polar | Secondary structure | SIFT prediction | Prediction score |
---|---|---|---|---|---|---|---|---|
rs1059575 | D57E | Neutral | P1S | 0.445 | 0.406 | S | TOLERATED | 1 |
Novel | S56S | - | - | - | - | - | TOLERATED | 0.05 |
rs17885437 | G74R | Disease | EC | 0.126 | 0.552 | C | DAMAGING | 0.01 |
Environment: The structural environment of the SNP calculated by the environment program, Area buried: Solvent accessibility score, Frac polar: Environmental polarity score, SIFT and Score: Ranges from 0 to 1, the amino acid substitution is predicted damaging if the score is ≤0.05, and tolerated if the score is >0.05.
SNP ID | Prediction result |
---|---|
rs1059575 | |
Novel | No prediction results |
rs17885437 |
PolyPhen-2 score (range from 0 to 1): Probably damaging (~ >0.80), possibly damaging (~ >0.60 to 0.80) and benign (~ ≤0.60).
SNP ID | Amino acid position | WT | MT | PH | Temp (˚C) | Stability | DDG value prediction Kcal/mol |
---|---|---|---|---|---|---|---|
rs1059575 | 57 | D | E | 7.0 | 25 | Decrease | −0.54 |
Novel | 56 | S | S | 7.0 | 25 | - | - |
rs17885437 | 74 | G | R | 7.0 | 25 | Decrease | −1 |
WT: Wild type amino acid, MT Mutant type amino acid, DDG: DG (New Protein)-DG (Wild Type) in Kcal/mol (DDG < 0: Decrease stability, DDG > 0: Increase stability), RI: Reliability index.
BLAST results showed high similarity to Homo sapiens HLA-DRB1 gene for all sequences (samples and control), with identical percentage between 92% - 93%, and these results showed great sequencing results (
Alignment showed the highly conserved target sequencing region for all sequences. Three SNPs were detected, first SNP was in sample (0016) and other two SNPs were within sample (0014) (
Differences | Glycine (native amino acid) | Arginine (mutant amino acid) |
---|---|---|
Schematic structure | ||
Size | Small in size | Bigger in size |
Charge | Natural in charge | Positively charge and it could make repulsion between the mutant residue and neighboring residues |
Flexibility | Most flexible of all residues | Can abolish this function which it might be necessary for the protein’s function |
Conservation | Highly conserved | Not observed at all, this mean in just some rare cases the mutation might occur without damaging the protein |
Properties | Calculates |
---|---|
- Molecular weight: | 30030.1 |
- Theoretical pI | 7.64 |
- Amino acid composition | Ala (A) 11 4.1% Arg (R) 19 7.1% Asn (N) 8 3.0% Asp (D) 9 3.4% Cys (C) 6 2.3% Gln (Q) 14 5.3% Glu (E) 17 6.4% Gly (G) 24 9.0% His (H) 6 2.3% Ile (I) 3 1.1% Leu (L) 27 10.2% Lys (K) 8 3.0% Met (M) 6 2.3% Phe (F) 16 6.0% Pro (P) 13 4.9% Ser (S) 21 7.9% Thr (T) 18 6.8% Trp (W) 5 1.9% Tyr (Y) 10 3.8% Val (V) 25 9.4% Pyl (O) 0 0.0% Sec (U) 0 0.0% |
- Total number of negatively charged residues (Asp + Glu) - Total number of positively charged residues (Arg + Lys) | 26 27 |
- Formula: - Total number of atoms: | C1344H2064N370O390S12 4180 |
- Extinction coefficients: (extinction coefficients are in units of M−1∙cm−1, at 280 nm measured in water). | 42,775 |
- Estimated half-life | 30 hours |
- Aliphatic index | 75.38 |
- Grand average of hydropathicity (GRAVY) | −0.260 |
Three tools were used to predict the functionality of three detected SNPs, results showed that two of three SNPs were predicted as tolerated and third one as damaging (
Protein sequence of target SNPs was submitted, results show stability change (decrease) for two SNPs (rs1059575, rs17885437) with deferments DDG values (
Protein sequence of just a predicted pathological SNP was submitted to Project Hope server, results show that there were wide physiochemical changes due to single variant from wide type (Glycine) to new type (Arginine) (
Homology modeling of target pathological SNP did used CPH model 3.2 server, and then model was visualized used chimera software, results shown the structural difference between wide and new amino acid in position 74 with 26 point contact (clash contact) of the mutant residue with neighbor atom, this contact point will increase the severity of damaging (
The study involved a group of five renal transplant known rejection patients on hemodialysis (plus one control), patients were dialyzing two to three times per week, and they were at different duration of renal transplant rejection from two to ten years when samples had been collected. Our main objective was to detect presence of SNPs through Sudanese genome of 23 pathological nsSNPs which identified located worldwide in this chromosome location through in silico study done by Hassan (2014).
Three SNPs were detected in this study using DNA sequencing technology, two of the three were known SNPs in dbSNPs (SNPs database) (rs1059575, rs17885437), and the third SNP was a novel SNP (not found in dbSNP). Then bioinformatics tools were used to determine the effect of three detected SNPs on the protein structure and function, we found that two were tolerant and one is damaging. The damaging SNP in HLA complex genes may affect renal transplantation rejection. From the above, the results are similar to Hassan’s results [
HLA typing using SNPs analysis is a suitable, accurate and cheap way to cover all types of HLA genes and could be used whole over the world. Damaging SNPs detections also could be an answer for unknown causes of many organ transplantation rejection cases. Results showed the power and impact of in silico tools on biomedical research and their ability to uncover the cause of genetic variations in different genetic diseases.
The authors declare that they have no conflicts of interest related to this work.
Mohamed M.Hassan,11,Sofia B.Mohamed,Mohamed A.Hussain,Amar A.Dowd, (2015) Deleterious Nonsynonymous SNP Found within HLA-DRB1 Gene Involved in Allograft Rejection in Sudanese Family: Using DNA Sequencing and Bioinformatics Methods. Open Journal of Immunology,05,222-232. doi: 10.4236/oji.2015.54018