Unique Polymorphisms at BCL11A, HBS1L-MYB and HBB Loci Associated with HbF in Kuwaiti Patients with Sickle Cell Disease.

Patients with sickle cell disease (SCD) in Kuwait have elevated HbF levels ranging from ~10-44%; however, the modulating factors are unclear. We investigated the association of single nucleotide polymorphisms (SNPs) at BCL11A, HBS1L-MYB and HBB with HbF levels in 237 Kuwaiti SCD patients, divided into 3 subgroups according to their HbF levels. Illumina Ampliseq custom DNA panel was used for genotyping and confirmed by arrayed primer extension or Sanger sequencing. In the BCL11A locus, the CC genotype of rs7606173 [χ2 = 16.5] and (GG) of rs10195871 [χ2 = 15.0] were associated with Hb-F1 and HbF-2 subgroups, unlike rs1427404-T [χ2 = 17.3], which showed the highest association across the three subgroups. HBS1L-MYB locus revealed 2 previously-described SNPs (rs66650371 [χ2 = 9.5] and rs35795442 [χ2 = 9.2]) and 2 previously-unreported SNPs, (rs13220662 [χ2 = 6.2] and rs1406811 [χ2 = 6.7]) that were associated with the HbF-3 subgroup, making this the key locus elevating HbF to the highest levels. HBB cluster variants were associated with lower levels of HbF (ß = -1.1). We report four previously-unpublished variants showing significant association with HbF. Each of the three quantitative trait loci affects HbF levels differently; unique SNPs, especially in HBS1L-MYB, elevate HbF to the highest levels.

Diagnosis of Sickle Cell Disease and HBB Haplotyping in the Era of Personalized Medicine: Role of Next Generation Sequencing.

Hemoglobin genotype and HBB haplotype are established genetic factors that modify the clinical phenotype in sickle cell disease (SCD). Current methods of establishing these two factors are cumbersome and/or prone to errors. The throughput capability of next generation sequencing (NGS) makes it ideal for simultaneous interrogation of the many genes of interest in SCD. This study was designed to confirm the diagnosis in patients with HbSS and Sß-thalassemia, identify any ß-thal mutations and simultaneously determine the ßS HBB haplotype. Illumina Ampliseq custom DNA panel was used to genotype the DNA samples. Haplotyping was based on the alleles on five haplotype-specific SNPs. The patients studied included 159 HbSS patients and 68 Sß-thal patients, previously diagnosed using high performance liquid chromatography (HPLC). There was considerable discordance between HPLC and NGS results, giving a false +ve rate of 20.5% with a sensitivity of 79% for the identification of Sßthal. Arab/India haplotype was found in 81.5% of ßS chromosomes, while the two most common, of the 13 ß-thal mutations detected, were IVS-1 del25 and IVS-II-1 (G>A). NGS is very versatile and can be deployed to simultaneously screen multiple gene loci for modifying polymorphisms, to afford personalized, evidence-based counselling and early intervention.

Alpha thalassemia genotypes in Kuwait.

BACKGROUND: The frequency of the alpha thalassemia trait is approximately 40% in the Kuwaiti population, but there has been no comprehensive study of the prevalent alleles. This is a report of patients who were referred for molecular diagnosis over a 20-year period. METHODS: This is a retrospective study of the α-globin genotypes obtained in the Hemoglobin Research Laboratory of the Department of Pediatrics, Kuwait University from 1994 to 2015. Genotyping was performed by a combination of PCR, allele-specific oligonucleotide hybridization and reverse dot blot hybridization (Vienna Lab Strip Assay). RESULTS: Four hundred samples were characterized and analyzed from individuals aged < 1 month to 80 years, with a median of 6 years from 283 unrelated families. Most (90.8%) were Kuwaiti nationals. The commonest genotype was homozygosity for the polyadenylation-1 mutation (αPA-1α/α PA-1α) in 33.3% of the samples, followed by heterozygosity (αα/α PA-1α) for the same mutation in 32.3%. PA-1 was therefore the most frequent allele (0.59). The frequency of the α0 (--MED) allele was 0.017. Rare alleles that were found in very low frequencies included α0 (--FIL) in a Filipino child, Hb Constant Spring, Hb Adana, and Hb Icaria. CONCLUSION: There is a wide variety of alpha thalassemia alleles among Kuwaitis, but nondeletional PA-1 is by far the most common cause of the moderate to severe HbH (ß4 tetramer) disease phenotype. The α0 (-MED) allele is also encountered, which has implications for premarital counseling, especially for the possibility of having babies with alpha thalassemia major (Barts hydrops fetalis).

High prevalence of protein tyrosine phosphatase non-receptor N22 gene functional variant R620W in systemic lupus erythematosus patients from Kuwait: implications for disease susceptibility.

BACKGROUND: Systemic lupus erythematosus (SLE) is an autoimmune inflammatory disease which involves the loss of self-tolerance with hyperactivation of autoreactive T- and B-cells. Protein tyrosine phosphatase non-receptor type 22 (PTPN22) encodes for lymphoid specific phosphatase (LYP) which is a key negative regulator of T lymphocyte activation. The aim of this study was to investigate the association between PTPN22 gene functional variant R620W and systemic lupus erythematosus (SLE) by comparing its prevalence in Kuwaiti SLE patients and controls. METHODS: The study included 134 SLE patients and 214 controls from Kuwait. The genotypes of PTPN22 gene functional variant R620W were determined by PCR-RFLP and confirmed by DNA sequence analysis in both SLE patients and the controls. RESULTS: A relatively high prevalence of the variant 620 W (T-allele) of the PTPN22 gene was detected in the SLE patients from Kuwait. 35.7% of the SLE patients had at least one variant allele (T-allele) compared to 15.9% in the controls. A statistically significant difference was detected in the frequency of variant genotypes, TT and CT between SLE patients and the controls (p < 0.0001). No association was detected between the PTPN22 gene variant and the Raynaud's phenomenon, renal involvement and severity of the SLE. CONCLUSIONS: The frequency of PTPN22 gene functional variant R620W reported in this study is amongst the highest compared to other world populations. A high prevalence of this variant in SLE patients in comparison to the healthy controls suggests its significant contribution in conferring susceptibility to SLE together with other factors.

Significant association of insertion/deletion polymorphism of the angiotensin-converting enzyme gene with rheumatoid arthritis.

OBJECTIVE: Only 30% of the genetic contribution to rheumatoid arthritis (RA) can be attributed to HLA genes, and other non-HLA genes may play a role in RA susceptibility. Angiotensin-converting enzyme (ACE) has been reported to be involved in pathogenesis of RA, and high levels of ACE have been documented in RA synovial fluid and pleural effusions. Since plasma and tissue levels of ACE are determined at the transcriptional level, we test the hypothesis that the genotype of ACE in RA patients may be a determining factor in pathogenesis. METHODS: Sixty patients with RA were recruited and clinically characterized according to disease duration, disease severity, disease activity, and American College of Rheumatology functional classes. ACE gene I/D polymorphism genotypes were determined in patients and healthy controls, using polymerase chain reaction. RESULTS: We found a significant overrepresentation of the DD genotype and the D allele in patients with RA; and we found that men with RA exhibited a higher frequency of the DD genotype and D allele compared to male controls. By logistic regression analysis the DD genotype confers a relative risk for development of RA of 3. CONCLUSION: Our study found an association between ACE deletion polymorphism and RA.

Hemoglobin F concentration as a function of age in Kuwaiti sickle cell disease patients.

OBJECTIVE: This study aimed to document the transition of hemoglobin (Hb) F levels from early childhood to adulthood in Kuwaiti sickle cell disease patients, investigating its relationship to sex, Hb genotype and coexistence of alpha-thalassemia trait. SUBJECTS AND METHODS: The following parameters were extracted from the patients' records: age, sex, Hb, mean corpuscular volume, mean corpuscular Hb, red blood cell count, Hb F, Hb S, Hb A(2) and alpha-globin genotype. Hb quantitation was performed with cation exchange HPLC, while alpha-globin genotype was determined by PCR. RESULTS: Records were available for 149 patients, made up of 94 SS and 55 Sbeta(0)thal; 83 males and 66 females, aged 3 months to 60 years (mean 10.5 +/- 1.8). The mean Hb F level in the whole population was 21.5 +/- 8.1% and was not significantly different between males and females, and SS or Sbeta(0)thal. When the age groups were analyzed, the Hb F level was highest (28.9 +/- 10.9%) in those below 5 years. Indeed, patients < or =2 years had a mean level of 31.9 +/- 13.0%. There was no significant difference in the Hb F levels in SS patients with or without coexistent alpha-thal trait. CONCLUSIONS: Kuwaiti sickle cell disease patients below 5 years of age have close to 30% Hb F and this is probably a major reason why they usually do not present before this age, unlike patients elsewhere who present within the first year of life.