New long noncoding RNA biomarkers and ceRNA networks on miR-616-3p in colorectal cancer: Bioinformatics-based study.

Background: Cancer development is aided by the role of long noncoding RNAs (lncRNAs) that act as competing endogenous RNAs (ceRNAs) absorbing microRNAs (miRNAs). We aimed to discover a novel regulatory axis in colorectal cancer (CRC) and potential biomarkers based on miR-616-3p. Materials and Methods: The gene expression omnibus database was mined for differentially expressed lncRNAs (DELs) and mRNAs. LncRNAs and mRNAs were predicted using the RegRNA and TargetScan databases. A combination of the ciBioPortal and Ensemble databases was used to locate the mRNAs. Cytoscape 3.7.1-built CeRNA networks. A quantitative real-time polymerase chain reaction (qRT-PCR) was utilized to confirm the expression levels of these RNA molecules. Statistical analyses were implemented by GraphPad Prism 9. Results: qRT-PCR showed (Linc01282, lnc-MYADM-1:1, and Zinc Finger Protein 347 [ZNF347]) were overexpressed whereas, (salt-inducible kinases 1 [SIK1], and miR-616-3p) were down regulated. Conclusion: These results identify unique, unreported lncRNAs as CRC prognostic biomarkers, as well as prospective mRNAs as new treatment targets and predictive biomarkers for CRC. In addition, our study uncovered unexplored ceRNA networks that should be studied further in CRC.

Calcium and dairy products in the chemoprevention of colorectal adenomas: a systematic review and meta-analysis.

The risk of transition to colorectal cancer (CRC) in advanced colorectal adenomas (ACAs) is about 2.5 times higher than the non-advanced ones. This systematic review and meta-analysis was performed to determine the effect of calcium and dairy products on the incidence of CAs and ACAs. Six databases were systematically searched and 37 relevant clinical trials and observational studies involving over 10,964 cases were selected for inclusion. The results showed that calcium consumption reduced the risk of CAs incidence by 8% (RR: 0.92; 95% CI: 0.89-0.96), and calcium intake as a food and dairy product reduced it about 21% (RR: 0.79; 95% CI: 0.72-0.86), and 12% (RR: 0.88; 95% CI: 0.78-0.98), respectively. However, calcium supplementation did not show a significant effect on CAs incidence (RR: 0.97; 95% CI: 0.89-1.05). Results also revealed that total calcium intake markedly reduced the risk of ACAs (RR: 0.79; 95% CI: 0.73-0.85) and the risk of recurrence of adenomas about 12% (RR: 0.88; 95% CI: 0.84-0.93). Our results suggest that natural sources of calcium such as dairy products and foods may have more effective role than supplementary calcium in terms of reducing the risk of incidence and recurrence of colorectal adenomas and advanced adenomas.

Constructing a novel competing Endogenous RNAs network based on NR3C1 and X-linked inhibitor of apoptosis protein genes reveals potential prognostic biomarkers in colorectal cancer.

Background: Long noncoding RNAs (lncRNAs) have been recognized as the main modulatory molecules in various cancers and perform as competing endogenous RNAs (ceRNAs). The nuclear hormone receptor superfamily of ligand-activated transcription factors (NR3C1) regulates numerous proliferative and metabolic processes such as tumorigenesis and metabolic diseases. Furthermore, X-linked inhibitor of apoptosis protein (XIAP) belongs to a family of the inhibitors of apoptosis proteins, is located downstream of the glucocorticoid receptor (GR or NR3C1) pathway, and cooperates with GR to suppress apoptosis. However, the underlying mechanisms of NR3C1 and XIAP in colorectal cancer (CRC) remain mainly unclear. This research aims to clarify the potential RNA biomarkers and to construct a novel ceRNA network in CRC. Materials and Methods: Multistep bioinformatics methods such as Lnc2cancer and miRDB databases were applied to identify candidate lncRNAs and miRNAs. The interaction energy between lncRNAs, NR3C1, and XIAP genes was analyzed by the LncRRIsearch database. Plus, microRNAs and lncRNA were evaluated via the Diana tools database to select microRNAs with the most binding scores. Quantitative reverse transcription-polymerase chain reaction (QRT-PCR) was applied to verify RNA molecules' expression levels and their association with the clinicopathological factors in 30 CRC tissues compared to 30 adjacent tissues. Results: QRT-PCR showed upregulation of KCNQ1OT1, NR3C1, and XIAP and downregulation of miR-421. The ceRNA network was constructed with 17 lncRNAs, 2 mRNAs, and 42 miRNAs. Thus, we explained the potential interactions between KCNQ1OT1 and miR-421 with NR3C1 and XIAP genes. Conclusion: Our study represents potential prognostic biomarkers and a new ceRNA network for further study in CRC.

Autozygosity-driven genetic diagnosis in consanguineous families from Italy and the Greater Middle East.

Autozygosity-driven exome analysis has been shown effective for identification of genes underlying recessive diseases especially in countries of the so-called Greater Middle East (GME), where high consanguinity unravels the phenotypic effects of recessive alleles and large family sizes facilitate homozygosity mapping. In Italy, as in most European countries, consanguinity is estimated low. Nonetheless, consanguineous Italian families are not uncommon in publications of genetic findings and are often key to new associations of genes with rare diseases. We collected 52 patients from 47 consanguineous families with suspected recessive diseases, 29 originated in GME countries and 18 of Italian descent. We performed autozygosity-driven exome analysis by detecting long runs of homozygosity (ROHs > 1.5 Mb) and by prioritizing candidate clinical variants within. We identified a pathogenic synonymous variant that had been previously missed in NARS2 and we increased an initial high diagnostic rate (47%) to 55% by matchmaking our candidate genes and including in the analysis shorter ROHs that may also happen to be autozygous. GME and Italian families contributed to diagnostic yield comparably. We found no significant difference either in the extension of the autozygous genome, or in the distribution of candidate clinical variants between GME and Italian families, while we showed that the average autozygous genome was larger and the mean number of candidate clinical variants was significantly higher (p = 0.003) in mutation-positive than in mutation-negative individuals, suggesting that these features influence the likelihood that the disease is autozygosity-related. We highlight the utility of autozygosity-driven genomic analysis also in countries and/or communities, where consanguinity is not widespread cultural tradition.

A Novel Cadherin 23 Variant for Hereditary Hearing Loss Reveals Additional Support for a DFNB12 Nonsyndromic Phenotype of CDH23.

BACKGROUND AND OBJECTIVES: Identification of the pathogenic mutations underlying hereditary hearing loss (HL) is difficult, since causative mutations in 60 different genes have so far been reported. METHODS: A comprehensive clinical and pedigree examination was performed on a multiplex family suffering from HL. Direct sequencing of GJB2 and genetic linkage analysis of 5 other most common recessive nonsyndromic HL (ARNSHL) genes were accomplished. Next-generation sequencing (NGS) was utilized to reveal the possible genetic etiology of the disease. RESULTS: NGS results showed a novel rare variant c.2977G>A (p.Asp993Asn) in the CDH23 gene. The variant, which is a missense in exon 26 of the CDH23 gene, fulfills the criteria of being categorized as pathogenic according to the American College of Medical Genetics and Genomics (ACMG) guideline. Electroretinography rejects the Usher syndrome in the family. CONCLUSIONS: The present study shows that an accurate molecular diagnosis based on NGS technologies largely improves molecular-diagnostic outcome and thus genetic counseling, and helps to clarify the recurrence risk in deaf families.

Therapeutic plasma exchange may adjust IL-6 and TGF-ß signals in relapsed MS patients peripheral blood.

OBJECTIVE: Effects of therapeutic plasma exchange (TPE) on immune cells and their cytokine production in MS, are unknown. Since interleukine-6 and tumor growth factor-ß have critical roles in MS immunopathogenesis, the impacts of TPE on the expression of these cytokines and their receptors on the surface of CD4+ T lymphocytes, were investigated. METHODS: Blood cells were obtained from 30 Relapsing-Remitting (RR) MS patients, before and after a complete TPE course. Cytokines mRNA and their receptor expression on the CD4+ T cells surface were assessed using real-time PCR and flowcytometry, respectively. RESULTS: TPE reduced symptom severity (P = .01) and the relief was higher in males than in females (P = .039). TPE also increased TGF-ß mRNA and decreased IL-6 receptor expressing cells frequency (P = .009 and P = .028, respectively). Moreover, the frequency of CD4+IL6R+ T cells was positively correlated with disease severity (P = .001). CONCLUSION: TPE impacts simultaneously on the TGF-ß mRNA and IL-6 receptor expression, and this may be a mechanism of improvement in MS relapse symptoms induced by the TPE.

Engineered zinc-finger nuclease to generate site-directed modification in the KLF1 gene for fetal hemoglobin induction.

Elevation of hemoglobin F (HbF) ameliorates symptoms of ß-thalassemia, as a common autosomal recessive disorder. In this study, the ability of an engineered zinc-finger nuclease (ZFN) system was assesed to disrupt the KLF1 gene to inhibit the γ to ß hemoglobin switching in K562 cells. This study was performed using a second generation integration-deficient lentiviral vector assigned to transient gene targeting. The sequences coding for zinc finger protein arrays were designed and subcloned in TDH plus as a transfer vector. Transduction of K562 cells was performed with the integrase minus lentivirus containing ZFN. The indel percentage of the transducted cells with lentivirus containing ZFN was about 29%. Differentiation of K562 cell line into erythroid cell lineage was induced with cisplatin concentration of 15 µg/mL. After differentiation, γ-globin and HbF expression were evaluated using real-time reverse-transcription polymerase chain reaction and hemoglobin electrophoresis methods. The levels of γ-globin messenger RNA were nine-fold higher in the ZFN treated cells compared with untreated cells 5 days after differentiation. Hemoglobin electrophoresis method showed the same results for HbF level measurement. Application of the ZFN tool to induce KLF1 gene mutation in adult erythroid progenitors might be a candidate to stimulate HbF expression in ß-thalassemia patients.

Effect of single nucleotide polymorphisms in SEPS1 and SEPP1 on expression in the protein level in metabolic syndrome in subjects with cardiovascular disease.

Metabolic syndrome (MetS) results from the interaction between environmental and genetic factors. Several previous studies considered the role of selenium in developing MetS. Two selenoproteins, selenoprotein S (SelS), and the Selenoprotein P (SePP) play an important role in antioxidative defense and therefore susceptibility to MetS. The involvement of SNPs in SEPP1 and SEPS1 have not been studied in MetS subjects. This study aims to investigate the association between the risk of MetS and four polymorphisms SEPS1 (rs28665122, rs4965373), SEPP1 (rs7579, rs3877899) in an Iranian population. The sample of this case-control study consisted of 132 Iranian patients with cardiovascular disease (71 MetS and 65 non-MetS subjects) from December 2015 to March 2016. Demographic data, medical history, and para-clinical were measured, and Taqman probes were used for allelic discrimination. The level of the SelS and the SePP were measured by the ELIZA method. No significant differences were found in the genotype frequencies of SEPS1 (rs4965373, rs28665122), SEPP1 (rs7579, rs3877899) in patients with MetS and the non-MetS group. The mean of SelS in MetS subjects with SEPS1 (rs4965373) GG genotype is significantly lower than the non-MetS group (4496.99 ± 3688.5 vs. 6148.6 ± 1127.0, P = 0.009). The mean of SePP in MetS subjects with SEPP1 (rs3877899) GG genotype is significantly lower than the non-MetS group (40.73 ± 8.44 vs.83.91 ± 21.33, P = 0.002). The mean of SePP in MetS subjects with SEPP1 (rs7579) GG genotype is lower than the non-MetS group (55.52 ± 16.7 vs. 109.48 ± 29.78, P = 0.01). In summary, the results of this study does not indicate significant differences in the SEPP1 (rs7579, rs3877899) and SEPS1 (rs4965373, rs28665122) genotypes between MetS and non-MetS subjects. However, the results show that the mean of expression of SelS and SePP decreased in the subjects with SEPP1 (rs7579) GG and SEPP1 (rs3877899) GG.

A Novel Pathogenic Variant in the CABP2 Gene Causes Severe Nonsyndromic Hearing Loss in a Consanguineous Iranian Family.

BACKGROUND AND OBJECTIVES: Hereditary hearing loss (HL) can originate from mutations in one of many genes involved in the complex process of hearing. CABP2 mutations have been reported to cause moderate HL. Here, we report the whole exome sequencing (WES) of a proband presenting with prelingual, severe HL in an Iranian family. METHODS: A comprehensive family history was obtained, and clinical evaluations and pedigree analysis were performed in the family with 2 affected members. After excluding mutations in the GJB2 gene and 7 other most common autosomal recessive nonsyndromic HL (ARNSHL) genes via Sanger sequencing and genetic linkage analysis in the family, WES was utilized to find the possible etiology of the disease. RESULTS: WES results showed a novel rare variant (c.311G>A) in the CABP2gene.This missense variant in the exon 4 of the CABP2gene meets the criteria of being pathogenic according to the American College of Medical Genetics and Genomics (ACMG) interpretation guidelines. CONCLUSIONS: Up to now, 3 mutations have been reported for the CABP2gene to cause moderate ARNSHL in different populations. Our results show that CABP2variantsalso cause severe ARNSHL, adding CABP2to the growing list of genes that exhibit phenotypic heterogeneity. Expanding our understanding of the mutational spectrum of HL genes is an important step in providing the correct clinical molecular interpretation and diagnosis for patients.

Distribution of CYP2D6 polymorphism in the Middle Eastern region.

Cytochrome P450 2D6 (CYP2D6) is an important drug-metabolizing enzyme involved in the pharmacokinetic metabolism of drugs. CYP2D6 gene is highly polymorphic, and the combination of its different alleles yields different phenotypes including extensive metabolizer (EM), intermediate metabolizer (IM), poor metabolizer (PM), and ultrarapid metabolizer (UM). Genotyping of the important alleles for this gene in different ethnicities is of particular importance for assessing the efficacy of various drugs. In this study, we reviewed the CYP2D6 allele and phenotype frequencies predicted from the genotypes of CYP2D6 in the Middle East area. Regardless of different ethnicities, the CYP2D6*41 allele frequency was shown to be higher than that of other reduced functional alleles. In addition, CYP2D6*4 was the most frequent nonfunctional allele in all studied populations in the Middle East. Taken together, our findings illustrated that the frequencies of PM or IM alleles and different genotypes harboring these alleles are relatively high in the Middle Eastern countries. Therefore, the study of CYP2D6 alleles for each patient to detect those that are at risk is of great importance to prevent adverse drug reactions through individualization therapy.

The association between genetic polymorphisms of the interleukin-10, tumor necrosis factor-alpha, and annexin A5 gene loci and restenosis after percutaneous coronary angioplasty and stenting.

BACKGROUND: Advances in the technology for percutaneous coronary angioplasty, such as coated stents, have reduced its complications, but restenosis remains an important clinical problem. The factors associated with an increased risk of restenosis include diabetes mellitus and multiple coronary artery disease. It is also possible that genetic factors play a role in restenosis although there are little data on this. We have investigated the association of three genetic markers of genes involved in inflammation leading to restenosis. MATERIALS AND METHODS: In this case-control study, 306 unrelated Iranian patients who were thought to have restenosis on clinical grounds were investigated. Based on the results of angiography, 104 patients were found to have >50% stenosis within an implanted stent, and these were allocated to the in-stent restenosis (ISR) group; 202 patients with no in-stent stenosis or stenosis ≤50% were allocated to the non-ISR (NISR) group. Demographic data were collected from medical records. Biochemical parameters were measured using routine methods. Genotypes of the interleukin-10 (IL-10), annexin A5 (AnxA5), and tumor necrosis factor-alpha (TNFα) loci were determined using real-time polymerase chain reaction and a high-resolution melting assay. RESULTS: Fasting blood glucose, serum triglycerides, and serum high-sensitivity C-reactive protein (hs-CRP) concentrations were higher in the ISR group than in the NISR group (P < 0.05), and a history of diabetes mellitus was significantly related to the presence of restenosis (P < 0.001). There were no significant differences in the frequency of the genetic polymorphisms of IL-10, AnxA5, and TNFα genes and the presence of ISR. CONCLUSION: After adjustment for clinical variables, the genetic polymorphisms at the IL-10, TNFα, and ANXA5 gene loci do not appear to be risk factors for >50% ISR in our population. However, our data suggested a significant association between diabetes mellitus, serum hs-CRP, stent type, and restenosis.

A novel homozygous DPH1 mutation causes intellectual disability and unique craniofacial features.

Biallelic mutations of the gene encoding diphthamide biosynthesis 1 (DPH1, NM_001383.3) cause developmental delay, dysmorphic features, sparse hair, and short stature (MIM *603527). Only two missense DPH1 mutations have been reported to date. Here, we describe a consanguineous family with two siblings both showing developmental delay, agenesis of the corpus callosum, dysmorphic facial features, sparse hair, brachycephaly, and short stature. By wholeexome sequencing, a homozygous frameshift mutation in DPH1 (c.1227delG, p.[Ala411Argfs*91]) was identified, which is likely responsible for the familial condition. The unique clinical features of the affected siblings are cleft palate and absent renal findings.

A homozygous NOP14 variant is likely to cause recurrent pregnancy loss.

Recurrent pregnancy loss is newly defined as more than two consecutive miscarriages. Recurrent pregnancy loss occurs in <5% of total pregnancies. The cause in approximately 40-60% of recurrent pregnancy loss cases remains elusive and must be determined. We investigated two unrelated Iranian consanguineous families with recurrent pregnancy loss. We performed exome sequencing using DNA from a miscarriage tissue and identified a homozygous NOP14 missense variant (c.[136C>G];[136C>G]) in both families. NOP14 is an evolutionally conserved protein among eukaryotes and is required for 18S rRNA processing and 40S ribosome biogenesis. Interestingly, in zebrafish, homozygous mutation of nop14 (possibly loss of function) resulting from retrovirus-mediated insertional mutagenesis led to embryonic lethality at 5 days after fertilization, mimicking early pregnancy loss in humans. Similarly, it is known that the nop14-null yeast is inviable. These data suggest that the homozygous NOP14 mutation is likely to cause recurrent pregnancy loss. Furthermore, this study shows that exome sequencing is very useful to determine the etiology of unsolved recurrent pregnancy loss.

Evaluation of the utility of peripheral blood vs bone marrow in karyotype and fluorescence in situ hybridization for myelodysplastic syndrome diagnosis.

OBJECTIVE: To clear the role of peripheral blood as a substitution for bone marrow in myelodysplastic syndrome and to evaluate the concordance between peripheral blood and bone marrow using karyotype and fluorescence in situ hybridization (FISH) methods. METHODS: We examined 35 bone marrow (BM) and peripheral blood (PB) samples from myelodysplastic syndrome (MDS) patient using karyotype and FISH. Karyotype method for BM and PB samples performed using the standard protocol with an exception for peripheral blood in which growth factor for cultivation was not used. FISH testing was performed using a panel of MDS-associated probes to detect 20q12, 20qter, 5q31, 5q33, 5p15 and chromosome 7 and 8 centromeres. RESULTS: Our results showed karyotypes of BM and PB are concordant in 74% of cases, while about 53% of these concordances were achieved from cases with normal karyotypes. However, the results of BM FISH were completely concordant with PB FISH. CONCLUSION: Although peripheral blood karyotype is not trustworthy for MDS diagnosis, examining peripheral blood, using the FISH method, could be useful for clinical monitoring.

Association of AHSG with alopecia and mental retardation (APMR) syndrome.

Alopecia with mental retardation syndrome (APMR) is a very rare autosomal recessive condition that is associated with total or partial absence of hair from the scalp and other parts of the body as well as variable intellectual disability. Here we present whole-exome sequencing results of a large consanguineous family segregating APMR syndrome with seven affected family members. Our study revealed a novel predicted pathogenic, homozygous missense mutation in the AHSG (OMIM 138680) gene (AHSG: NM_001622:exon7:c.950G>A:p.Arg317His). The variant is predicted to affect a region of the protein required for protein processing and disrupts a phosphorylation motif. In addition, the altered protein migrates with an aberrant size relative to healthy individuals. Consistent with the phenotype, AHSG maps within APMR linkage region 1 (APMR 1) as reported before, and falls within runs of homozygosity (ROH). Previous families with APMR syndrome have been studied through linkage analyses and the linkage resolution did not allow pointing out to a single gene candidate. Our study is the first report to identify a homozygous missense mutation for APMR syndrome through whole-exome sequencing.

Identification a novel mononucleotide deletion mutation in GAA in pompe disease patients.

BACKGROUND: Mutations in the acid alpha-glucosidase (GAA) gene usually lead to reduced GAA activity. In this study, we analyzed the mutations of GAA and GAA enzyme activity from one sibling suspected Pompe disease and their first-degree relatives. MATERIALS AND METHODS: In this cross-sectional study, GAA enzyme activity assay was assessed using tandem mass spectrometry. Polymerase chain reaction and Sanger sequencing were performed for GAA analysis. RESULTS: GAA enzyme activity was significantly decreased in patients compared to the normal range (P = 0.02). Two individuals showed ten alterations in the GAA sequence, in which one of them (c. 1650del G) has not been previously described in the literature. A single Guanine deletion (del-G) was detected at codon 551 in exon 12. CONCLUSION: According to the literature, the detected change is a novel mutation. We hypothesized that the discovered deletion in the GAA might lead to a reduced activity of the gene product.

Genetic disruption of the KLF1 gene to overexpress the γ-globin gene using the CRISPR/Cas9 system.

BACKGROUND: ß-thalassemia comprises a major group of human genetic disorders involving a decrease in or an end to the normal synthesis of the ß-globin chains of hemoglobin. KLF1 is a key regulatory molecule involved in the γ- to ß-globin gene switching process directly inducing the expression of the ß-globin gene and indirectly repressing γ-globin. The present study aimed to investigate the ability of an engineered CRISPR/Cas9 system with respect to disrupting the KLF1 gene to inhibit the γ- to ß-hemoglobin switching process in K562 cells. METHODS: We targeted three sites on the KLF1 gene, two of which are upstream of codon 288 in exon 2 and the other site being in exon 3. RESULTS: The average indel percentage in the cells transfected with CRISPR a, b and c was approximately 24%. Relative quantification was performed for the assessment of γ-globin expression. The levels of γ-globin mRNA on day 5 of differentiation were 8.1-, 7.7- and 1.8-fold in the cells treated with CRISPR/Cas9 a, b and c, respectively,compared to untreated cells. The measurement of HbF expression levels confirmed the same results. CONCLUSIONS: The findings obtained in the present study support the induction of an indel mutation in the KLF1 gene leading to a null allele. As a result, the effect of KLF1 on the expression of BCL11A is decreased and its inhibitory effect on γ-globin gene expression is removed. Application of CRISPR technology to induce an indel in the KLF1 gene in adult erythroid progenitors may provide a method for activating fetal hemoglobin expression in individuals with ß-thalassemia or sickle cell disease.

Reciprocal 22q11.2 Deletion and Duplication in Siblings with Karyotypically Normal Parents.

The 22q11.2 locus is known to harbor a high risk for structural variation caused by non-allelic homologous recombination, resulting in deletions and duplications. Here, we describe the first family with one sibling carrying the 22q11 deletion and the other carrying the reciprocal duplication. FISH and SNP array analysis of the parents show a maternal origin for both deletion and duplication, without indications of balanced deletions/duplications or mosaicism. We hypothesize that germline mosaicism in the mother underlies the deletion and duplication, which would implicate a high recurrence risk for her offspring.

A novel homozygous mutation in HSF4 causing autosomal recessive congenital cataract.

Cataract is defined as opacity in the crystalline lens and congenital cataract occurs during the first year of life. Until now, mutations of more than 50 genes in congenital cataract have been reported with various modes of inheritance. Among them, HSF4 mutations have been reported in autosomal dominant, autosomal recessive and age-related forms of cataract. The inheritance patterns of these mutations depend on their mutational positions in HSF4: autosomal dominant or recessive mutations are respectively found either in a DNA-binding domain or in (or downstream of) hydrophobic repeats. Here we report a novel homozygous HSF4 mutation (c.521T>C, p.Leu174Pro) in two affected sibs of an Iranian consanguineous family using whole exome sequencing. The mutation is predicted as highly pathogenic by in silico analysis (SIFT, Polyphen2 and MutationTaster) and is not found in any of control databases. This mutation is located in a hydrophobic repeat of the HSF4 protein, which is consistent with the mode of inheritance as an autosomal recessive trait.

Comparison of different methods for erythroid differentiation in the K562 cell line.

OBJECTIVE: To compare methods for erythroid differentiation of K562 cells that will be promising in the treatment of beta-thalassemia by inducing γ-globin synthesis. RESULTS: Cells were treated separately with: RPMI 1640 medium without glutamine, RPMI 1640 medium without glutamine supplemented with 1 mM sodium butyrate, RPMI 1640 medium supplemented with 1 mM sodium butyrate, 25 µg cisplatin/ml, 0.1 µg cytosine arabinoside/ml. The highest differentiation (84 %) with minimum toxicity was obtained with cisplatin at 15 µg /ml. Real-time RT-PCR showed that expression of the γ-globin gene was significantly higher in the cells differentiated with cisplatin compared to undifferentiated cells (P < 0.001). CONCLUSIONS: Cisplatin is useful in the experimental therapy of ß-globin gene defects and can be considered for examining the basic mechanism of γ-reactivation.