Implication of efflux pumps and ERG11 genes in resistance of clinical Trichosporon asahii isolates to fluconazole.

Introduction. Trichosporon asahii has been recognized as an opportunistic agent having a limited sensitivity to antifungal treatment.Hypothesis/Gap Statement. Molecular mechanisms of azole resistance have been rarely reported for Trichosproron asahii. Similar to other fungi, we hypothesized that both ERG11 gene mutation and efflux pumps genes hyper-expression were implicated.Aim. The current work aimed to study the sensitivity of clinical T. asahii isolates to different antifungal agents and to explore their resistance mechanisms by molecular methods including real-time PCR and gene sequencing.Methods. The sensitivity of T. asahii isolates to fluconazole, amphotericin B and voriconazole was estimated by the Etest method. Real-time PCR was used to measure the relative expression of Pdr11, Mdr and ERG11 genes via the ACT1 housekeeping gene. Three pairs of primers were also chosen to sequence the ERG11 gene. This exploration was followed by statistical study including the receiver operating characteristic (ROC) curve analysis to identify a relationship between gene mean expression and the sensitivity of isolates.Results. In 31 clinical isolates, the resistance frequencies were 87, 16.1 and 3.2 %, respectively, for amphotericin B, fluconazole and voriconazole. Quantitative real-time PCR demonstrated that only Mdr over-expression was significantly associated with FCZ resistance confirmed by univariate statistical study and the ROC curve analysis (P <0.05). The ERG11 sequencing revealed two mutations H380G and S381A in TN325U11 (MIC FCZ=8 µg ml-1) and H437R in TN114U09 (MIC FCZ=256 µg ml-1) in highly conserved regions (close to the haem-binding domain) but their involvement in the resistance mechanism has not yet been assigned.Conclusion. T. asahii FCZ resistance mechanisms are proven to be much more complex and gene alteration sequence and/or expression can be involved. Only Mdr gene over-expression was significantly associated with FCZ resistance and no good correlation was observed between FCZ and VCZ MIC values and relative gene expression. ERG11 sequence alteration seems to play a major role in T. asahii FCZ resistance mechanism but their involvement needs further confirmation.

Use of FreeStyle Libre Flash Monitor Register in the Netherlands (FLARE-NL1): Patient Experiences, Satisfaction, and Cost Analysis.

In patients with diabetes mellitus (DM), adequate glucose control is of major importance. When treatment schemes become more complicated, proper self-management through intermittent self-measurement of blood glucose (SMBG), among others, becomes crucial in achieving this goal. In the last decade, continuous glucose monitoring (CGM) has been on the rise, providing not only intermittent information but also information on continuous glucose trends. The FreeStyle Libre (FSL) Flash CGM system is a CGM system mainly used for patients with DM and is designed based on the same techniques as early CGMs. Compared with earlier CGMs, the FSL is factory calibrated, has no automated readings or direct alarms, and is cheaper to use. Although less accurate compared with the gold standard for SMBG, users report high satisfaction because it is easy to use and can help users monitor glucose trends. The Flash Monitor Register in the Netherlands (FLARE-NL) study aims to assess the effects of FSL Flash CGM use in daily practice. The study has a before-after design, with each participant being his or her own control. Users will be followed for at least 1 year. The endpoints include changes in HbA1c, frequency and severity of hypoglycemias, and quality of life. In addition, the effects of its use on work absenteeism rate, diabetes-related hospital admission rate, and daily functioning (including sports performance) will be studied. Furthermore, cost-benefit analysis based on the combination of registered information within the health insurance data will be investigated. Ultimately, the data gathered in this study will help increase the knowledge and skills of the use of the Flash CGM in daily practice and assess the financial impact on the use of the Flash CGM within the Dutch healthcare system.

Performance of the FreeStyle Libre Flash glucose monitoring system in patients with type 1 and 2 diabetes mellitus.

OBJECTIVE: To evaluate the performance of the FreeStyle Libre Flash continuous glucose monitoring (FSL-CGM) system against established central laboratory methods. RESEARCH DESIGN AND METHODS: 20 subjects (8 type 1 diabetes mellitus, 12 type 2 diabetes mellitus) were analyzed. FSL-CGM sensor measurements (inserted in arm and abdomen) were compared with capillary blood glucose results analyzed with StatStrip as semigold standard. The glucose response after a standardized oral glucose load was measured by FSL-CGM and capillary samples analyzed by perchloric acid hexokinase (PCA-HK) method, StatStrip and FSL test strip (FSLC), and a commonly used CGM system (iPro2). RESULTS: FSL-CGM arm sensor readings showed 85.5% of paired readings falling within Clarke Error Grid (ISO 15197:2013) zone A when compared with StatStrip. For FSL-CGM abdomen and FSLC, these percentages were 64% and 98%, respectively. The overall correlation of FSL-CGM in the arm and the StatStrip indicates a performance with lower results with the FSL-CGM in the arm than expected based on the StatStrip in the lower glucose ranges, and higher results than expected in the higher ranges. Following a standardized glucose load, a slower rise in glucose level was observed for FSL-CGM arm as compared with PCA-HK, StatStrip, FSLC, and iPro2 during the first 45-60 min after glucose load ingestion. CONCLUSIONS: Certain matters need attention while using the FSL-CGM in daily life including the observed lower values in the lower ranges, and the underestimation of the effect of a meal on glucose response. These effects of such deviations can partly be overcome by optimizing the available user instructions. TRIAL REGISTRATION NUMBER: TC5348; results.

Molecular basis of pyruvate kinase deficiency among Tunisians: description of new mutations affecting coding and noncoding regions in the PKLR gene.

INTRODUCTION: Pyruvate kinase (PK) deficiency is one of the most common hereditary nonspherocytic hemolytic anemias worldwide with clinical manifestations ranging from mild to severe hemolysis. However, investigation of this enzymopathy is lacking in Tunisia. We report here a pioneer investigation of PK deficiency among Tunisian cases referred to our laboratory for biological analysis of unknown cause of hemolytic anemia. METHODS: Two hundred and fifty-three patients with unknown cause of hemolytic anemia have been addressed to our laboratory in order to investigate for red blood cells genetic disorders. Red cell enzyme activities were measured by standard methods, and molecular analysis was performed by DNA sequencing. The interpretation of mutation effect and the molecular modeling were performed by using specific software. RESULTS: Six different PKLR mutations were found (c.966-1G>T; c.965+1G>A; c.721G>T; c.1163C>A; c.1456C>T; c.1537T>A), among which four are described for the first time. Genotype-phenotype correlations for the novel missense mutations were investigated by three-dimensional structure analysis. CONCLUSION: This study provides important data of PK deficiency among Tunisians. It might be followed by a large neonatal screening to determine the spectrum of PK mutations and identify potential deficient patients for an early medical follow-up.

Analysis of virulence factors and in vivo biofilm-forming capacity of Yarrowia lipolytica isolated from patients with fungemia.

Yarrowia lipolytica is ubiquitous in the environment, opportunistic, and might be considered as one of the causative agents of catheter-related candidemia. Our work aimed to study some virulence factors of Y. lipolytica such as hydrolases production and biofilm formation with comparison to the most frequent Candida specie in human disease. In sum, 58 clinical isolates of Y. lipolytica, 16 C. glabrata, and 12 C. albicans were collected from Intensive care unit (ICU). All were tested for enzymatic production and biofilm formation. All tested isolates of C. albicans and C. glabrata were able to degrade casein, and 98.2% of Y. lipolytica showed caseinase activity but no gelatinase activity was detected in all isolates. Y. lipolytica strains showed significantly lower (3.4%) in vitro phospholipase activity than C. albicans and C. glabrata (P < .05). No significant differences of the hemolytic activity were detected between the three species (P > .05). Concerning biofilm formation, and unlike the results obtained on polystyrene plate, the number of adhered and biofilm cultivable cells obtained by Y. lipolytica after 168 hours of catheter subcutaneous implantation is significantly greater and tends to be more compact and structured hyphal layer. Although C. albicans remains the most pathogenic yeast, development of selective ability of Y. lipolytica to adhere, to form a biofilm on catheter medical devices, and to produce phospholipase and hemolytic enzyme is of particular interest, and it is strongly recommended to be vigilant in the use of medical implanted medical devices, particularly in ICU.

Rare hemoglobin variants in Tunisian population.

During the last 30 years, many studies concerning hemoglobinopathies were realized among Tunisians. More than twenty different thalassemic alleles were detected on the ß-globin gene, and less are affecting the α-globin genes. Unusual hemoglobin (Hb) variants other than Hb S, Hb C, and Hb O-arab, which are the most frequent variants in Tunisia, were also detected. Eight Tunisian subjects were studied at phenotypic and molecular levels. Hematological indices and hemoglobin (Hb) pattern were performed by alkaline electrophoresis and isoelectric focusing (IEF),and the Hb fractions were quantitated by cation exchange HPLC. On genomic level, coding regions were amplified by polymerase chain reaction (PCR) followed by a sequencing of the purified PCR products using the dye terminator method. Seven uncommon Hb variants were detected and described for the first time among Tunisians. HbA2-Tunis [δ46(CD5), Gly → Glu, GGG → GAG] is the newly described δ-chain variant in our laboratory, and some other variants (Hb Constant Spring, G San Jose, and Hb J-Bangkok) are very uncommon in the Mediterranean region. We present here an updated review of the Hb variants detected among Tunisians. Twenty-one rare Hb variants were detected affecting the α1-, α2-, δ-, γ-, and ß-globin genes, leading in some cases to a severe phenotype especially when the stability is completely altered. The ethnical history of Tunisia could explain this important variability of the observed rare Hb variants.

Reconstitution of regulatory T-cell subsets after allogeneic hematopoietic SCT.

Previous studies on regulatory T-cell (Treg) reconstitution after allogeneic hematopoietic SCT (HSCT) have suggested that, within the GVHD process, imbalance between effector T cells and Tregs may be more important than the absolute numbers of circulating Tregs. No study has analyzed naive vs memory Treg reconstitution in a longitudinal cohort with large numbers of patients. The reconstitution of total and subsets of Treg was prospectively analyzed by flow cytometry in 185 consecutive recipients at 3, 6, 12 and 24 months after allogeneic HSCT. The levels of total, naive and memory Tregs increased, mainly within the memory subset, but remained lower than healthy controls up to 2 years after transplantation. Reduced-intensity conditioning and peripheral blood (PBSC) as the source of stem cells were associated with better 3-month reconstitution. In multivariate analysis, PBSC, recipient age ⩽25 and no anti-thymoglobulin in the conditioning regimen were associated with a better Treg reconstitution. Naive Treg long-term reconstitution was mainly influenced by recipient age. Whereas prior acute GVHD impaired Treg reconstitution, Treg subsets (absolute numbers and frequencies relative to CD4(+) T-cell subsets) at 3, 6 and 12 months after HSCT were not associated with the occurrence of a later episode of chronic GVHD.

Frequency of three polymorphisms of the CCL5 gene (rs2107538, rs2280788 and rs2280789) and their implications for the phenotypic expression of sickle cell anemia in Tunisia.

The pro-inflammatory context of sickle cell disease promotes the liberation of cytokines such as CCL5, encoded by a gene located on chromosome 17. Herein, the occurrence of three variations of CCL5 in sickle cell anemia (SCA) and their relations to two major complications - painful crisis and presence of infections - were investigated. 100 SCA Tunisian patients and 100 healthy subjects were included in the case control study. Then the sample of patients was divided into two groups according to the presence or absence of each complication. The polymorphisms, namely g.-403G>A, g.-28C>G and g.In1.+1T>C, were analyzed by PCR/sequencing. Our findings show the presence of eight genotypes, namely GG, GA and AA of g.-403G>A, CC, CG and GG of g.-28C>G, and TT and TC of g.In1.+1T>C. The frequencies of studied single nucleotide polymorphisms (SNPs) and haplotypes in SCA patients do not differ significantly from healthy control group results. There is also no significant association between the analyzed polymorphisms and complications as for painful crisis and presence of infections (p > 0.05). Altogether, our data support the conclusion that the three polymorphisms of CCL5, namely g.-403G>A, g.-28C>G and g.In1.+1T>C, do not seem to be involved in the clinical variability of SCA in Tunisia.

Three new mutations account for the prevalence of glucose 6 phosphate deshydrogenase (G6PD) deficiency in Tunisia.

A previous study on G6PD deficiency carried out on Tunisian population, led to the finding of seven different mutations with the prevalence of G6PD A- variant. This present study reports 23 new unrelated deficient subjects studied at the molecular level to determine the mutation that causes G6PD deficiency. Using PCR-SSCP of coding regions followed by direct sequencing of abnormal pattern, three new mutations were detected. Two of them are polymorphic intronic mutations. The first is IVS-V 655C-->C/T, found in four female subjects with mild deficiency of class III variant. The second is IVS-VIII 43 G-->A, found in three male subjects with mild deficiency of class III variant. The third mutation is in the exon region so that it changes the primary structure of the molecule. It is cited for the first time and named G6PD Tunisia. This variant affects the exon 7 of the gene at genomic position 15435 G→T. Its cDNA position is 93 G→G/T, it changes arg 246 to leu. This mutation was found in one heterozygote female with deficiency of class II who have had hemolytic anemia due to ingestion of fava beans. Finally, G6PD Med variant, reported before in three cases, was also found in five other cases (four heterozygote females and one male hemizygote). These findings first enlarge the spectre of mutations to be ten variant mutations, characterizing the Tunisian population and also contribute with hemoglobin gene research in our laboratory to trace the whole genetic map of Tunisian population.

Candida glabrata strain relatedness by new microsatellite markers.

We investigated six microsatellite markers to type 85 unrelated and 118 related isolates of Candida glabrata from 36 patients. Three new markers were selected from the complete sequence of CBS138 and three previously described markers, RPM2, MTI and ERG3 were used. We found a genetic diversity of 0.949 by combining four of them. By applying the new microsatellite markers GLM4, GLM5 and GLM6 we were able to discriminate 29 isolates, originally identified by the more established markers, RPM2, MTI and ERG3. When epidemiologically closely related isolates from 36 patients were typed, 25 patients (72%) exhibited identical or highly related multilocus genotypes. We noted a microvariation in 4 of the patients. This minor change of one locus could be explained by a single step mutation. Since one of these patients had not received antifungal treatment; thus, the relationship between genome variation and antifungal therapy remains controversial. We can conclude from our analysis of these new microsatellite markers that they are highly selective and therefore should be considered as a useful typing system for differentiating related and unrelated isolates of C. glabrata, as well as being able to detect microvariation.

Antifungal susceptibility of bloodstream Candida isolates in Sfax hospital: Tunisia.

UNLABELLED: Invasive candidiasis has emerged as an important nosocomial infection, causing significant morbidity and mortality especially among critically ill patients. The aim of our study was to determine specie distribution and resistance profiles of Candida species isolated from blood cultures. MATERIALS AND METHODS: We conducted a retrospective study of all episodes of candidemia diagnosed in our laboratory from January 2006 to May 2009. The susceptibility to antifungal agents of all Candida isolates was tested by using a Sensititre(®) YeastOne panel. RESULTS: A total of 130 Candida isolates were recovered from blood cultures. Candida tropicalis was the most frequent specie (37.7%), followed by C. albicans (22.3%), C. glabrata (19.2%), and C. parapsilosis (12.2%). All the isolates were inhibited by ≤1 µg/ml of amphotericin B and ≤2 µg/ml of caspofungin. For fluconazole, 7.3% of clinical isolates were resistant. It was most active against C. parapsilosis (100% susceptible), C. albicans (95.8% susceptible), and C. tropicalis (94% susceptible). All of the fluconazole-susceptible isolates were susceptible to voriconazole, as were 83.3% of the fluconazole-susceptible-dose-dependent isolates. Among fluconazole-resistant isolates, 85.7% were susceptible to voriconazole. CONCLUSIONS: In our institution, C. tropicalis was the most frequent specie isolated from the bloodstream. Caspofungin had an excellent in vitro activity against Candida isolates and was the drug of choice among fluconazole-resistant isolates.

Epidemiological survey of vulvovaginal candidosis in Sfax, Tunisia.

Vulvovaginal candidosis (VVC) is a common infection of the female genital tract affecting 75% women at least once in their lifetime. The aim of this study was to determine the incidence and potential risk factors associated with VVC and recurrent vulvovaginal candidosis (RVVC). A prospective study of women with vaginitis symptoms was conducted over 2 years in the regional clinic of population and family education in Sfax. A discriminant analysis was used to evaluate the association between the incidence of Candida vaginitis and potential risk factors. Sporadic and recurrent VVC were documented respectively in 48% and 6.1%. The most frequent factors associated with positive Candida culture were employed women, uncontrolled diabetes, history of genital infection and intrauterine device contraception. Increased episode numbers of VVC and condom/spermicidal contraception were positively associated with recurrences. Candida albicans was the predominantly isolated species (76.3%) followed by Candida glabrata (19.3%). Infection with C. glabrata occurred in 34% and 17.5% of patients with RVVC and VVC respectively. The discriminant investigation had provided further insights into the basis for prevention and control of RVVC. Increased prevalence of C. glabrata in patients with RVVC and observed risk factors should be taken into consideration to achieve success in the management of this infection.

Microsatellite analysis and susceptibility to FCZ of Candida glabrata invasive isolates in Sfax Hospital, Tunisia.

We have noted that, during the last few years, there has been a redistribution of the most common Candida species with an increase in non-C. albicans Candida species, particularly Candida glabrata. In many countries, the high frequency of Candida glabrata shows the highest resistance rates. The main objective of this investigation was to analyze the genotypic variability of invasive C. glabrata isolates recovered over a period of six years and assess their in vitro susceptibility to fluconazole to determine the possible existence of relationships between genotype and susceptibility. We collected 50 invasive C. glabrata isolates (21.4%) from January 2001 to December 2007. The in vitro susceptibility profiles as determined by the E-test method showed that 8.3% of the isolates were resistant to fluconazole. The typing with three microsatellite markers RPM2, MTI and ERG3 demonstrated 12 multilocus genotypes distributed irregularly with a predominance of G1 (38%). A cluster (G9) was found among isolates collected in the same ward, at the same time period, suggesting cross transmission. Eleven of 13 patients who had previously been colonized by C. glabrata, were infected by their colonizing strains. However, we noted after prolonged treatment with fluconazole that there was an increase of the MIC for an isolate from one patient and in another patient, the selection of a more resistant variant. In our study, we didn't find an association between genotype and susceptibility to fluconazole. In conclusion, the predominance of some genotypes could be explained by nosocomial transmission or a selective ecological advantage rather than an emergence of a resistant isolate.

A silent composite hemoglobinopathy characterized by gene sequencing.

We report the case of a 35-year-old Tunisian women with a chronic anemia non investigated for a long time. Laboratory analysis using advanced technology of DNA sequencing revealed a compound heterozygote for Hb O Arab and cd 39 beta degrees-thalassemia. It's the first time that such a genotype has been characterized by gene sequencing.

[Polymorphisms of the UDP-glucuronosyltransferase 1-A1 gene in Tunisian population]. / Polymorphismes alleliques du gene UDP-glucuronosyltransferase 1A1 dans une population Tunisienne.

The UDP-glucuronosyltransferase 1A1 gene that encode the enzyme UGT1A1 responsible for glucuronidation undergoes several variations that may affect the enzymatic activity or expression and which are the cause of metabolic disorders related to the glucuronidation of bilirubin, such as Gilbert's syndrome and Crigler Najjar's syndrome. Among the most common variations, there is the repeat polymorphism A (TA) n TAA in TATA box and biallelic mutation G211A in exon 1. We consider in this work to determine their frequencies in a healthy population. The polymorphism A (TA) n TAA showed that genotype [TA7/TA7] was described as being associated with Gilbert's syndrome and was encountered in 11% of the population studied. This percentage is close to the value described in the Caucasian population, estimated at 10%. Concerning the polymorphism G211A, our results show that the mutated allele is encountered in 15.7% of our study population. This frequency differs greatly from that reported for Caucasians and Afro-Americans but it is similar to that perceived at the Japanese. All these results suggest that the Tunisian population appears to be heterogeneous view UGT1A1 gene mutation status. Regarding the origins and distribution of such polymorphisms in our population, the study reveals a high haplotypic haplotype (TA) 6-G considered ancestral. The comparison of the haplotype structure generally leads to the development of a hypothetical tree of the origin and spread of different haplotypes.

A review of molecular techniques to type Candida glabrata isolates.

Candida glabrata has emerged as a common cause of fungal infection causing mucosal and systemic infections. This yeast is of concern because of its reduced antifungal susceptibility to azole antifungals such as fluconazole. A clear understanding of the epidemiology of Candida infection and colonisation required a reliable typing system for the evaluation of strain relatedness. In this study, we discuss the different molecular approaches for typing C. glabrata isolates. Recent advances in the use of molecular biology-based techniques have enabled investigators to develop typing systems with greater sensitivities. Several molecular genotypic approaches have been developed for fast and accurate identification of C. glabrata in vitro. These techniques have been widely used to study diverse aspects such as nosocomial transmission. Molecular typing of C. glabrata could also provide information on strain variation, such as microvariation and microevolution.

[A clinical respiratory evaluation of dysphagic patients with chronic aspirations]. / Evaluation respiratoire clinique des patients présentant des fausses routes chroniques dans le cadre de troubles de la d gélutition.

INTRODUCTION: The basical respiratory state of dysphagic patients with chronic aspirations is not well-known. OBJECTIVE: to determine if chronic aspirations are associated with chronic clinical respiratory complications. MATERIALS AND METHODS: A prospective study was performed on 76 patients with swallowing disorders; The patients who aspirate are determined by swallowing videofluoroscopy. All patients answer to a questionnaire analysing their general and respiratory handicap. And a respiratory clinical examination was performed. The clinical symptoms between the group of aspirating patients and non aspirating patients were analysed. RESULTS: The global handicap and the respiratory handicap, caused by swallowing dysfunction, are more important for the aspirating patients (p < 0.05). The aspirating patients are more often symptomatic (p < 0.05); They present more functional and physical respiratory signs (p < 0.05); it seems that they present more often respiratory infections. CONCLUSION: Chronic aspirations are associated with a chronic handicapping respiratory disease, that is still to be described with para-clinical exams.

Molecular characterization of erythrocyte glucose-6-phosphate dehydrogenase deficiency in Tunisia.

Screening of G6PD deficiency was carried out on 79 unrelated subjects (32 females and 47 males), all coming from out consultation. DNA from deficient subject (11 females and 30 males) was analyzed for the presence of G6PD mutation. Known mutations were studied by the appropriate restriction enzyme digestion of fragment amplified by PCR. Where the mutation could not be identified in this way, the samples were subjected to SSCP analysis and abnormal fragments were sequenced. Through these methods, seven different mutations have been identified. Among deficient females, eight had the African variant A-(tow of them were homozygous) and three had the Mediterranean variant, one of them was homozygous and have had a haemolytic crisis after ingestion of fava beans showing at birth manifestation of neonatal jaundice. Among deficient males, four were hospitalized and transfused after a haemolytic crisis due to ingestion of fava beans. All of them have had manifestation of neonatal jaundice. Of them, one carried the Mediterranean variant and three others had the African variant A-. Among the remaining deficient males, 15 had A-variant, two had the Aurès mutation. SSCP analysis of nine mild deficient males, revealed the presence of the association of 1311 CT/93 TC in two subjects, a newly described silent mutation in the exon 12 associated with the polymorphism in the intron 11 93 TC in one subject and tow single intronic base deletion. The first is IVS V 17 (-C) found in two subjects and the second is IVS VIII 43 (-G) encountered in four subjects.

Haplotype diversity and linkage disequilibrium at human G6PD: recent origin of alleles that confer malarial resistance.

The frequencies of low-activity alleles of glucose-6-phosphate dehydrogenase in humans are highly correlated with the prevalence of malaria. These "deficiency" alleles are thought to provide reduced risk from infection by the Plasmodium parasite and are maintained at high frequency despite the hemopathologies that they cause. Haplotype analysis of "A-" and "Med" mutations at this locus indicates that they have evolved independently and have increased in frequency at a rate that is too rapid to be explained by random genetic drift. Statistical modeling indicates that the A- allele arose within the past 3840 to 11,760 years and the Med allele arose within the past 1600 to 6640 years. These results support the hypothesis that malaria has had a major impact on humans only since the introduction of agriculture within the past 10,000 years and provide a striking example of the signature of selection on the human genome.

Two fetal hemoglobin variants affecting the same residue: Hb F-Emirates [G gamma 59(E3)Lys-->Glu] and Hb F-Sacromonte [G gamma 59(E3)Lys-->Gln].

Two fast-moving fetal hemoglobin variants were discovered in hematologically normal newborn babies; the first originated in the United Arab Emirates and the second in France. The structural study, carried out by miniaturized techniques of protein chemistry, showed that these two mutations affected the same residue of the G gamma chain, the lysine at position 59(E3) was replaced by glutamic acid in Hb F-Emirates, and by glutamine in Hb F-Sacromonte.