Globin Gene Cluster Research Articles

Genetic Polymorphisms Associated with Fetal Hemoglobin (HbF) Levels and F-Cell Numbers: A Systematic Review of Genome-Wide Association Studies.

Elevated fetal hemoglobin (HbF), which is partly controlled by genetic modifiers, ameliorates disease severity in β hemoglobinopathies. Understanding the genetic basis of this trait holds great promise for personalized therapeutic approaches. PubMed, MedRxiv, and the GWAS Catalog were searched up to May 2024 to identify eligible GWAS studies following PRISMA guidelines. Four independent reviewers screened, extracted, and synthesized data using narrative and descriptive methods. Study quality was assessed using a modified version of the Q-Genie tool. Pathway enrichment analysis was conducted on gene lists derived from the selected GWAS studies. Out of 113 initially screened studies, 62 underwent full-text review, and 16 met the inclusion criteria for quality assessment and data synthesis. A total of 939 significant SNP-trait associations (p-value < 1 × 10-5) were identified, mapping to 133 genes (23 with overlapping variant positions) and 103 intergenic sequences. Most SNP-trait associations converged around BCL11A (chr.2), HBS1L-MYB, (chr.6), olfactory receptor and beta globin (HBB) gene clusters (chr.11), with less frequent loci including FHIT (chr.3), ALDH8A1, BACH2, RPS6KA2, SGK1 (chr.6), JAZF1 (chr.7), MMP26 (chr.11), COCH (chr.14), ABCC1 (chr.16), CTC1, PFAS (chr.17), GCDH, KLF1, NFIX, and ZBTB7A (chr.19). Pathway analysis highlighted Gene Ontology (GO) terms and pathways related to olfaction, hemoglobin and haptoglobin binding, and oxygen carrier activity. This systematic review confirms established genetic modifiers of HbF level, while highlighting less frequently associated loci as promising areas for further research. Expanding research across ethnic populations is essential for advancing personalized therapies and enhancing outcomes for individuals with sickle cell disease or β-thalassemia.

Identification of a rare [Gγ(Aγδβ)0] -thalassemia using tandem mass spectrometry.

Thalassemias are a group of inherited monogenic disorders characterized by defects in the synthesis of one or more of the globin chain subunits of the hemoglobin tetramer. Delta-beta (δβ-) thalassemia has large deletions in the β globin gene cluster involving δ- and β-globin genes, leading to absent or reduced synthesis of both δ- and β-globin chains. Here, we used direct globin-chain analysis using tandem mass spectrometry for the diagnosis of δβ-thalassemia. Two cases from unrelated families were recruited for the study based on clinical and hematological evaluation. Peptides obtained after trypsin digestion of proteins extracted from red blood cell pellets from two affected individuals and their parents were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Mass spectrometric analysis revealed a severe reduction in δ, β, and Aγ globin proteins with increased Gγ globin protein in the affected individuals. The diagnosis of Gγ(Aγδβ)0 -thalassemia in the homozygous state in the affected individuals and in the heterozygous state in the parents was made from our results. The diagnosis was confirmed at the genetic level using multiplex ligation-dependent probe amplification (MLPA). Our findings demonstrate the utility of direct globin protein quantitation using LC-MS/MS to quantify individual globin proteins reflecting changes in globin production. This approach can be utilized for accurate and timely diagnosis of hemoglobinopathies, including rare variants, where existing diagnostic methods provide inconclusive results.

Beta Globin Gene Cluster Haplotypes in Beta Thalassemia in the Kurdistan Region of Iraq

β-thalassemia is a prevalent inherited red cell disorder in the Kurdistan region of Iraq. To determine the chromosomal background of the frequent β-thalassemia mutations in the latter region, we investigated the β-globin gene cluster haplotypes in 202 β-thalassemia chromosomes. Haplotypes analysis utilized restriction fragment length polymorphism-PCR of seven restriction sites through the β-globin gene cluster. It was observed that IVS-II-1 (G > A) was mainly associated with haplotype III (68.8%), IVS-1-110 (G > A), codon 8/9 (+G) and codon 44 (-C) with haplotype I (in 90.0%, 100%, and 62.5% respectively), IVS-1-6 (T > C) with haplotype VI (97.4%), codon 8 (-AA) with haplotype IV (75%), codon 5(-CT) and IVS1.1 (G > A) with haplotype V (55.6% and 58.3% respectively), while codon 39 (C > T) and IVS1.5 (G > C) were mainly associated with haplotype VII (85.7% and 75% respectively). These observations support the notion that while some mutations may have originated in the Kurdistan region, others were more likely brought in by gene flow from neighboring countries or the Indian subcontinent. The association of some β-thalassemia defects with more than one haplotype may be due to mutations or recombination events.

Genome-wide association study identifies novel candidate malaria resistance genes in Cameroon.

Recent data suggest that only a small fraction of severe malaria heritability is explained by the totality of genetic markers discovered so far. The extensive genetic diversity within African populations means that significant associations are likely to be found in Africa. In their series of multi-site genome-wide association studies (GWAS) across sub-Saharan Africa, the Malaria Genomic Epidemiology Network (MalariaGEN) observed specific limitations and encouraged country-specific analyses. Here, we present findings of a GWAS of Cameroonian participants that contributed to MalariaGEN projects (n = 1103). We identified protective associations at polymorphisms within the enhancer region of CHST15 [Benjamin-Hochberg false discovery rate (FDR) < 0.02] that are specific to populations of African ancestry, and that tag strong eQTLs of CHST15 in hepatic cells. In-silico functional analysis revealed a signature of epigenetic regulation of CHST15 that is preserved in populations in historically malaria endemic regions, with haplotype analysis revealing a haplotype that is specific to these populations. Association analysis by ethnolinguistic group identified protective associations within SOD2 (FDR < 0.04), a gene previously shown to be significantly induced in pre-asymptomatic malaria patients from Cameroon. Haplotype analysis revealed substantial heterogeneity within the beta-like globin (HBB) gene cluster amongst the major ethnic groups in Cameroon confirming differential malaria pressure and underscoring age-old fine-scale genetic structure within the country. Our findings revealed novel insights in the evolutionary genetics of populations living in Cameroon under malaria pressure with new significant protective loci (CHST15 and SOD2) and emphasized the significant attenuation of genetic association signals by fine-scale genetic structure.

Evaluation of in silico predictors on short nucleotide variants in HBA1, HBA2, and HBB associated with haemoglobinopathies.

Haemoglobinopathies are the commonest monogenic diseases worldwide and are caused by variants in the globin gene clusters. With over 2400 variants detected to date, their interpretation using the American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) guidelines is challenging and computational evidence can provide valuable input about their functional annotation. While many in silico predictors have already been developed, their performance varies for different genes and diseases. In this study, we evaluate 31 in silico predictors using a dataset of 1627 variants in HBA1, HBA2, and HBB. By varying the decision threshold for each tool, we analyse their performance (a) as binary classifiers of pathogenicity and (b) by using different non-overlapping pathogenic and benign thresholds for their optimal use in the ACMG/AMP framework. Our results show that CADD, Eigen-PC, and REVEL are the overall top performers, with the former reaching moderate strength level for pathogenic prediction. Eigen-PC and REVEL achieve the highest accuracies for missense variants, while CADD is also a reliable predictor of non-missense variants. Moreover, SpliceAI is the top performing splicing predictor, reaching strong level of evidence, while GERP++ and phyloP are the most accurate conservation tools. This study provides evidence about the optimal use of computational tools in globin gene clusters under the ACMG/AMP framework.

P135: THE CLINGEN HEMOGLOBINOPATHY VARIANT CURATION EXPERT PANEL

Background: Accurate and consistent interpretation of sequence variants is integral to the delivery of safe and reliable diagnostic genetic services. To standardize the interpretation process, in 2015, the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) published a joint guideline based on different lines of evidence for the classification of sequence variants in Mendelian diseases. The generality of this guideline necessitates the application of expert judgment when evaluating and weighing evidence for variant interpretation. The Clinical Genome Resource (ClinGen) assembles Variant Curation Expert Panels (VCEPs) to perform gene- and disease-specific modifications of the ACMG/AMP framework. The ClinGen Hemoglobinopathy VCEP was created collaboratively between the ITHANET portal and the Global Globin Network of the Human Variome Project towards comprehensive annotation of all variants related to hemoglobinopathies. Aim: The adaptation of the ACMG/AMP variant interpretation guidelines of use in hemoglobinopathies. Methods: The Hemoglobinopathy VCEP focuses on the review and annotation of variants located in the globin gene clusters, namely α-globin locus (NG_000006), which includes genes HBA1, HBA2 and HBZ, and β-globin locus (NG_000007) which includes genes HBB, HBD, HBG1, HBG2 and HBE and the regulatory element LCRB. Using a consensus approach and guidance by the ClinGen Sequence Variant Interpretation Working Group, the Hemoglobinopathy VCEP has prepared a pre-final version of the specified ACMG/AMP criteria for hemoglobinopathies. Results: The Hemoglobinopathy VCEP developed disease-specific rules for sequence variant classification based on evidence criteria that assess variant frequency, variant types and disease causality, protein domains and mutational hotspots implicated in disease, clinical manifestations, segregation, in silico predictions and functional evidence. Conclusions: For the first time, the Hemoglobinopathy VCEP will provide a standardised classification of the pathogenicity of variants related to hemoglobinopathies. The Hemoglobinopathy VCEP specifications were approved by ClinGen in April 2021 (Step 2 approval), which initiated the process of further validation and adaptation with known globin gene variants in a pilot study (toward Step 3 approval).

The Effect of Alpha Thalassemia, HbF and HbC on Haematological Parameters of Sickle Cell Disease Patients in Ibadan, Nigeria.

BackgroundSickle cell disease is a protean disease with limited data on Nigeria’s phenotypic and genetic variants. This study was conducted to provide baseline data on these variants by characterising the existing forms of sickle cell disease and correlating these with basic haematological parameters.MethodsAdult and paediatric patients with SCD were recruited from a tertiary health centre in Nigeria. Patients were age and sex-matched with healthy controls. Blood samples were obtained for Full Blood Count, phenotyping by High-Performance Liquid Chromatography, and genotyping for alpha thalassemia by multiplex Gap-polymerase chain reaction. Data analysis was done using IBM SPSS statistics version 23.ResultsA total of 130 patients with sickle cell disease and 117 controls were studied. Alpha thalassemia in the study population was due to a 3.7kb deletion in the alpha-globin gene cluster at a prevalence of 45.4% in the patients and 47% in the controls. The prevalence of the various existing forms of SCD genotype was: Homozygous S without alpha gene deletion (HbSS)- 39.2%; HbSC - 10.8%; HbSSα+1- 35.4%; HbSSα+2 - 6.9% and HbSF- 7.7%. In the control population, HbAA without alpha gene deletion had a prevalence of 42.7%, HbAAα+1 was 25.6%, HbAA α+2 was 6%, HbAS- 7.7%, HbAS α+1 – 11.1%, HbAS α+2 - 2.6%, HbAC – 2.6% and HbAC α+1 – 1.7%. HbA2 was significantly elevated in HbSS individuals with two alpha gene deletions but reduced in normal controls (HbAA) with alpha gene deletions. HbF and HbA2 were negatively correlated with each other (r= −0.587, p < 0.001). Individuals with the HbSC genotype followed by HbSSα+2 had the best haematological parameters.ConclusionsHaematological parameters vary with haemoglobin genotype. The C haemoglobin and homozygous alpha-thalassemia deletion had a better ameliorating effect on SCD haematological parameters than the F haemoglobin in this population. The effect of alpha thalassemia on some haematological parameters in SCD patients are reversed in normal controls.

Large β Globin Gene Cluster Deletions and Implications on Transcription Factor Regulation in Hemoglobin Switching - Α Novel ε Gγ Αγ Deletion with Intact LCR

The εγδβ thalassemias (EGDBT) are infrequently occurring deletions of the β globin gene cluster on chromosome 11. EGDBTs can be an unsuspected cause of severe neonatal anemia that resolves during the first year of life into a normal Hb A2 thalassemic phenotype, presumed to be due to an imbalance of β-like chains during development. First described in 1972, they are categorized into two groups, both with loss of the five DNase I hypersensitivity sites upstream of the β gene (HBB) called the locus control region (LCR). In group 1 deletions, all or most of the β globin gene cluster including HBB is deleted; in group 2 deletions, removal of the LCR silences an intact HBB.Large deletions that do not involve LCR but remove the embryonic (HBE) and fetal genes (HBG2/HBG1) [LCR(ε Gγ Aγ) 0δβ] have not been reported. It is not known whether a deletion with intact LCR as well as δ and β genes would similarly manifest as neonatal anemia that resolves into a normocytic phenotype with normal hemoglobin fraction percentages after infancy.Four individuals with an LCR(ε Gγ Aγ) 0δβ deletion have been evaluated. These include monochorionic, diamniotic twin girls whose newborn screen (NBS) returned significant for Hb A percentage greater than Hb F for both twins. NBS was repeated two weeks later with the same result. The newborns were induced late pre-term (36 weeks and 6 days) after an uncomplicated pregnancy, including no features of twin to twin transfusion. Both infants had no anemia and bilirubin levels remained in the low to low-intermediate risk zones. Neither twin received transfusions or phototherapy during admission.At a follow up visit at 20 months of age a hemoglobin electrophoresis was performed which showed borderline Hb A2 and no Hb F in both twins (Table 1). Ferritin levels were low-normal in Twin A (14 µg/L) and low in Twin B (9 µg/L). Both infants were found to have a heterozygous large deletion affecting HBE, HBG2, HBG1, and HBBP1 loci by Multiplex Ligation-dependent Probe Amplification (MLPA), a method that uses multiple DNA probe pairs to determine the size of the DNA segment deleted. MLPA deletion/duplication testing on the α genes was negative. No β globin variants were identified by DNA sequencing. An evaluation was performed on the 25 year-old healthy mother and showed similar findings including the heterozygous large deletion. Mother and infants continue to be asymptomatic with normal red cell indices for age (Table 1).The fourth was an unrelated 40-year-old female with mild anemia screened by hemoglobin electrophoresis for obstetric evaluation who was found to have borderline Hb A2 (see table 1). MLPA showed a heterozygous deletion involving the same loci. Again, no β globin variants were identified by DNA sequencing.All four cases showed the same MLPA pattern except one non-specific probe commonly altered in common gamma gene conversion events. Long range sequencing performed on one case confirmed a single contiguous 32,599 bp deletion that matched the MLPA data (g.5262276-5294875). The detected deletion has not been reported in literature and differs from EGDBT mutations in that the LCR is intact. As the LCR controls expression of the entire gene cluster, this mutation is expected to display different phenotypic features than classic EGDBT. HBD and HBB are not deleted and therefore adequate transcription of Hb A2 and Hb A are expected with no imbalance of α/β chains after the neonatal period. It is postulated this finding explains the decreased Hb F levels in the NBS test result. The Hb A2 increase is likely a product of this deletion and does not indicate β-thalassemia.The heterozygous absence of HBE and HBG2/HBG1 may result in upregulation of HBD and HBB expression through loss of FKLF and FKLF-2 binding. In addition, HBB-EKLF binding would have less competition as the gamma promoter elements have been heterozygously deleted.In summary, in contrast to classic EGDBTs which cause transient severe neonatal anemia and resolve to normal Hb A2 thalassemic indices, this novel LCR(ε Gγ Aγ) 0δβ deletion was associated with normal CBC values with an absence of severe neonatal anemia, inverted Hb F/Hb A percentages at birth and borderline Hb A2 levels. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

HBB gene cluster haplotype diversity in sickle cell anemia patients of Chhattisgarh, India

Sickle cell anemia (SCA) is one of the hereditary hemoglobin disorders in Indian populations. An exceptionally high prevalence of SCA is observed in the populations of Chhattisgarh. Restriction fragment length polymorphism (RFLP) haplotypes of the beta globin (HBB) gene cluster are important as population data, anthropological purpose for tracing migration of SCA allele and predicting the severity of SCA disease. The purpose of this study was to elucidate the HBB haplotypes and their correlation with clinical and hematological profile of SCA patients of Chhattisgarh population. The HBB gene cluster haplotypes were determined in 190 SCA patients by the polymerase chain reaction-restriction fragment length polymorphism method. Medical records of patients were reviewed to obtain pertinent clinical features, hemoglobin fractions, and other biochemical variables. Among the analyzed patients, 74% had Arab-Indian (AI) haplotype, followed by 21% atypical haplotypes. Senegal, Benin, and Cameroon types of HBB haplotypes represented 3%, 1%, and 1% of the patients, respectively. Comparison of various biochemical and hematological variables and clinical complications among various haplotypes did not reveal significant differences. The high frequency of atypical haplotypes observed may have been generated by single and double crossing-over between AI haplotype and normal HBB haplotype. Considering the Indian population???s genetic structure and diversity, the results of our study should be considered as introductory, and our study can serve as a possible tool for additional studies of SCA in India.

A polygenic score for acute vaso-occlusive pain in pediatric sickle cell disease

AbstractIndividuals with monogenic disorders can experience variable phenotypes that are influenced by genetic variation. To investigate this in sickle cell disease (SCD), we performed whole-genome sequencing (WGS) of 722 individuals with hemoglobin HbSS or HbSβ0-thalassemia from Baylor College of Medicine and from the St. Jude Children's Research Hospital Sickle Cell Clinical Research and Intervention Program (SCCRIP) longitudinal cohort study. We developed pipelines to identify genetic variants that modulate sickle hemoglobin polymerization in red blood cells and combined these with pain-associated variants to build a polygenic score (PGS) for acute vaso-occlusive pain (VOP). Overall, we interrogated the α-thalassemia deletion −α3.7 and 133 candidate single-nucleotide polymorphisms (SNPs) across 66 genes for associations with VOP in 327 SCCRIP participants followed longitudinally over 6 years. Twenty-one SNPs in 9 loci were associated with VOP, including 3 (BCL11A, MYB, and the β-like globin gene cluster) that regulate erythrocyte fetal hemoglobin (HbF) levels and 6 (COMT, TBC1D1, KCNJ6, FAAH, NR3C1, and IL1A) that were associated previously with various pain syndromes. An unweighted PGS integrating all 21 SNPs was associated with the VOP event rate (estimate, 0.35; standard error, 0.04; P = 5.9 × 10−14) and VOP event occurrence (estimate, 0.42; standard error, 0.06; P = 4.1 × 10−13). These associations were stronger than those of any single locus. Our findings provide insights into the genetic modulation of VOP in children with SCD. More generally, we demonstrate the utility of WGS for investigating genetic contributions to the variable expression of SCD-associated morbidities.

RETROSPECTIVE ANALYSIS OF ALPHA GLOBIN COPY NUMBER VARIATIONS DETERMINED BY MLPA IN THE TRAKYA REGION

Objective: Alpha thalassemia is a common type of hemoglobinopathy that occurs as a result of deletions or point mutations in the alpha globin gene cluster. The molecular analysis of alpha thalassemia is challenging due to the presence of genes with high sequence similarities in alpha globin gene clusters and pseudogenes. As well as in all genetic diseases, determining the causative mutation types of alpha thalassemia and their frequencies have critical importance for accurate genetic screening and prevention strategies. Material and Method: In our study, alpha globin copy number variations determined by the Multiplex Ligation-dependent Probe Amplifcation (MLPA) method were examined retrospectively with suspicion of alpha thalassemia in 35 female and 43 male patients tested in the Genetic Diseases Diagnosis Center of the Medical Genetics Department at Trakya University Faculty of Medicine. Results: The most common deletion among our patients was the −α3.7 (35.3%), followed by the -α20.5 (10.3%) deletion. The -αSEA deletion was detected in three patients while 4 out of 78 cases were found to have the -αMED deletion. In three patients, a heterozygous large deletion and in one case HS40 regulatory region deletion were detected. In 14 (18%) of the patients, α globin triplications were detected. The -α4.2 deletion was detected in only one of our patients. Conclusion: Our study is the first to report the presence of eight different alpha globin copy number changes and 13 different alpha globin genotypes in the Trakya region.

Epigenetic Insights and Potential Modifiers as Therapeutic Targets in β-Thalassemia.

Thalassemia, an inherited quantitative globin disorder, consists of two types, α– and β–thalassemia. β–thalassemia is a heterogeneous disease that can be asymptomatic, mild, or even severe. Considerable research has focused on investigating its underlying etiology. These studies found that DNA hypomethylation in the β–globin gene cluster is significantly related to fetal hemoglobin (HbF) elevation. Histone modification reactivates γ-globin gene expression in adults and increases β–globin expression. Down-regulation of γ–globin suppressor genes, i.e., BCL11A, KLF1, HBG-XMN1, HBS1L-MYB, and SOX6, elevates the HbF level. β–thalassemia severity is predictable through FLT1, ARG2, NOS2A, and MAP3K5 gene expression. NOS2A and MAP3K5 may predict the β–thalassemia patient’s response to hydroxyurea, a HbF-inducing drug. The transcription factors NRF2 and BACH1 work with antioxidant enzymes, i.e., PRDX1, PRDX2, TRX1, and SOD1, to protect erythrocytes from oxidative damage, thus increasing their lifespan. A single β–thalassemia-causing mutation can result in different phenotypes, and these are predictable by IGSF4 and LARP2 methylation as well as long non-coding RNA expression levels. Finally, the coinheritance of β–thalassemia with α–thalassemia ameliorates the β–thalassemia clinical presentation. In conclusion, the management of β–thalassemia is currently limited to genetic and epigenetic approaches, and numerous factors should be further explored in the future.

Identification of thalassemia gene cluster deletion by long‐read whole‐genome sequencing (LR‐WGS)

At present, a variety of molecular detection methods are obtained to diagnose thalassemia accurately. Although exome sequencing or specific panels have been widely used in clinical diagnosis of genetic diseases, the positive rate is about 25%-30%. Because the detection range is limited to exons and splice sites, and the read length is usually 100-150bp, there are limitations in the detection of globin gene clusters with pseudogenes. In this study, seven thalassemia patients were selected to perform whole-genome sequencing (WGS) with long read at 400bp to make accurate detection for thalassemia deletions. And we used PCR and Sanger sequencing to confirm the gene deletions in the patients. WGS analysis detected a rare 172kb deletion on the α-globin gene cluster at chr16: 57009-330001, 19kb deletion at chr16: 215396-234699, 11kb deletion at chr16:220861-231981; and 27kb deletion on the β-globin gene deletion at chr11: 5222878-5250288, 21.4kb deletion at chr11: 5236361-5257771, 78.9kb deletion at chr11: 5191121-5270050. All the seven patients carried heterozygous deletions, including three in α-gene cluster, three in β-gene cluster, and one in both globin clusters. Our results indicate that long-read WGS will be beneficial to the diagnosis of genetic diseases with pseudogenes or highly duplicated sequences and will enable clinical geneticists to inform high-risk couples and provide prenatal diagnosis.

A New Gene Deletion in the α-Like Globin Gene Cluster as the Molecular Basis for the Rare α-Thalassemia-1 {–/αα) in Blacks: HbH Disease in Sickle Cell Trait

A novel deletion of at least 26 kilobase of DNA, including both a-globin genes, the Ψα- and -globin genes, but sparing the functional ς-gene was found in a 10-year-old black boy with HbH disease and sickle cell trait. This particular deletion has not previously been described in blacks. Its existence makes it likely that the absence of Hb Barts hydrops fetalis in blacks is due to the rarity of the chromosome lacking two a-globin genes rather than a result of early embryonic death due to the failure to synthesize embryonic hemoglobins because of deletion of functional ς-globin genes. © 1986 by Grune & Stratton, Inc.

Detection of an α-Globin Fusion Gene Using Real-Time Polymerase Chain Reaction-Based Multicolor Melting Curve

Genetic recombination between homologous sequences on the human globin gene clusters can lead to the creation of fusion genes. In this study, we report the detection of an α-globin fusion gene by using real-time polymerase chain reaction (qPCR)-based multicolor melting curve analysis (MMCA). The carriers of this fusion gene had a mild α-thalassemia phenotype with a normal hemoglobin (Hb) value and borderline hematological indices. Sequence analysis revealed that the mutant gene was the result of a fusion between the α2 and ψα1 genes. Our results indicate that the MMCA has the ability to detect the fusion gene, which is helpful for genetic counseling in thalassemia prevalent areas.

Hemoglobinopathies and Sickle Cell Disease

Key Points Inherited hemoglobin disorders account for most common single-gene defect in humans.Thalassemias are caused by mutations in the globin gene cluster, resulting in a decrease in globin chain synthesis while structural hemoglobinopathies usually result from a point mutation in the α- or β-globin gene that causes a functional abnormality.High-performance liquid chromatography has become the test of choice for the diagnosis of thalassemias and hemoglobinopathies because it reliably separates different hemoglobins.Newborn screening has helped with early diagnosis and management, thus decreasing morbidity and improving longevity.Blood transfusion, and appropriate use of iron chelating agents are mainstay of the treatment. Use of appropriate antibiotic prophylaxis starting from the neonatal period and appropriate vaccinations have decreased morbidity and improved survival in patients with sickle cell disease.

Clinical utility of combined preimplantation genetic testing methods in couples at risk of passing on beta thalassemia/hemoglobin E disease: A retrospective review from a single center.

Thalassemia and hemoglobinopathy is a group of hereditary blood disorder with diverse clinical manifestation inherited by autosomal recessive manner. The Beta thalassemia/Hemoglobin E disease (HbE/βthal) causes a variable degree of hemolysis and the most severe form of HbE/βthal disease develop a lifelong transfusion-dependent anemia. Preimplantation genetic testing (PGT) is an established procedure of embryo genetic analysis to avoid the risk of passing on this particular condition from the carrier parents to their offspring. Preimplantation genetic testing for chromosomal aneuploidy (PGT-A) also facilitates the selection of embryos without chromosomal aberration resulting in the successful embryo implantation rate. Herein, we study the clinical outcome of using combined PGT-M and PGT-A in couples at risk of passing on HbE/βthal disease. The study was performed from January 2016 to December 2017. PGT-M was developed using short tandem repeat linkage analysis around the beta globin gene cluster and direct mutation testing using primer extension-based mini-sequencing. Thereafter, we recruited 15 couples at risk of passing on HbE/βthal disease who underwent a combined total of 22 IVF cycles. PGT was performed in 106 embryos with a 3.89% allele drop-out rate. Using combined PGT-M and PGT-A methods, 80% of women obtained satisfactory genetic testing results and were able to undergo embryo transfer within the first two cycles. The successful implantation rate was 64.29%. PGT accuracy was evaluated by prenatal and postnatal genetic confirmation and 100% had a genetic status consistent with PGT results. The overall clinical outcome of successful live birth for couples at risk of producing offspring with HbE/βthal was 53.33%. Conclusively, combined PGT-M and PGT-A is a useful technology to prevent HbE/βthal disease in the offspring of recessive carriers.

High Hemoglobin F in Sickle Cell Disease: Waning Protection with Age

Background: Sickle cell disease (SCD) is a monogenetic inherited red cell disorder with pleomorphic clinical manifestations. Hemoglobin F (HbF) concentration is the major genetic modifier of clinical expression and levels between 10% and 20% have been found to improve survival and decrease vaso-occlusive complications (VOCs). As survival for patients with SCD has increased dramatically in the past 20 years, we hypothesized that SCD-related complications may increase with age, even in "high F" individuals. Recent data suggest that individuals with the Arab-Indian haplotype and/or HbS-δβ thalassemia, who have HbF of 15% to 25%, still suffer from VOCs. SCD patients with HBS and deletional hereditary persistence of fetal hemoglobin (HPFH) have high levels of HbF (&amp;gt;30%) in a pancellular distribution, and absence of symptoms as suggested by prior studies. Here, we report on a cohort of patients with HbSS/Sβ0, with HbF &amp;gt;14%, either hydroxyurea naïve or treated for ≤ 6 months. Methods: We report on a cohort of 11 adults with SCD and HbF levels &amp;gt; 14%, who had never been on hydroxyurea, or were treated for ≤6 months. Charts were reviewed for VOCs, opioid therapy, acute chest syndrome (ACS), avascular necrosis (AVN), retinopathy, pneumonia, leg ulcers, splenomegaly/infarction, stroke, pulmonary hypertension, cardiomyopathy/infarction, chronic kidney disease, priapism and miscarriage. All events were confirmed by supportive laboratory or imaging data. HbF levels for each patient are represented as lifetime average values. In 10/11 patients, cellular distribution of HbF by flow cytometry (CytoFlex, Beckman Coulter, CA) was performed following fixation, permeabilization, and incubation of red blood cells with a monoclonal antibody against Hb-F, directly conjugated with FITC (Cat # MHFH01, Life Technologies, MD). Beta globin dosage genetic analysis was performed on a subset of patients (8/11) which used multiplex-polymerase chain amplification of fragments specific for portions of the epsilon, gammaG, gammaA, delta, and beta globin genes, which is commonly associated with deletional variant of HPFH (Quest Diagnostics, VA). Results: Our cohort consisted of 11 patients, 3 males, age range 23 -72 years, one of which (age 72) is deceased. Genotypes included 10 HbSS, four with alpha thalassemia trait, and one HbS/β0. Mean HbF levels ranged from 14 % to 29.50%, SD ± 4.76. Table 1 shows patients ordered in ascending age with clinical complications for each patient. Notably, all patients experienced VOCs regardless of age or HbF level, 91% were prescribed opioids, 46% of whom were on chronic opioid therapy, 40 % had leg ulcers 36% had AVN and 27% had ACS . The number of complications per patient increased with age, despite maintenance of high HbF. Available flow cytometry and genetic data revealed heterocellular distribution of HbF in 8 patients, and pancellular distribution in 2 patients: patient (#2) and patient (#10). Patient (#2) is positive for deletional HPFH with genetic confirmation of heterozygous positive deletion in the beta globin gene cluster. Genetic data is not available for patient (#10) at this time. Interestingly, both patients still exhibited VOCs, complicated by yearly hospitalizations, required intermittent opioid therapy and with age, developed AVN. Laboratory data are notable for minimal levels of hemolysis. Discussion: Most individuals with SCD living in high resources countries now reach adulthood. Therefore the disease profile that was previously studied and reported mostly in the pediatric population is evolving. High levels of HbF appear to delay the onset of complications, but do not completely prevent them, perhaps due to a heterocellular distribution of HbF or non-deletional HPFH. In our cohort, two patients with confirmed pancellular distribution of HbF, one of which with confirmed deletional HPFH, were not fully protected from the clinical complications of SCD, suggesting alternative pathogenic mechanisms for VOCs may be at play. Therefore, individuals with HbF concentrations of as high as ~30 % may still suffer from pain, hospitalizations for VOC's, and vasculopathic changes, such as leg ulcers. The number of complications increases with age despite maintenance of high HbF. Therefore, additional therapy and close vigilance is necessary when treating sickle cell patients with high levels of HbF, especially with increasing age. Disclosures Reyes Gil: Albert Einstein College of Medicine: Patents &amp; Royalties: Patent application pending for NETs AI software. Ogu:Vertex Pharmaceuticals: Consultancy. Minniti:Doris Duke Foundation: Research Funding.

Long Range Sequencing Shows Improved Resolution in the Detection of Beta Globin Cluster Variants

Introduction: Globin gene sequencing has long been impeded by repetitive regions, homology and structural variation. Currently, long-read sequencing technology has been limited by high cost and error rate. These challenges can be mitigated by sample multiplexing of targeted capture sequencing and the use of consensus reads, respectively. This ongoing study analyzes the utility of long range sequencing technology in identifying historically problematic beta globin cluster mutations. Materials and methods: Long range sequencing was performed on complex beta globin gene cluster cases utilizing the Sequel platform (Pacific Biosciences, Menlo Park, California, United States). Sample input was 1 microgram of DNA extracted from whole blood. Circular Consensus Sequence (CCS) FASTQ sequences files were mapped to reference genome (GRCh37/hg19) using NGMLR, followed by annotation of variants using a custom bioinformatics workflow. Comparison to multiplex ligation-dependent probe amplification (MLPA), Sanger sequencing, array CGH, short-range NGS (300 bp), hemoglobin protein, and clinical data was performed. Results: CCS read length varied from 1 to 10 kb, average 3.5. Analysis of the beta globin cluster region showed impressive superiority and comprehensiveness to Sanger, MLPA, array CGH and short range sequencing technology: 1) single nucleotide variants (SNVs) were identified to a sensitivity and specificity of 99.5%; 2) large structural variants (SVs) such as large deletions, duplications, insertions, crossovers and fusions spanning many kilobases were characterized in the heterozygous, homozygous and compound heterozygous state to a precise genomic coordinate (table 1); 3) phasing SNVs identified Hb S haplotypes (Central African region (CAR), Benin, Senegal, Arab-Indian and Cameroon). Conclusion: 1) Long range sequencing holds significant promise in genotyping hemoglobin disorders. This method improves efficiency with potential for a single test offering with increased resolution, particularly in large deletion analysis. Complex cases greatly benefited from improved range and resolution. 2) The decreased requirement of blood volume for extensive testing is of particular interest for the pediatric population. 3) Genotyping of Hb S patients detected by positive newborn screening will help predict need for prophylaxis or therapy by assessing variant zygosity, phasing and haplotypes. In conclusion, we believe that long range sequencing has immense potential to contribute extensively to thalassemia and hemoglobinopathy diagnostics and is far superior to the currently available technologies for beta globin cluster analysis. Disclosures Jen: Celgene: Employment.

Genetic Analysis, Prenatal Diagnosis and Preimplantation Genetic Diagnosis of Taiwanese Deletion β-Thalassemia

To perform genetic analysis, prenatal diagnosis and preimplantation genetic diagnosis (PGD) in a family with a rare deletional β- thalassemia. Hematological parameters of the peripheral blood collected from all the family members were analyzed by whole blood cell analysis and capillary zone electrophoresis (CZE). Polymerase chain reaction-reverse dot blot (PCR-RDB) was used to identify 17 common β- thalassemia gene mutations, the multiplex ligation-dependent probe amplification (MLPA) and gap-polymerase chain reaction (gap-PCR) were used to identify β- globin gene cluster deletions. Chorionic villus sample or umbilical cord blood was obtained for prenatal diagnosis. Oligo-cells from blastocyst biopsy were collected for preimplantation genetic diagnosis by whole genome amplification and next generation sequencing. The proband was a carrier of Taiwanese deletion β- thalassemia, two fetuses were both thalassemia majors. The PGD results showed that 6 of 11 tested embryos could be choose for transplantation. The Taiwanese deletion is a rare type deletion of β- globin gene cluster, and it can lead to thalassemia intermedia or thalassemia major when compounded with other β- globin gene mutation. PGD is another choice for thalassemia couples.