Whole-exome sequencing indicates FLG2 variant associated with leg ulcers in Brazilian sickle cell anemia patients

Although sickle cell anemia results from homozygosity for a single mutation at position 7 of the β-globin chain, the clinical aspects of this condition are very heterogeneous. Complications include leg ulcers, which have a negative impact on patients’ quality of life and are related to the severity of the disease. Nevertheless, the complex pathogenesis of this complication has yet to be elucidated. To identify novel genes associated with leg ulcers in sickle cell anemia, we performed whole-exome sequencing of extreme phenotypes in a sample of Brazilian sickle cell anemia patients and validated our findings in another sample. Our discovery cohort consisted of 40 unrelated sickle cell anemia patients selected based on extreme phenotypes: 20 patients without leg ulcers, aged from 40 to 61 years, and 20 with chronic leg ulcers. DNA was extracted from peripheral blood leukocytes and used for whole-exome sequencing. After the bioinformatics analysis, eight variants were selected for validation by Sanger sequencing and TaqMan® genotyping in 293 sickle cell anemia patients (153 without leg ulcers) from two different locations in Brazil. After the validation, Fisher’s exact test revealed a statistically significant difference in a stop codon variant (rs12568784 G/T) in the FLG2 gene between the GT and GG genotypes ( P = 0.035). We highlight the importance of rs12568784 in leg ulcer development as this variant of the FLG2 gene results in impairment of the skin barrier, predisposing the individual to inflammation and infection. Additionally, we suggest that the remaining seven variants and the genes in which they occur could be strong candidates for leg ulcers in sickle cell anemia. Impact statement To our knowledge, the present study is the first to use whole-exome sequencing based on extreme phenotypes to identify new candidate genes associated with leg ulcers in sickle cell anemia patients. There are few studies about this complication; the pathogenesis remains complex and has yet to be fully elucidated. We identified interesting associations in genes never related with this complication to our knowledge, especially the variant in the FLG2 gene. The knowledge of variants related with leg ulcer in sickle cell anemia may lead to a better comprehension of the disease’s etiology, allowing prevention and early treatment options in risk genotypes while improving quality of life for these patients.

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Genetic Predictors of Hemoglobin F Response to Hydroxyurea in Sickle Cell Anemia

Blood ◽

10.1182/blood.v120.21.241.241 ◽

2012 ◽

Vol 120 (21) ◽

pp. 241-241 ◽

Cited By ~ 1

Author(s):

Vivien A Sheehan ◽

Thad A Howard ◽

Aniko Sabo ◽

Uma Nagasaswamy ◽

Jacy R Crosby ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Fetal Hemoglobin ◽

Acid Oxidation ◽

Genetic Modifiers ◽

Low Responders ◽

Genetic Predictors ◽

Whole Exome

Abstract Abstract 241 Although they ostensibly have a monogenetic disease, individuals with sickle cell anemia exhibit wide variability in their laboratory and clinical phenotype, suggesting additional genetic modifiers exist beyond the sickle mutation. One of the most powerful and reproducible disease modifiers is fetal hemoglobin (HbF) level. The most widely used and safest method for increasing innate fetal hemoglobin levels in patients with sickle cell anemia is hydroxyurea. While hydroxyurea has disease modulating effects outside of HbF induction, the majority of its benefit is directly related to the %HbF produced in response to the drug. Unfortunately, the amount of the HbF produced in response to hydroxyurea is highly variable between individuals, with induced HbF levels ranging from 5 to >30% even for compliant patients on similar dosing regimens at the maximum tolerated dose (MTD). Hydroxyurea is an ideal target for pharmacogenomics investigation, since there is strong concordance of HbF response to hydroxyurea within sibling pairs, and amount of HbF produced at MTD is a quantifiable and objective phenotype. To address the hypothesis that genetic modifiers influence pharmacological induction of HbF, we investigated pediatric patients treated prospectively with hydroxyurea. We analyzed patients enrolled in the HUSTLE (NCT NCT00305175) and SWiTCH (NCT 00122980) studies (n=183); all patients received an identical dose escalation regimen to MTD and had the most reliable HbF phenotypes available. To best identify genetic modifiers of hydroxyurea induction of HbF, we categorized study subjects according to their HbF response. Of a total cohort of 183 treated subjects, we identified 55 pediatric patients who represented the extreme ends of HbF response to hydroxyurea: 30 high responders (final HbF >30% and >25% change from %HbF at baseline) and 25 low responders (final HbF <20%, and <15% change from %HbF at baseline). There was no significant difference between high and low responders by age. Baseline fetal hemoglobin was similar between the two groups, suggesting that genetic predictors of drug response will differ from genetic regulators of endogenous HbF production. Absolute neutrophil count and hydroxyurea dose at MTD did not differ between high and low responders, evidence of uniform treatment. We performed whole exome sequencing on these 55 subjects and achieved 20X coverage in 95% of exonic sequences. In our statistical analysis, we compared nucleotide polymorphisms between low responders and high responders. Univariate testing identified ten nonsynonymous polymorphisms with p-values below 1×10−3, six of which are shown below. Additional candidate mutations with higher p-values were selected for further analysis based on known function in hematopoiesis or cell cycle arrest (Table). Whole exome sequencing genotyping was verified by TaqMan PCR or Sanger sequencing. Together, these variants represent excellent candidate genetic mutations to explain differences in HbF responses. These data represent the first examples of genetic predictors of HbF response to hydroxyurea using whole exome sequencing. Gene db SNP ID Amino acid change Function Direction of HbF response P-value PPP1R15A rs11541192 Gly312Ser Cell stress recovery Higher 1.87 × 10−4 HSD17B4 rs28943594 Met710Val Fatty acid oxidation Lower 4.16 × 10−4 HSD17B4 rs28943589 Lys122Asn Fatty acid oxidation Lower 4.18 × 10−4 FLVCR1 rs11120047 Ala52Pro Erythroid maturation Higher 4.21 × 10−4 LAMA5 rs6143021 His2036Arg Glomerular filtration Higher 4.21 × 10−4 ATP4A rs2733743 Val265Ala Iron absorption Higher 4.23 × 10−4 PPP1R15A rs611251 Val199Ala Cell stress recovery Higher 1.05 × 10−3 AKAP12 rs10872670 Lys19Glu Cell senescence Lower 1.28 × 10−3 SLC17A4 rs11754288 Ala372Thr Anion transporter Lower 1.86 × 10−3 RREB1 rs115093903 Leu983Ser Erythroid maturation Lower 2.31 × 10−3 DCHS2 rs61746132 Pro2676Leu Unknown Higher 2.90 × 10−3 SALL2 rs61743453 Pro840Arg Transcription factor Higher 9.17 × 10− Disclosures: Off Label Use: Hydroxyurea is FDA approved for adults but not children with sickle cell anemia.

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Whole Exome Sequencing Identifies Novel Genes for Fetal Hemoglobin Response to Hydroxyurea in Children with Sickle Cell Anemia

PLoS ONE ◽

10.1371/journal.pone.0110740 ◽

2014 ◽

Vol 9 (10) ◽

pp. e110740 ◽

Cited By ~ 20

Author(s):

Vivien A. Sheehan ◽

Jacy R. Crosby ◽

Aniko Sabo ◽

Nicole A. Mortier ◽

Thad A. Howard ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Fetal Hemoglobin ◽

Novel Genes ◽

Whole Exome

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Exome Sequencing of Extreme Phenotypes Suggests Novel Candidate Genes As Modifiers of Leg Ulcer in Sickle Cell Anemia

Blood ◽

10.1182/blood-2018-99-115192 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2351-2351

Author(s):

Gabriela Queila de Carvalho Siqueira ◽

Galina Ananina ◽

Murilo Guimarães Borges ◽

Bruno Batista de Souza ◽

Iscia Teresinha Lopes Cendes ◽

...

Keyword(s):

Exome Sequencing ◽

Candidate Genes ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Conflicts Of Interest ◽

Leg Ulcer ◽

Clinical Aspects ◽

P Value ◽

Single Mutation ◽

Extreme Phenotypes

Abstract Background: Despite resulting from homozygosity of a single mutation at position 6 of the beta-globin locus, the clinical aspects of sickle cell anemia (SCA) are very heterogeneous. Leg ulcer is one of the many resulting complications, exerting a quite negative impact in the quality of life of patients and is related to the severity of the disease. The pathogenesis of such complication is complex and still not well explained. Objective: Seeking for novel genes associated with leg ulcer in SCA, we proposed exome sequencing of extreme phenotypes in a sample of the Brazilian population. Methods: Our sample consisted of 40 unrelated patients with SCA from the Hematology and Hemotherapy Center of the University of Campinas (Unicamp), Campinas, SP, Brazil. The cohort was composed by 20 patients who didn't have leg ulcer, with ages varying from 40 to 61 years (it is unusual the appearance of this disorder at such age) and 20 patients with chronic leg ulcer. DNA was collected from peripheral blood leukocytes and submitted to exome sequencing. Capture and enrichment were performed with the Nextera Rapid Capture Exome kit (Illumina) and samples were loaded in the Illumina HiSeq2500. The bioinformatics process was based on the GATK Pipeline and manual filters in vcftools and excel. Briefly, we prioritized the variants according to some conditions, like score quality, depth, Hardy-Weinberg equilibrium and minor allele frequency greater than 0.1. Subsequently, through PLINK, we selected the variants with p value less than 0.05 from Fisher association test. The annotation was made through wAnnovar. We excluded the synonymous variants, remaining with 244 variants at this point. Finally, the deleterious variants were selected, in addition to those not classified by SIFT, PolyPhen2 and FATHMM. Twenty-one variants remained, all of them in genes beyond the usual set of a priori biological candidate genes for this phenotype. We picked up 6 variants with the lowest p values: rs4857302 (CRYBG3), rs3782489 (KRT77), rs11800462 (TNFRSF25), rs13428956 (FOXD4L1), rs201853154 (UBTFL1) and rs11454536 (VWDE). We suggest that these variants and their genes could potentially be related to the development of leg ulcer in SCA. However, they should all be validated in other populations for confirmation. Disclosures No relevant conflicts of interest to declare.

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Whole-exome sequencing for RH genotyping and alloimmunization risk in children with sickle cell anemia

Blood Advances ◽

10.1182/bloodadvances.2017007898 ◽

2017 ◽

Vol 1 (18) ◽

pp. 1414-1422 ◽

Cited By ~ 37

Author(s):

Stella T. Chou ◽

Jonathan M. Flanagan ◽

Sunitha Vege ◽

Naomi L. C. Luban ◽

R. Clark Brown ◽

...

Keyword(s):

Risk Factor ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Whole Exome ◽

Key Points

Key PointsWES can be applied for precise RH genotyping, detection of new or uncommon variants, and determination of RHD zygosity. An altered RH genotype is a risk factor for Rh alloimmunization in patients with sickle cell anemia.

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Updated leg ulcer pathway: improving healing times and reducing costs

British Journal of Nursing ◽

10.12968/bjon.2019.28.20.s21 ◽

2019 ◽

Vol 28 (20) ◽

pp. S21-S26

Author(s):

Leanne Atkin ◽

Alison Schofield ◽

Anita Kilroy-Findley

Keyword(s):

Quality Of Life ◽

Leg Ulcer ◽

Leg Ulcers ◽

Nursing Time ◽

Venous Leg Ulcers ◽

Reduced Costs ◽

Reducing Costs

Regardless of the amount of literature and evidence on leg ulcer management, there are still significant variations in treatment. Implementing a standardised leg ulcer pathway to ensure patients are appropriately and timely assessed could help reduce nursing time and overall costs, while improving healing outcomes and patients' quality of life. Such a pathway was introduced in Lincolnshire and Leicestershire, UK, to treat venous leg ulcers (VLUs). The results showed improved healing times, reduced costs and fewer nurse visits, among other findings.

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Up-Regulation of Mir-21 and Mir-130a and Serum Leptin Levels on Leg Ulcers Development in Sickle Cell Anemia

Blood ◽

10.1182/blood-2019-132043 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 975-975

Author(s):

Thais Helena Chaves Batista ◽

Rodrigo Marcionilo Santana ◽

Marcondes José de Vasconcelos Costa Sobreira ◽

Gabriela da Silva Arcanjo ◽

Diego Arruda Falcao ◽

...

Keyword(s):

Sickle Cell ◽

Sickle Cell Anemia ◽

Leg Ulcer ◽

Acute Chest Syndrome ◽

Control Group ◽

Blood Collection ◽

Leg Ulcers ◽

Serum Leptin ◽

Ulcer Group ◽

And Control

Introduction: Leg ulcers (LUs) are a cutaneous complication of sickle cell anemia (SCA), whose etiology is considered multifactorial. In the search for new candidates for modulators of SCA clinical events, recent evidence suggests the significant role of mechanisms related to post-transcriptional regulation, especially microRNAs (miRNAs). Thus, the analysis of miRNAs miR-21 and miR-130a differential expression in patients with SCA becomes an interesting approach, since both act in the regulation of several biological mechanisms related to the pathophysiology of LU, especially the tissue repair process. In addition, these miRNAs have already been related to the regulation of serum leptin levels, a strong angiogenic pleiotropic hormone that acts in the healing process of skin lesions. Therefore, the aim of the study was to investigate the influence of miR-21 and miR-130a and serum leptin levels on the development of LUs in SCA patients. Methods: After analyzing medical records, 60 SCA patients were selected. Patients who presented some of the main clinical manifestations that may have etiology due to the underlying disease (for example: osteonecrosis, stroke, priapism and acute chest syndrome) were not included. Patients with a history of LU were considered cases, and those who did not develop this complication (n=20), were considered control (median age: 26 years, range: 19-61, 50% males). The control group was called "HbSS-Control" and the case group was divided into two subgroups: Active leg ulcer group, composed of 19 patients with active LU at the time of blood collection (median age: 35 years, range: 24-56, 68% males), and healed leg ulcer group, composed of 21 patients with healed LU at the time of blood collection (median age: 34 years, range: 22-52, 43% males). In addition, it was analyzed a group of 10 donors with normal hemoglobin profile (median age: 25 years, range: 20-30, 50% males), identified as "HbAA-Control". Expression levels of miRNAs extracted from peripheral blood, using mirVanaTM PARIS Kit (Invitrogen™) were evaluated by RT-qPCR technique utilizing TaqMan® probes. Serum leptin levels of the patients were evaluated employing the ELISA method (Human Leptin ELISA Kit, Millipore®). Mann-Whitney and Kruskal-Wallis tests were applied to compare continuous variables. Results: Up-regulation of both miRNAs was observed in the active leg ulcer group in contrast to the healed leg ulcer (miR-21: P<0.0001, Figure 1A, Fold change [FC]=14,2; miR-130a: P=0.0004, FC=18,8, Figure 1B) and Control-HbSS groups (miR-21: P<0.0001, FC=34,4, Figure 1A; miR-130a: P=0.0006, FC=15,3, Figure 1B) and the HbAA-Control group (miR-21: P<0.0001, FC=5,8, Figure 1C; miR-130a: P=0.0009, FC=10,9, Figure 1D). However, there was no significant difference between the healed leg ulcer, HbSS-Control and HbAA-Control groups (miR-21: P=0.1829, Figure 1E; miR-130a: P=0.3537; Figure 1F). Furthermore, the active leg ulcer group had lower serum leptin levels when compared to the healed leg ulcer and Control-HbSS groups (P=0.0058; Figure 2A). The levels of leptin in the healed leg ulcer group did not differ from the Control-HbSS group (P=0.5929; Figure 2B). Conclusion: Our results demonstrated an inverse relation between the miRNAs miR-21 and miR-130a expression with serum leptin levels, suggesting that the up-regulation of these miRNAS may be related to the chronicity and healing of LUs in individuals with SCA through decreased of serum leptin levels. Disclosures No relevant conflicts of interest to declare.

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Whole Exome Sequencing in Patients With Ossification of the Spinal Ligaments

10.21203/rs.3.rs-406569/v1 ◽

2021 ◽

Author(s):

Jiliang Zhai ◽

Jiahao Li ◽

Yu Zhao ◽

Xing Wei

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Vital Role ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Whole Exome ◽

Clinical Presentations ◽

Spinal Ligaments ◽

Neurological Pathology

Abstract Background The clinical presentations of ossification of spinal ligament (OSL) are majorly myelopathy and/or radiculopathy, with serious neurological pathology resulting in paralysis of extremities and disturbances of motility (motor function) lowering the quality of life. Currently, studies find that missense single nucleotide polymorphisms (SNPs) play a vital role in the susceptibility for complex diseases. Methods In the present study, we used whole exome sequencing (WES) method to explore variants in exomes and found out novel potential responsible genes for OSL. Genomic DNA was extracted from ligamentum flavum collected from 5 OSL patients and 5 control patients. Whole-exome sequencing was then performed, while variation forecasts and conservation examination were subsequently assessed. Results 8 common SNP variants were exhibited in all 5 subjects of OSL, presented in the genes of GRHL2, CUL3, WHAMM, IL17RD, POM121L12, SLC26A8 and PTPN23. After screened with numbers of samples and additional screenings with deleterious Polyphen2_HDIV_score, Polyphen2_HVAR_score and SIFT, 4 common SNP variants were displayed in 4 subjects of OSL, presented in the genes of KRT84, KIF1B, NRAP and CETN1. 7 common SNP variants were existed in 3 subjects of OSL, presented in the genes of CCT3, ANLN, ESRRB, SRBD1, ODF3L1, BRAT1 and RBP3. Conclusion We found out novel potential variants in several genes, especially WHAMM, KIF1B and ESRRB, which represented potentially pathogenic mutations in patients with OSL.

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Quality of Life in Egyptian Children and Adolescents with Sickle Cell Anemia

Egyptian Journal of Pediatrics ◽

10.12816/0049216 ◽

2017 ◽

Vol 34 (1-2) ◽

pp. 109-118

Author(s):

Mohamed Abdel-Mohsen Ellabody ◽

Hisham Ahmed Ramy ◽

Naglaa Fathy Mahmoud

Keyword(s):

Quality Of Life ◽

Children And Adolescents ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Egyptian Children

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77P Measurement of health quality of life in the multicenter study of hydroxyurea in sickle cell anemia

Controlled Clinical Trials ◽

10.1016/0197-2456(94)90205-4 ◽

1994 ◽

Vol 15 (3) ◽

pp. 120

Author(s):

F. Barton ◽

M. Terrin ◽

S. Charache ◽

M. Koshy ◽

P. Swerdlow ◽

...

Keyword(s):

Quality Of Life ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Multicenter Study ◽

Health Quality ◽

Health Quality Of Life

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Qualidade de vida dos portadores de doença falciforme

Revista de Enfermagem UFPE on line ◽

10.5205/1981-8963-v13i2a237424p424-430-2019 ◽

2019 ◽

Vol 13 (2) ◽

pp. 24

Author(s):

Kamila Tuany Lacerda Leão Lima ◽

João Otávio Ferreira Pereira ◽

Paulo Roberto De Melo Reis ◽

Keila Correia De Alcântara ◽

Flávia Melo Rodrigues

Keyword(s):

Quality Of Life ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Functional Capacity ◽

Sickle Cell Anemia ◽

Physical Aspect ◽

Cell Disease ◽

Calidad De Vida ◽

Sf 36

RESUMOObjetivo: avaliar a qualidade de vida de portadores de células falciformes atendidos pelo Programa de Anemia Falciforme. Método: trata-se de um estudo quantitativo, descritivo e analítico em 20 portadores de anemia falciforme e 40 indivíduos não falciformes. Coletaram-se os dados por meio de entrevistas no período entre fevereiro a maio de 2015. Avaliou-se a qualidade de vida por meio de SF-36 e WHOQOL-BREF. Apresentaram-se os resultados em forma de tabelas. Resultados: constata-se que a maioria dos pacientes com doença falciforme se declara como negros e castanhos e com baixo nível de escolaridade; os aspectos físicos e a capacidade funcional tiveram os piores resultados e, com a idade, o aspecto físico se torna mais comprometido. Mostrou-se, pelo questionário SF-36, que, entre os escores, os indivíduos com DF apresentavam dor, capacidade funcional, vitalidade, aspectos físicos, emocionais e de saúde mental como os mais prejudicados em relação ao grupo de pacientes sem DF. Conclusão: apresentou-se, pela avaliação WHOQOL-BREF, comprometimento significativo da qualidade de vida física e geral entre os pacientes com DF; já os participantes com doença falciforme sofrem um impacto negativo na qualidade de vida, o que interfere e influencia a saúde dessas pessoas. Descritores: Qualidade de Vida; Doença Crônica; Perfil de Impacto da Doença; Anemia Falciforme; Anemia Hemolítica; Eritrócitos. ABSTRACT Objective: to evaluate the quality of life of sickle cell patients treated by the Sickle Cell Anemia Program. Method: this is a quantitative, descriptive and analytical study in 20 patients with sickle cell anemia and 40 non-sickle individuals. Data was collected through interviews between February and May 2015. Quality of life was evaluated through SF-36 and WHOQOL-BREF. Results were presented in the form of tables. Results: the majority of patients with sickle cell disease declare themselves as black and brown with a low level of schooling; the physical aspects and the functional capacity had the worst results and, with age, the physical aspect becomes more compromised. The SF-36 questionnaire showed that, among the scores, individuals with FD presented pain, functional capacity, vitality, physical, emotional and mental health aspects as the most impaired in relation to the group of patients without SCD. Conclusion: the WHOQOL-BREF evaluation showed a significant impairment of physical and general quality of life among patients with DF; participants with sickle-cell disease have a negative impact on quality of life, which interferes with and influences the health of these people. Descritores: Quality of Life; Chronic Disease; Sickness Impact Profile; Sickle Cell Anemia; Anemia Hemolytic; Erythrocytes.RESUMEN Objetivo: evaluar la calidad de vida de portadores de células falciformes atendidos por el Programa de Anemia Falciforme. Método: se trata de un estudio cuantitativo, descriptivo y analítico en 20 portadores de anemia falciforme y 40 individuos no falciformes. Se recogieron los datos a través de entrevistas en el período entre febrero a mayo de 2015. Se evaluó la calidad de vida por medio de SF-36 y WHOQOL-BREF. Se presentaron los resultados en forma de tablas. Resultados: se constata que la mayoría de los pacientes con enfermedad falciforme se declara como negros y castaños y con bajo nivel de escolaridad; los aspectos físicos y la capacidad funcional tuvieron los peores resultados y, con la edad, el aspecto físico se vuelve más comprometido. Se mostró, por el cuestionario SF-36, que entre los escores, los individuos con DF presentaban dolor, capacidad funcional, vitalidad, aspectos físicos, emocionales y de salud mental como los más perjudicados en relación al grupo de pacientes sin DF. Conclusión: se presentó, por la evaluación WHOQOL-BREF, un compromiso significativo de la calidad de vida física y general entre los pacientes con DF; ya los participantes con enfermedad falciforme sufren un impacto negativo en la calidad de vida, lo que interfiere e influye en la salud de esas personas. Descritores: Calidad de Vida; Enfermedad Crónica; Perfil de Impacto de la Enfermedad; Anemia de Células falciformes; Anemia Hemolítica; Eritrocitos.

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