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• W2766474110 abstract "Mediterranean stomatocytosis/macrothrombocytopenia is a rare autosomal recessive disorder caused by enhanced gastrointestinal absorption of and reduced excretion of plant sterols (sitosterolaemia). It is characterised by short stature, chronic abdominal discomfort, splenomegaly and laboratory features of chronic haemolysis along with the presence of stomatocytic red cells and macrothrombocytopenia on blood films. The rarity as well as protean manifestations of this disorder make it a challenging diagnosis for clinicians as well as pathologists. A 12-year-old north Indian male, born from a non-consanguineous marriage, was on intermittent follow-up for a presumptive diagnosis of hereditary spherocytosis (HS). His 8-year-old sister too had features of chronic haemolysis with documented cholelithiasis. Clinical findings in both siblings included intermittent jaundice, failure-to-thrive (height and weight 79% and 46% of expected, respectively, i.e., <3rd centile for age) and intermittent epistaxis. Peripheral blood showed the presence of spherocytes, variable numbers of stomatocytes, reticulocytosis and thrombocytopenia. Incubated osmotic fragility (iOFT) was increased in both. However, the flow cytometric eosin-5′-maleimide (EMA) dye-binding test was normal (mean channel fluorescence ratio 1.09; reference value≤0.8). The study was approved by the institutional ethics committee of PGIMER, Chandigarh. Samples were collected after obtaining consent of the patient according to approved human subjects' guidelines. In light of the EMA results, the patient's complete clinical and laboratory records were reviewed. Past history revealed multiple hospital admissions, starting when he was 2 years old for sub-acute intestinal obstruction which was managed conservatively. A 5 mm stone in the common bile duct was noted which passed spontaneously. Later hospital visits were for diarrhoea, failure to gain weight and height, giardiasis, fever, jaundice. Hepatosplenomegaly 4 and 7 cm below right and left costal margins, respectively, were noted. Investigations were negative for malaria, Wilson's disease and chronic liver disease, including viral and autoimmune hepatitis. Based on a single positive report of anti-smooth muscle actin (SMA) antibody, he had received prednisolone and azathioprine at 12 years of age. The boy's haemoglobin was 117 g/L, total leukocyte count 10.1 × 109/L, red blood cell count 3.66 × 1012/L, mean corpuscular volume 109.2 fL, mean corpuscular haemoglobin 32 pg, mean corpuscular haemoglobin concentration 29.3 g%, red cell distribution width 19.2% and corrected reticulocyte count 9.8%. The automated platelet count (impedance method) was 5 × 109/L. On a blood film, red cells showed moderate anisopoikilocytosis with a predominant population of stomatocytes and reticulocytosis (Fig. 1A,B). Platelets were reduced (∼50-60 × 109/L on smear estimates) with anisocytosis and several giant forms (Fig. 1C). Mean platelet volumes for the siblings ranged from 11.3 to 12.9 fL (reference range 7.5–11.5). Biochemical investigations revealed total bilirubin 1.77 mg% with conjugated fraction 0.26 mg%, aspartate aminotransferase 89 U/L, alanine aminotransferase 189 U/L and alkaline phosphatise 189 U/L. Prothrombin time was 13.8 s. Direct Coombs test, G6PD deficiency screening, urine and plasma haemoglobin levels and haemoglobin high-performance liquid chromatography were normal or negative. Flow cytometry based OFT showed increased lysis of red cells with 9.2% residual cells [reference range (RR) 52.3 ± 20.5%]. Serology for HIV, hepatitis B surface antigen, hepatitis C, A and E were negative. Thyroid function was normal: T3 1.71 (0.8–2 ng/mL), T4 12.48 (4.8–12.7 μg/dL) and thyroid-stimulating hormone (TSH) 1.79 (0.27–4.2 μIU/mL). A duodenal biopsy revealed intraepithelial lymphocytosis with crypt hyperplasia. However, anti-tissue transglutaminase (tTG) antibody was 1.3 U/mL (RR <5 U/mL). Serum IgA levels 174 (80–280 mg%), IgG levels 1115 (1248.11 ± 274.31 mg%), ceruloplasmin 25 mg/dL, and urine copper 70 μg/L were normal. Kayser–Fleischer rings were not observed on evaluation of the eye. Bone marrow examination was reported as megakaryocytic thrombocytopenia with a lack of significant dyserythropoiesis. Autoimmune lymphoproliferative syndrome was excluded by immunophenotyping (double-negative T-cells 1.78% of all CD3+ TCR αβ+ T-lymphocytes). A liver biopsy was performed twice; at 8 years it showed non-specific changes and at 12 years chronic hepatitis was reported. Autoimmune markers: anti-nuclear antibodies (ANA), anti-mitochondrial antibodies (AMA), anti-liver kidney microsomal antibodies (LKM) and anti-parietal cell antibodies (PCA) were negative. Anti-SMA was positive in 1:20 dilutions once. X-ray for bone age was reduced to 11–13 years at 15 years of age. The patient's sister had chronic mild anaemia (haemoglobin varying 10–11 g/L), reticulocytosis (3.0–8.5%) and thrombocytopenia (platelet counts between 28–101 × 109/L). Stomatocytes and spherocytes predominantly constituted all the red cells (Fig. 1D). Haematological investigations (except for iOFT and flow OFT), serum ceruloplasmin and anti-tTG antibody, viral and autoimmune markers were normal or negative. The ovaries were not visualised in a pelvic ultrasonogram, and buccal smear for Barr body as well as karyotyping revealed mosaicism for Turner syndrome [45,X/46,XX (1:1)]. Endocrinological testing revealed luteinising hormone ≤0.1 (2.4–12.6 mIU/mL), follicle-stimulating hormone 3.78 (3.5–12.5 mIU/mL), 17β-estradiol 29.81 (12.5–166 pg/mL), prolactin 6.49 (4.79–23.3 ng/mL), testosterone basal ≤0.081 (0.2–2.9 nmol/L), T3 1.81 (0.8–2 ng/mL), T4 9.51 (4.8–12.7 μg/dL), and TSH 4.41 (0.27–4.2 μIU/mL). The echocardiography was normal. Peripheral blood film of the younger brother aged 7 years revealed stomatocytosis, reticulocytosis and macrothrombocytopenia as well. His clinical complaints were restricted to failure to thrive. Screening of parents revealed only few stomatocytes. Haematological parameters for siblings and parents are compiled in Table 1.Table 1Haematological parameters of the index case, siblings and parentsIndex caseYounger sisterYounger brotherMotherFatherAge/Sex19/M13/F7/M30/F35/MHb g/dL12.49.110.312.012.9PCV%41.229.934.437.139.7RBC/μL3.603.213.593.874.44MCV fL114.593.395.796.089.5MCH pg34.428.228.731.129.2MCHC %30.130.33032.432.6RDW cv%18.82017.617.016.9Reticulocyte %6.812.67.32.82.2Platelets/μL500021,0008000147,000113,000RBCsStomatocytes ++, few spherocytesStomatocytes ++, macrocytes, polychromasia +Stomatocytes ++, few spherocytesStomatocytes (∼30%)Few stomatocytesPlateletsPlatelets reduced with few giant formsPlatelets moderately reduced with few giant formsPlatelets reduced with few giant formsPlatelets adequate with few giant formsPlatelets adequateHb, haemoglobin; MCH, mean corpuscular haemoglobin; MCHC, mean corpuscular haemoglobin concentration; MCV, mean corpuscular volume; PCV, packed cell volume; RBCs, red blood cells; RDW, red cell distribution width. Open table in a new tab Hb, haemoglobin; MCH, mean corpuscular haemoglobin; MCHC, mean corpuscular haemoglobin concentration; MCV, mean corpuscular volume; PCV, packed cell volume; RBCs, red blood cells; RDW, red cell distribution width. The clinical manifestations in the family raised the suspicion of syndromic stomatocytosis with macrothrombocytopenia. The implicated genes in this condition are known to be ABCG5 and ABCG8, both having 13 exons and transcript lengths of 2740 and 2665 bp, respectively, making Sanger sequencing non-viable as a diagnostic approach. Therefore, next-generation sequencing (NGS) was attempted in the sister to characterise the molecular defect. Genomic DNA was extracted by the QIAamp DNA Blood Midi Kit (Qiagen, Germany). DNA was quantified on NanoDrop 2000 spectrophotometer (ThermoFisher Scientific Inc, USA) and Qubit 2.0 Fluorometer (Invitrogen, USA). The quality of DNA library was assessed on a 2100 Bioanalyzer System (Agilent Technologies, USA) and sequenced on a MiSeq Sequencing System using the TruSight One Sequencing Panel (Illumina, USA). Variant calling, annotation and reporting were performed using MiSeq Reporter and VariantStudio v2.1 software and visualised using the Integrated Genome Viewer. NGS revealed a homozygous mutation in exon 6 of the ATP-binding cassette, subfamily G, member 5, ABCG5 [c.727C>T (p.Arg243Ter), rs119479066] at 18X sequencing depth (Fig. 2). This R243X mutation in homozygous state has been previously described in two Asian-origin South African sisters with sitosterolaemia, and their parents were found to be heterozygous.1Lee M.H. Lu K. Hazard S. et al.Identification of a gene, ABCG5, important in the regulation of dietary cholesterol absorption.Nat Genet. 2001; : 2779-2783Google Scholar Sanger sequencing validated all three siblings as being homozygous and parents as heterozygous for the same mutation (Fig. 3). Other stomatocytic syndromes, dehydrated hereditary stomatocytosis (PIEZO1 gene) and overhydrated hereditary stomatocytosis (RHAG gene) were also excluded by NGS data analysis.Fig. 3Chromatograms showing ABCG5 [c.727C>T (p.Arg243Ter), rs119479066] mutation to be homozygous in siblings and heterozygous in parents.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Haplotype analysis of the ABCG5/ABCG8 genes in our patients was attempted. For this, we analysed in our NGS data 14 single nucleotide polymorphisms (SNPs) that have been previously described informative variants in haplotype analysis for ABCG5/ABCG8 by Pandit et al. Our patients' data as well as the NGS results available from 29 other unrelated subjects were reviewed for these 14 SNPs. All three affected siblings had an identical haplotype (CCGGATGGCCCCGC) with homozygosity for all the 14 SNPs in comparison to 29 other unrelated subjects. In addition, our haplotype noted was distinct from the Caucasian sitosterolaemia families and healthy African-American cohorts.2Pandit B. Ahn G.S. Hazard S.E. Gordon D. Patel S.B. A detailed Hapmap of the Sitosterolemia locus spanning 69 kb; differences between Caucasians and African-Americans.BMC Med Genet. 2006; 7: 13Crossref PubMed Scopus (18) Google Scholar The R243X mutation occurred at a conserved CpG site and based on the haplotype analysis it appears likely to have occurred independently. Since this is the only family with Mediterranean stomatocytosis/macrothrombocytopenia available to us, it is difficult to conclusively establish a founder haplotype at present. Sitosterolaemia is rare and clinically typical cases reported in the literature have not often had a genetic confirmation. There is one prior morphological description of sitosterolaemia described from India.3Agrawal P. Parashar S. Tyagi N. Sitosterolemia with extensive xanthomas in two siblings in India: a case report.IOSR J Dent Med Sci. 2015; 14: 41-44Google Scholar Our report represents the first molecular diagnosis of this disorder in India using NGS technology which was subsequently validated by Sanger sequencing in all the family members. The link between sitosterolaemia and stomatocytosis was described relatively recently. The haematological manifestations are macrothrombocytopenia, haemolytic anaemia with features of stomatocytosis and reticulocytosis.4Su Y. Wang Z. Yang H. et al.Clinical and molecular genetic analysis of a family with sitosterolemia and co-existing erythrocyte and platelet abnormalities.Haematologica. 2006; 91: 1392-1395PubMed Google Scholar, 5Wang G. Wang Z. Liang J. Cao L. Bai X. Ruan C. A phytosterolemia patient presenting exclusively with macrothrombocytopenia and stomatocytic hemolysis.Acta Haematol. 2011; 126: 95-98Crossref PubMed Scopus (19) Google Scholar, 6Kaya Z. Niu D.M. Yorulmaz A. Tekin A. Gürsel T. A novel mutation of ABCG5 gene in a Turkish boy with phytosterolemia presenting with macrotrombocytopenia and stomatocytosis.Pediatr Blood Cancer. 2014; 61: 1457-1459Crossref PubMed Scopus (14) Google Scholar, 7Rees D.C. Iolascon A. Carella M. et al.Stomatocytic haemolysis and macrothrombocytopenia (Mediterranean stomatocytosis/macrothrombocytopenia) is the haematological presentation of phytosterolaemia.Br J Haematol. 2005; 130: 297-309Crossref PubMed Scopus (107) Google Scholar Platelet aggregation abnormalities, including absent response to ristocetin and variable responses with other agonists, have been described. This could not be tested in our patients, since the platelet counts were below the minimum 100 × 109/L required for conventional light transmission aggregometry. Our patients lacked the classical primary manifestations of sitosterolaemia, including xanthomas, arthralgias or premature artherosclerosis, etc; the clinical phenotype was largely restricted to abnormal haematological features8Yoo E. Sitosterolemia: a review and update of pathophysiology, clinical spectrum, diagnosis, and management.Ann Pediatr Endocrinol Metab. 2016; 21: 7-14Crossref PubMed Scopus (22) Google Scholar leading to diagnostic delay. Wang et al. also reported patients with primarily haematological manifestations.9Wang Z. Cao L. Su Y. et al.Specific macrothrombocytopenia/hemolytic anemia associated with sitosterolemia.Am J Hematol. 2014; 89: 320-324Crossref PubMed Scopus (52) Google Scholar They raised the concern that the majority of patients with sitosterolaemia were initially misdiagnosed as other chronic haemolytic anaemias/thrombocytopenias, leading to inappropriate therapies, as in our case. NGS-based diagnosis helps in characterising the defective gene and instituting appropriate management in such patients. Sitosterolaemia is resistant to standard statin-based treatment, though may respond to ezetimibe, a sterol absorption inhibitor that favourably increases platelet count and decreases mean platelet volume.10Othman R.A. Myrie S.B. Mymin D. et al.Ezetimibe reduces plant sterol accumulation and favorably increases platelet count in sitosterolemia.J Pediatr. 2015; 166: 125-131Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar In conclusion, the major diagnostic lesson from our family is that Mediterranean stomatocytosis/macrothrombocytopenia should be considered in patients with large platelets and unexplained mild haemolysis. Stomatocytes should be actively searched for and their persistence documented. The iOFT can help corroborate the morphology; however, it can also mislead, as in our case. NGS has significantly helped to reduce the time spent in investigating clinical conditions with protean manifestations which can puzzle clinicians and laboratory investigators alike. A critical evaluation of the blood film and phenotype is vital to identify the causative mutation(s). This patient profile truly illustrates the idiom: ‘The eyes observe what the mind knows’. The authors show gratitude towards all the technical and clinical staff involved in the study. This study was funded from Institutional funds. MJ and AA received Senior Research Fellowship from Indian Council of Medical Research, New Delhi, India. The authors state that there are no conflicts of interest to disclose." @default.
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• W2766474110 date "2017-12-01" @default.
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• W2766474110 title "First report of Mediterranean stomatocytosis/macrothrombocytopenia in an Indian family: a diagnostic dilemma" @default.
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