Investigating haemoglobinopathies

Carrier frequencies of thalassaemia alleles (%) Regionβ-Thalassaemiaα 0 -Thalassaemiaα + -Thalassaemia Americas 0–30–50–40 Eastern Mediterranean 2–180–21–60 Europe 0–191–20–12 Southeast Asia 0–111–303–40 Sub-Saharan Africa 0–12010–50 Western Pacific 0–1302–60 Weatherall D, et al. Inherited Disorders of Hemoglobin. In: Disease Control Priorities in Developing Countries. 2nd ed. New York: Oxford University Press; 2006: Available from: 7% of the world’s population are carriers of haemoglobin disorders

Types of haemoglobinopathies Thalassaemias – result from an imbalance in  and  globin gene production, most commonly due to –Point mutations in the  gene –Deletion of one or more  genes Haemoglobin variants - result from point mutation in the  or  genes leading to amino acid substitution, producing a different haemoglobin, sometimes with different properties

l Heterozygous: –Thalassaemia trait/minor Mild/no microcytic anaemia l Homozygous: –Thalassaemia major Marked anaemia (usually transfusion dependence) Iron overload Transfusion complications

Clinical impact of thalassaemia major l Transfusion dependence l Iron overload –Cardiac complications –Endocrine (diabetes, hypothyroidism, hypogonadism, hypoparathyroidism) l Transfusion complications (eg hepatitis C) l Osteoporosis – bone disease

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Mutations in thalassaemia  thalassaemia –200 point mutations in the  globin gene –Deletions are rare  thalassaemia –Deletion of one  globin gene on an allele Common, many ethnic groups –Deletion of both  globin genes on an allele Less common, some ethnic groups –Occasional point mutations

Alpha thalassaemia genotypes

 globin under-production leads to excess of other haemoglobins  globin under-production leads to excess of other haemoglobins

Microcytosis l Thalassaemia trait –Anisocytosis, poikilocytosis –Target cells l Iron deficiency –Hypochromic red cells –Pencil cells l Anaemia of chronic disease (eg rheumatoid arthritis) – upregulation of hepcidin, functional iron deficiency (poor release of iron from enterocytes, hepatocytes) l Very rare: sideroblastic anaemia

Iron deficiency

Thalassaemia trait

Cellulose acetate and citrate agar gel electrophoresis A2 C S F A Barts H C S A F

Thalassaemia trait - phenotype  thalassaemia trait –Variable microcytosis, mild anaemia  thalassaemia –Single gene deletion: none/microcytosis –Two gene deletion: mild anaemia/variable microcytosis –Three gene deletion (Hb H disease) Variable –Four gene deletion Hb Barts hydrops fetalis l Compound heterozygotes –Sickling disorders (HbSS, HbSC, HbS-  -thal

Interpreting the haemoglobin EPG An elevated HbA 2 is diagnostic of  thalassaemia trait Hb H inclusions are diagnostic of  thalassaemia trait l Elevated Hb F –May be seen in  thalassaemia trait –Other disorders of erythropoiesis –Pregnancy –Hereditary persistence of fetal haemoglobin l Abnormal bands –Haemoglobin E (with or without  thalassaemia), Lepore Matters because of compound heterozygosity with  thalassaemia trait –Hb S, C Matter because can contribute to sickling –Other D, O, etc, etc Often don’t matter Some produce unstable haemoglobins Can’t be easily characterised on standard HbEPG

Lab Testing l FBC l Characterisation of abnormal haemoglobins –Haemoglobin electrophoresis –HPLC –Supravital staining for H inclusions l Iron studies –Ferritin –Transferrin/TIBC –Transferrin saturation –Serum iron l (inflammatory markers)

Common problems in Hb EPG interpretation l H inclusions are rare –  thalassaemia cannot be excluded The findings of  thalassaemia may be masked by iron deficiency (reduction in Hb A 2 ) Rare problem of normal Hb A 2  thalassaemia trait l Unexplained elevated Hb F

Solutions to difficult Hb EPG results l Family studies l Repeat testing when iron replete l DNA testing –  globin gene PCR testing –  globin gene sequencing

When does it matter? l Pre-pregnancy l Early pregnancy –DNA testing is rapid if the mutations are known –DNA testing is slow and may not yield a result if the mutation is not known –CVS possible at 11 weeks –Second trimester amniocentesis –Genetic counselling takes time and causes anxiety l The role for screening?

Important patterns l Microcytosis in early pregnancy –Partner should have FBC, iron studies and Hb EPG (to exclude both thalassaemia trait and a sickling disorder). –Don’t wait for the woman’s results l Microcytosis (in a male or female) –Test the partner –Test other family members – it may help someone else l Microcytosis in both partners –Is there a risk of Hb H disease or Hb Barts hydrops fetalis –“masked”  thalassaemia major l Hb S or Hb E trait –Think of compound heterozygosity –Test the partner and other family members