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Principles of Molecular Disease: Lessons from the Hemoglobinopathies Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital The effect of mutations on protein function • mutations resulting in a loss of function of the protein • mutations resulting in a gain of function of the protein • mutations resulting in a novel property by the protein • mutations resulting in gene expression at the wrong time or place Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital Loss-of-function mutations • deletion of the entire gene (and eventually also contiguous genes) examples: microdeletion syndromes, monosomies (Turner), a-thalassemias • chromosomal rearrangements • nonsense mutations or frameshift mutations resulting in a premature stop codon • missense mutations may also abolish protein function Severity of disease ~ amount of function lost Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital Gain-of-function mutations = mutations that enhance one or more of the normal functions of the protein • mutations that enhance one normal function of the protein f.e.: the G380R mutation in FGFR3 causing achondroplasia • mutations that increase the production of a normal protein in its normal environment f.e.: trisomy 21 (Down syndrome) duplication of PMP22 in Charcot-Marie-Tooth disease type 1A chromosomal duplications in cancer Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital Novel property mutations = mutations that give a novel property to the protein without necessarily altering its normal function these mutations are rather infrequent since most amino acid substitutions are either neutral or detrimental to the function or stability of the protein a classic example is sickle cell disease Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital Mutations associated with heterochronic or ectopic gene expression = mutations that alter the regulatory regions of a gene to cause inappropriate expression, at an abnormal time or place Examples: • oncogene mutations in cancer • hereditary persistence of fetal hemoglobin (continued expression of the g-globin gene) Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital Genetic Disorders of the Central Nervous System – International Module on Pediatric Rehabilitation G Mortier – Department Medical Genetics – Ghent University Hospital Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital The Hemoglobinopathies • most common single-gene disorders in humans • more than 5% of the world’s population is carrier of an abnormal globin gene Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital The Hemoglobins • hemoglobin (Hb) is the oxygen carrier in vertebrate red blood cells • the Hb A molecule contains 4 subunits: 2 a-chains and 2 b-chains • each subunit is composed of : - a polypeptide chain (globin) - a prosthetic group (heme) which is an iron-containing pigment that combines with oxygen • globin structure highly conserved Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital The Hemoglobins • hemoglobin (Hb) is the oxygen carrier in vertebrate red blood cells • the Hb A molecule contains 4 subunits: 2 a-chains and 2 b-chains • each subunit is composed of : - a polypeptide chain (globin) - a prosthetic group (heme) which is an iron-containing pigment that combines with oxygen • globin structure highly conserved • Hb A (adult hemoglobin): a2 b2 • and five other hemoglobins Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital identical chromosome 16 differ only in 10/146 AA chromosome 11 In adult life: >97% HbA 2% HbA2 <1% HbF common ancestral gene Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital Globin switching Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital 6 kb Chr 11 LCR e Gg Ag yb d b • expression of b-globin gene controlled by nearby promoter and LCR • locus control region (LCR): required for the expression of all the genes in the b-globin cluster • deletions of LCR results in egdb- thalassemia Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital Three broad groups of mutations that affect hemoglobin 1. mutations that alter the structure of the globin protein 2. mutations that decrease the synthesis of one or more globin chains 3. mutations that impair the globin developmental switching Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital 1. Hemoglobin Structural Variants • usually due to point mutations in one of the globin genes • more than 400 abnormal hemoglobin variants have been described • only about 50% are clinically significant • three classes: - mutants that cause hemolytic anemia - mutants that alter oxygen transport - mutants that reduce the abundance of the globin chain (thalassemias) Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital Hemoglobin Structural Variants that cause Hemolytic Anemia Pathogenesis - the mutant makes the Hb tetramer unstable loss-of-function mutations f.e.: Hb Hammersmith (b-chain Phe42Ser mutation) - the mutant gives the globin chain an unusual rigid structure novel property mutations f.e.: sickle cell globin; HbC Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital Sickle Cell Disease • sickle cell hemoglobin (HbS) was the first abnormal Hb detected • HbS: singe nucleotide substitution results in Glu6Val mutation in b-chain • sickle cell disease is due to homozygosity for the Glu6Val mutation (AR condition) • sickle cell trait refers to the heterozygous state • common in equatorial Africa; 1/600 African Americans is born with the disease • about 8% of African Americans are heterozygous • heterozygotes are protected against malaria Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital Sickle Cell Disease • HbS has a normal ability to bind oxygen in normal circumstances • in deoxygenated blood HbS is less soluble as normal Hb • in conditions of low oxygen tension, the HbS molecules aggregate which distort the RBC to a sickle shape. These misshapen erythrocytes are less deformable than normal and cannot squeeze in single file through capillaries, thereby blocking blood flow and causing local ischemia Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital 1. Hemoglobin Structural Variants • usually due to point mutations in one of the globin genes • more than 400 abnormal hemoglobin variants have been described • only about 50% are clinically significant • three classes: - mutants that cause hemolytic anemia - mutants that alter oxygen transport - mutants that reduce the abundance of the globin chain (thalassemias) Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital Hemoglobin Structural Variants that alter oxygen transport • these mutations have little or no effect on hemoglobin stability • mutations in the region of the heme pocket will affect the heme-globin bond in a way that makes the iron resistant to the enzyme methemoglobin reductase. The result is accumulation of methemoglobin in the blood with cyanosis as a consequence. f.e. Hb Hyde Park (b-chain His92Tyr mutation) • heterozygotes are cyanotic but usually asymptomatic. The homozygous state is presumably lethal. Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital 2. Hemoglobin Synthesis Disorders (The Thalassemias) • collectively the most common human single-gene disorders! • carriers have an protective advantage against malaria • > qalassa (sea): first discovered in persons of Mediterranean origin • imbalance in the normal ratio of a : b chains - reduction in synthesis of globin chains - instability of globin chains • excess of normal chains will damage the RBCs (hemolytic anemia) • defect in Hb synthesis will cause hypochromic, microcytic anemia Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital The a - Thalassemias • decreased to absent a-globin production • affect the formation of both fetal and adult Hb • in the absence of a-globins: - Hb Bart’s: g4 Homotetrameric Hb that are ineffective oxygen carriers - Hb H: b4 • hydrops fetalis in severe forms (due to severe hypoxia) • anemia in mild forms (precipitation of Hb inclusions in RBCs damage in spleen destruction) Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital The a - Thalassemias • most commonly due to deletion of the a-globin genes Misalignment with Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital The a - Thalassemias clinical condition number of functional a-genes a-globin gene genotype a-chain production Normal 4 aa/aa 100% Silent carrier 3 aa/a- 75% a-thalassemia trait 2 a-/a- or aa/- - * 50% 1 a-/- - 25% 0 - -/- - 0% (mild anemia, microcytosis) Hb H (b4) disease (moderately severe hemolytic anemia) Hydrops fetalis (Hb Bart’s g4) * Carriers frequent in Southeast Asia Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital The a - Thalassemias • most commonly due to deletion of the a-globin genes • other forms rather rare: - form due to the ZF deletion (named after individual ZF) - the ATR-X syndrome Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital a – Thalassemia due to the ZF deletion transcribed from the opposite strand a2-gene is silenced due to the generation of antisense RNAs from the truncated LUC7L gene wild-type antisense transcripts do also exist and play a role in regulation of gene expression (f.e. X inactivation)! Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital The ATR-X syndrome • association of mental retardation and a-thalassemia • due to mutations in the X-linked ATRX gene • ATRX codes for a chromatin remodeling protein • the ATRX protein functions in trans to activate the expression of the a-globin genes and other genes important for normal brain function • partial loss-of-function mutations result in modest reduction of a-globin synthesis • somatic (more severe) mutations in ATRX cause the a-thalassemia myelodysplasia syndrome (if germline: hydrops fetalis!) Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital The ATR-X syndrome • X-linked form of syndromic mental retardation • prevalence: 1-9/1.000.000 ? • 95% severe to profound mental retardation • usually no speech • 30% seizures; 75% microcephaly (postnatal onset) • spastic paraparesis may be present • facial dysmorphism • 80% genital abnormalities (undescended testes to ambiguous genitalia) • 65% short stature; 20% heart defects • Hb H inclusions in 90% present, usually mild anemia (Hb H inclusions only appear after 30%-40% reduction in a-globin synthesis) Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital The ATR-X syndrome Erythrocytes after incubation in briljant cresyl blue. Hb H inclusions in three cells give them a golf ball like appearance From Gibbons R. Orphanet Journal of Rare Diseases 2006;1:15 Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital The b - Thalassemias • decreased b-globin production • onset not apparent until a few months after birth • same pathophysiology as in a-thalassemias: precipitation of the excess a-chains results in hypochromic, microcytic anemia • increased levels of HbA2 (a2d2) and HbF (a2g2) • usually due to single-base pair substitutions (rather than deletions) • persons with two abnormal alleles are frequently compound heterozygous • two b-thalassemia alleles: usually thalassemia major (severe anemia) • one b-thalassemia allele: thalassemia minor (mild anemia, no clinic) • simple versus complex b-thalassemia Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital of the 3’ end of the gene >> Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital Point mutations that cause b-thalassemia are distributed throughout the gene. They affect virtually every process required for the production of normal b-globin. Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital Posttranscriptional modifications of mRNA Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital RNA splicing mutations in b – Thalassemias (1) Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital RNA splicing mutations in b – Thalassemias (2) Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital RNA splicing mutations in b – Thalassemias (3) Very frequent in Southeast Asia Hb E: example of a single nucleotide substitution that affects both RNA splicing and the coding sequence Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital Complex b – Thalassemias (egdb) thalassemia (illustrates importance of LCR) (db) thalassemia (Agdb) thalassemia Hereditary persistence of fetal hemoglobin Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital 3. Globin Developmental Switching Disorders • hereditary persistence of fetal hemoglobin • group of clinically benign conditions • production of higher levels of Hb F than is seen in (db) thalassemia • they impair the perinatal switch from g-globin to b-globin synthesis Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital Fig 12.1 Postgraduate course Human Genetics – 06/12/08 Geert Mortier, MD, PhD – Center for Medical Genetics – Ghent University Hospital