Transcript Dia 1 - BeSHG

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