Hbb

c.[20A>T;220G>A]

Figure 1.20 Sequence variation in HBB. The DNA sequence of HBB has been found to be remarkably diverse, with many nonsynonymous variants resulting in amino acid changes. Single amino acid substitutions have been reported in at least 138 out of 146 codons in p globin with over 335 different single nucleotide changes as well as concurrent variants, insertions, and deletions (Huisman et al. 1996). The portion of the figure showing sequences, genomic location, and SNPs was adapted from a screenshot of the UCSC Genome Browser (Kent et al. 2002) (http://genome.ucsc.edu/) (Human March 2006 Assembly). Screenshot of the Database of Hemoglobin Variants and Thalassemias (http://globin.bx.psu.edu/hbvar/menu.html) (Patrinos et al. 2004), reprinted with permission.

Single nucleotide variant

Example: Hb Kansas

- A to C substitution changes codon 102 from AAC to ACC, resulting in Asn to Thr substitution

- leads to reduced oxygen affinity of Hb

Example: Hb Luton

- A to T substitution changes codon 89 from CAC to CTC, resulting in His to Leu substitution

- leads to increased oxygen affinity of Hb and polycythaemia

Example: Hb S

- A to T substitution changes codon 6 from GAG to GTG, resulting in Glu to Val substitution

- leads to sickle haemoglobin

Example: Hb Koln

- G to A substitution changes codon 98 from GTG to ATG, resulting in Val to Met substitution

- leads to unstable Hb, anaemia with Heinz body formation

More than one single nucleotide change

New variant (mutation) arising on background of existing variation or due to crossover event

Example: Hb C-Harlem

- G to A substitution coding DNA position 220 in addition to sickle variant

- red cells sickle

Fusion Hb

Example: Hb Lepore

- hybrid sequence starting as S chain then becoming p chain; hybrid gene under control of S promoter

- due to unequal cross over between misaligned chromosomes

Variant affecting initiation codon

Deletions

Termination codon variant

Example: Hb M-Boston

- C to T substitution changes codon 58 from CAC to TAC, resulting in His to Tyr substitution

- affects oxygen transport

Deletions

Example: Hb Gun Hill

- HBB:c.274_288del

- deletion of sequence CTG CAC TGT GAC AAG results in loss of Leu-His-Cys-Asp-Lsy

- leads to unstable Hb, impaired binding and chronic haemolysis

Example: Hb Grady

- HBA1:pThr119_Pro120insGluPheThr

- insertion of Glu Phe and Thr residues between codons 118 and 119 leads to elongation of a chain

- no significant change in Hb function

Deletion/insertions

Example: Hb Montreal

- HBB:c.220_228delinsAlaArgCysGln

- deletion of Asp Gly Leu at positions 73-76 with insertion of Ala Arg Cys Gln

- resulst in unstable Hb; initially described in patient with haemolytic anaemia

Termination codon variant

Example: Hb Constant Spring

- a chain 172 amino acids (normal 142aa) as a result of T to C substitution in stop codon; a globin synthesis reduced

- commonest non deletion variant leading to a thalassaemia

Example: Hb Thionville

- Substitution of glutamic acid for valine as first residue in the processed a chain assoicated with retention of the initiator methionine residue

Variants leading to frameshift

Example: Hb Wayne

- HBA2:c.420delA

- first reported frameshift mutation in man

- results from 'A' nucleotide deletion at third position of codon 139 in HBA2 or HBA1

Unaffected

ACC UCC AAA UAC CGU UAA Thr Ser Lys Tyr Arg Term

Hb Wayne ACC UCC AAU ACC GUU AAG CUG GAG CCU CGG UAG Thr Ser Asn Thr Val Lys Leu Glu Pro Arg Term

Figure 1.21 Overview of structural variants of haemoglobin and their molecular basis at the DNA level. Numbers of haemoglobin variants known for a and p chains are shown on the central pie chart and under major headings classifying types of variation. The vast majority are single nucleotide changes. Numbers of variants and selected examples derived from Old (2006).

such as Hb C (common in West Africa and areas of the Mediterranean) and Hb E (India, South East Asia) were named on the basis of letters of the alphabet, while more recently described variants have names derived from the place of discovery (Hb Leidin, Hb Lepore).

An online database of sequence variants leading to haemoglobin variants, thalassaemias, and haemoglobinopathies 'Human Hemoglobin Variants and Thalassemias' is available (http://globin.bx.psu.edu/ hbvar) (Giardine et al. 2007). In most cases haemoglobin variants are not associated with disease, however some can affect the normal functioning of the haemoglobin molecule in a variety of ways (Fig. 1.21). The variant haemoglobin may be unstable, precipitating within the red blood cells and leading to haemolytic anaemia as seen with Hb Koln (Carrell et al. 1966), or oxygen affinity may be reduced (Hb Kansas) (Reissmann et al. 1961) or increased (Hb Luton) (Williamson et al. 1992). Major physiological changes can result from single amino acid substitutions, as seen with Hb M Boston in which a critical amino acid involved in haem binding within a pocket created by the globin chain is changed from histidine to tyrosine due to a C to T nucleotide substitution (HBA2:c.175C>T). The iron becomes locked in an oxidized state and unable to participate in oxygen transport (Gerald et al. 1957; Pulsinelli et al. 1973). Clinically this is manifest as methaemoglobinaemia and cyanosis. While the examples discussed to date result from single nucleotide substitutions, more rarely other DNA sequence diversity can lead to structural haemoglobin variants. These include multiple single nucleotide changes (Hb C-Harlem), nucleotide deletions (Hb Gun Hill) or insertions (Hb Grady), or fusion events combining different globin chains (for example Hb Lepore) (Fig. 1.21).

Thalassaemias result from a defect or imbalance in synthesis of one or more of the globin molecules (Weatherall 2001, 2004a, 2004b; Weatherall and Clegg 2001). The mechanisms whereby changes in DNA may result in altered globin synthesis and thalassaemia are diverse, involving the processes of transcription, RNA processing and translation. a thalassaemia can arise from a variety of different genetic causes, ranging from single base changes in the DNA sequence of genes encoding a globin (HBA! and HBA2) to small and large scale deletions; over 80 such events have been identified with large deletions most common (Box 1.15). Single base changes (point mutations) and small deletions or insertions most commonly account for ß thalassemia, with over 200 different DNA sequence variants (mutations) identified in the ß globin gene HBB. In terms of global health, ß thalassemia represents the greater burden of disease and public health challenge (Box 1.16).

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