Figure 1.27 A regulatory SNP creates a new promoter. Schematic of a globin cluster and flanking region showing multispecies conserved sequences (MCSs) and DNase hypersensitive sites (DHSs) together with location of regulatory sequence variant (de Gobbi et al. 2006).

1.3.10 Tandem repeats

DNA sequence within the a and p globin loci was recognized to vary between individuals irrespective of a particular disease phenotype. Specific DNA sequences recognized by particular restriction enzymes were found to be polymorphic, providing genetic markers for early linkage studies. Repetitive DNA sequences are seen ranging from simple dinucleotide repeats to more complex repeating units spanning hundreds of nucleotides. Repeat units are tandemly arranged 'head to tail' without intervening sequence, constituting 'tandem repeats' which may be highly polymorphic within and between populations (Box 1.21; reviewed in detail in Chapter 7).

'Microsatellites' comprise arrays less than 100 base pairs (bp) in length made up of simple repeats up to 6 bp in length. Microsatellites are common, constituting an estimated 3% of the human genome with over one million such loci (Lander et al. 2001; Ellegren 2004). Sequencing of the terminal two megabases (Mb) (1000 kb) of chromosome 16p revealed 322 simple repeats, constituting 1.8% of the sequence and some 35 625 bp (Daniels et al. 2001). A variety of microsatellites are recognized in the a and P globin loci, notably dinucleotide repeats such as (AC) n (Poncz et al. 1983; Shen et al. 1993). Use of microsatellite markers including dinucleotide, trinucleotide, and tetranucleotide repeats allowed linkage maps (Section 2.2.2) of chromosome 16 to be established (Kozman et al. 1995).

Longer tandem repeats are also seen which can be hypervariable between individuals. Such 'minisatellites' are typically between 100 bp and 20 kb in size with repeat units between 7 and 100 bp. A hypervariable region was recognized 8 kb downstream of HBA1 in both patients with thalassaemia and unaffected individuals, which comprised between 70 and 450 repeats of a 17 bp sequence 'ACACGGGGGGAACAGCG' (Higgs et al. 1981; Jarman et al. 1986). Some 100 kb upstream of the a globin gene cluster a further minisatellite is seen in which there are between five and 55 repeats of a 57 bp sequence (Jarman and Higgs 1988). These and other minisatellites, together with restriction enzyme fragment length polymorphisms in the globin loci, were found to be highly variable among diverse populations providing markers for genetic population studies (Higgs et al. 1986).

'Satellite' DNA comprises very large arrays spanning hundreds to thousands of kilobases of DNA. Such sequences are commonly encountered across the genome, notably at centromeres (a region visible as a constriction during metaphase typically in the middle of chromosomes; see Box 1.4) as well as pericentromeric and telomeric regions (repetitive DNA found at the end of chromosomes seen as simple repeats of the sequence TTAGGG; Box 7.3) where DNA is typically in a compressed transcriptionally inactive state (heterochroma-tin). Satellite DNA is recognized in the terminal region of chromosome 16p but it is in the pericentromeric region of chromosome 16p11 that large tracts of alpha satellite DNA have been found (Martin et al. 2004).

1.3.11 Mobile DNA elements and chromosomal rearrangements

Mobile DNA elements are segments of DNA that can transport or duplicate themselves (transpose) to other genomic regions. They are commonly found across the human genome although almost exclusively such DNA segments now represent a genomic 'fossil record' of past

Box 1.21 Tandemly repeated DNA

Tandemly repeated DNA sequences in a head to tail configuration occur commonly across the genome and they are typically polymorphic in nature due to expansion or contraction of the number of repeating units. In some cases they show extreme levels of variation allowing useful approaches such as DNA

fingerprinting to uniquely identify a particular individual (Section 7.4.1) (Jeffreys et al. 1985). Classification is possible on the basis of increasing size into microsatellite, minisatellite and satellite DNA.

events with only a very small number of full length elements remaining competent for transposition. The biology of mobile DNA elements is reviewed in detail in Chapter 8 where the major classes, retrotransposons (which transpose via an RNA copy) and DNA transposons are discussed. Retrotransposons include long interspersed elements (LINEs) and short interspersed elements (SINEs) of which some are autonomous, such as the LINE 1 family, while others such as Alu elements are dependent on active L1 elements encoding the proteins required for retrotransposition. Alu elements are characteristic DNA sequences some 300 bp in length which are very common, constituting some 10% of human genomic sequence with an estimated one million copies present. They are polymorphic and have proved highly informative in population genetic analysis and studies of evolutionary history.

Sequencing of the terminal 2 000 000 bp of chromosome 16, which includes the a globin gene cluster, revealed a very gene-rich region with 100 confirmed and 20 predicted genes. The sequence also contained a high proportion of Alu repeats (nearly 20%) with 1442 such elements identified, constituting the majority of the SINEs identified (Daniels et al. 2001). LINEs over this region were present less commonly than the genomic average, with 279 described (5% of the sequence); elsewhere within the region, 126 long terminal repeat (LTR) retrotransposons were found (2.5% of sequence).

The high sequence homology of Alu repeats predisposes to unequal homologous recombination leading to a range of chromosomal rearrangements including deletions, duplications, and translocations. Such an event was recognized approximately 105 kb from the 16p subtelomeric region leading to an interstitial rearrangement (deletion or translocation) which led to loss of the MCS-R2 (HS-40) upstream regulatory element and a thalassaemia (Flint et al. 1996). Homologous recombination due to a crossover event between Alu elements is also recognized, leading for example to a 62 kb deletion and a thalassaemia (denoted aaRA) (Nicholls et al. 1987). Indeed, Alu elements are found to be present at many of the breakpoints around the a globin gene locus (Nicholls et al. 1987).

1.3.12 Monosomy and trisomy of the terminal end of chromosome 16p

Transfer of chromosomal regions between two non-homologous chromosomes as a result of breakage and reattachment results in chromosomal rearrangements, a structural change described as a translocation (Box 1.22).

Study of the thalassaemias has provided examples of specific translocation events leading to partial monosomy or trisomy. This is illustrated by the case of a 3-year-old child who presented with moderate global developmental delay and a mild hypochromic

Box 1.22 Translocations

Translocations may result after breaks involving two chromosomes, for example during meiotic recombination involving mispaired chromosomes, with part of one chromosome becoming detached and reattaching to another non-homologous chromosome. There may be no net gain or loss of genetic material, in which case the translocation is said to be balanced, or there may be, in which case it is unbalanced. Reciprocal translocations between non-homologous chromosomes are relatively common and usually spontaneous leading to stable balanced exchanges. Robertsonian translocations are the most common recurrent type of translocation and specifically involve the acrocentric chromosomes (chromosomes 13, 14, 15, 21, and 22) in which breaks in the very short p arm lead to fusion of the remaining long arms (Section 3.4.4). Most often this involves exchanges between chromosomes 13 and 14, and 14 and 21 - for the individual there is little phenotypic consequence but for their offspring there is a risk of monosomy or trisomy. Translocations are reviewed in Section 3.4.

microcytic anaemia (Buckle et al. 1988). She was found to have a thalassaemia trait with a moderate degree of mental retardation together with mildly dysmorphic features. Her father had no cytogenetic abnormalities detected (his chromosomal complement of 23 chromosome pairs denoted 46,XY), however her mother had a balanced reciprocal translocation involving the long arm of chromosome 10 (10q26.13) and short arm of chromosome 16 (16p13.3). This is denoted t(10,16) (q26.13;p13.3) giving the nomenclature 46,XX,t(10,16) (q26.13;p13.3). For her mother there was no apparent phenotypic consequence as there was no net gain or loss or genetic material. However, the affected child inherited a 'derived' copy of chromosome 16 lacking the terminal region of 16p beyond band 13.3 (denoted 16p13.3^pter) and having in its place 10q26.13^qter, together with a normal copy of chromosome 16 from her father, meaning that she had partial monosomy for the terminal region of 16p beyond 16p13.3 which includes the a globin gene cluster (Fig. 1.28). DNA studies confirmed that the affected child did not inherit the maternal copy of chromosome 10 bearing the terminal region of chromosome 10q; rather she inherited two normal copies of chromosome 10 which meant that in combination with the terminal portion of 10q present on the rearranged chromosome 16 she inherited, the child had partial trisomy for chromosome 10q26.13^qter. This study provided evidence supporting the physical location of the HBA genes within the terminal region of 16p13.3^pter.

Other examples involving partial monosomy for the terminal portion of chromosome 16p have been reported. A Nigerian family was described in which the mother had a balanced translocation involving the subtelomeric regions of the short arms of chromosomes 1 and 16 which were cytogenetically invisible but had significant consequences. Her son inherited the derived copy of chromosome 16 leading to partial monosomy and a thalassaemia; his sister inherited a normal copy of chromosome 16 but the derived copy of chromosome 1 (Lamb et al. 1989). Both children had some dysmorphic features and mental retardation (Box 1.23).

Trisomy involving the terminal end of chromosome 16p is also seen, for example trisomy distal to 16p12 was found to be present in a 7-week-old infant presenting with a number of congenital malformations including cleft palate, talipes (club foot), and hypospadias (a urinary tract abnormality) (Wainscoat et al. 1981). This resulted from a maternal reciprocal translocation involving the short arms of chromosomes 14 and 16 (14p11 and 16p12) denoted t(14;16)(p11;p12). As would be predicted with additional copies of the HBA genes being present, an excess of a globin chain production was seen.

The consequences of possession of an abnormal number of chromosomes (aneuploidy) or portions of chromosomes (segmental aneusomy) are reviewed in more detail in Chapter 3. Loss of a single chromosome (monosomy) involving autosomal chromosomes

Box 1.23 Alpha thalassaemia and mental retardation

The relationship between a thalassaemia and mental retardation was recognized some years earlier in some patients with Hb H disease (Weatherall et al. 1981) and is now classified as a thalassaemia/ mental retardation, deletion type (ATR16) (OMIM 141750). Wilkie and colleagues reported a series of patients with a thalassaemia, mental retardation, and a range of dysmorphic features - all had deletions involving the terminal end of 16p (16p13.3)

but of variable extent with four cases due to unbalanced translocations (Wilkie et al. 1990). Among the de novo truncations, one case involved a substantial 2 Mb terminal deletion which was found to be stabilized at the chromosomal breakpoint by the addition of telomeric (TTAGGG)n repeats (Lamb et al. 1993), a mechanism found to be present in a series of similar truncations of chromosome 16 involving 16p13.3 (Flint et al. 1994).

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