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`GENENTECH 2010
`GENZYME V. GENENTECH
`IPR2016-00383
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`Cell
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`racial groups, these crossovers have probably oc-
`curred independently on a number of occasions. Al-
`though there are two major regions of homology con-
`taining the 011 and 012 genes, it is not yet clear whether
`recombination has occurred at many places through-
`out these regions, or whether it is confined to only a
`few “hotspots."
`The nondeletion forms (as defined by restriction
`mapping of genomic DNA) of oz thalassemia are char-
`acterized by reduced production of oz globin, although
`both at genes are intact. Quantitation of oz mRNA and
`assessment of the relative proportions of the tran-
`scripts of the a1 and a2 loci by Berk-Sharp analysis
`suggest considerable heterogeneity at the molecular
`level (Higgs et al., PNAS 78, 5833-5837, 1.981; Lieb-
`haber and Kan, J. Clin. Invest. 68, 439-466, 1981;
`Orkin and Goff, Cell 24, 345-351, 1981).
`in some
`instances only one of the pair of at-globin genes is
`active; sequence analysis of a case of this type has
`shown a 5 bp deletion in the 5' IVS1 splice junction
`that may interfere with normal RNA processing (Orkin
`et al., PNAS 78, 5041-5045, 1981).
`In others both
`(X loci are active, although at a reduced (and variable)
`level. Some of these conditions may be transcriptional
`mutations, although others will probably turn out to be
`nonsense mutations and unstable at variants, as has
`been found to be the case in some of the ,8 thalasse-
`mias. The oi-globin chain-termination mutants, such as
`hemoglobin Constant Spring, have the phenotype of
`oc* thalassemia because the oz“ mRNA is very unsta-
`ble. This probably results from nucleolytic degradation
`following destabilization of the mRNA during read-
`through of the normally untranslated 3’ end. These
`insights into the molecular pathology of 01 thalassemia
`emphasize its extraordinary molecular diversity.
`Some interesting questions for the population ge-
`neticist arise from these observations. What, for ex-
`ample, are the selective factors that have allowed the
`deletion forms of of‘ thalassemia to reach a prevalence
`of 30%-40% in parts of West Africa and even as high
`as 90% in some pockets in India? Why is a° thalas-
`semia so rare in these populations when it
`is very
`common in Southeast Asia. where there is also a high
`incidence of the deletion forms of a* thalassemia?
`Why is it that in eastern Saudi Arabia, where well over
`50% of the population are affected with both deletion
`and nondeletion 01+ thalassemia,
`01° thalassemia is
`unknown? Why in the vast populations of Southeast
`Asia is there apparently only one oi°-thalassemia hap-
`lotype, whereas in the Mediterranean, where ’oz° thal-
`assemia is much less common, there are at least
`three?
`
`A rather different story is emerging for the ,8 thal-
`assemias. With the exception of a variety due to a
`deletion of the 3' end of the /3‘-globin gene that is
`found in some Indians, the H thalassemias seem to
`result largely from mutations that interfere with mRNA
`processing,
`translation or stability, or occasionally
`
`from highly unstable ,8-globin variants. Nonsense mu-
`tations, notably those of codons 17 and 39 of the B-
`globin mRNA, have been found to be the basis for a
`number of /3° thalassemias (Chang and Kan, PNAS
`76, 2886-2889, 1979; Orkin and Goff, JBC 256,
`9782-9784, 1981). One example of ,8” thalassemia
`due to a frameshift mutation has been found (Orkin
`and Goff, JBC 256, 9782-9784, 1981). Several
`cases of B1 thalassemia are due to a single nucleotide
`substitution (G to A) in IVS1 cf the B gene (Spritz et
`al., PNAS 78, 2455-2459, 1981 ; Westaway and Wil-
`liamson, NAR 9, 1777-1788, 1981). This has been
`cloned into an SV40—pBR328 vector and introduced
`into HeLa cells, and the RNA produced by the trans-
`fected cells has been analyzed by S1 nuclease map-
`ping and cDNA sequencing (Busslinger et al., Cell 27,
`289-298, 1981). By creating an alternative splicing
`junction, this mutation apparently leads to the produc-
`tion of about 90% of abnormally spliced ,8-globin
`mRNA with an early stop coclon; however, some
`mRNA is spliced normally, and hence a 8+ thalasse-
`mia phenotype results. In other cases, a G to A sub-
`stitution in the 5'-end IVS1 or IVS2 splice junctions
`appears to lead to a more severe processing defect,
`and hence to the phenotype of ,8° thalassemia (Baird
`et al., PNAS 78, 4218-4221, 1981; Orkin et al.,
`Nature, in press). A variety of ,8° thalassemia found in
`Kurdish Jews is characterized by a highly unstable [3
`mRNA, although themechanism is not known (Maquat
`et al., Cell 27, 543-553, 1981). While such studies
`have clarified the molecular basis for some types of
`,8+ and ,8° thalassemia, they have not yet, with one
`possible exception, unearthed a good candidate for a
`primary mutation affecting transcription. The excep-
`tion is the single-base change (C to G) 87 nucleotides
`preceding the CAP site and just upstream from the
`CCAAT box in the 5'—flanking region of a [i—thalasse-
`mia gene (Orkin et al., Nature, in press). Evidence that
`this is the actual cause of the ,8 thalassemia phenotype
`has yet to be obtained. The impression gained so far
`is that mutations that completely block B-gene tran-
`scription are extremely rare,
`in contrast with those
`defects that affect processing of the primary tran-
`script, or stability or translation of ,8-globin mRNA.
`The ,8-thalassemic mutations appear to be in linkage
`disequilibrium with a limited group of restriction en-
`zyme polymorphisms within and flanking the ,8-gene
`cluster (Orkin et al., Nature, in press). In the Mediter-
`ranean population there are nine common B-gene
`haplotypes, each with a different associated 6-thal-
`assemlc mutation. The different haplotypes and mo-
`lecular variants of ,8 thalassemia vary widely in fre-
`quency, although their distribution among ,8 thalas-
`semic patients is largely similar to that in the nonaf-
`fected members of the population from which these
`patients were derived. The reasons for these curious
`relations remain to be determined.
`
`The 8,8 thalassemias and some forms of HPFH have
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`disclosed yet another pattern of molecular defects.
`These conditions, which are classified into Gy and
`G1/Ay forms depending on the structure of the hemo-
`globin F that is produced, involve deletions of different
`sizes of the 6-67-Ay—5—fi globin-gene cluster on the
`short arm of chromosome 1 1 (figure). In G1/A}/8,8 thal-
`assemia the deletions start 3'
`to the /S‘ gene and
`remove the whole of the B and part of the 8 genes, but
`leave the 5' end of the 8 gene intact (Bernards et al.,
`Gene 6, 265-280, 1979; Ottolenghi et al., Nature
`278, 654-657, 1979). Two deletions responsible for
`G",/Ay HPFH extend farther in the 5' direction and
`remove all of the 6 gene, one finishing in the region of
`the Alu repeat sequence, which lies 5’ to the 6 locus,
`while another extends 5 kb farther upstream (Mears
`et al., PNAS 75, 1222-1226, 1978; Bernards and
`Flavell, NAR 8, 1521-1534, 1980; Tuan et al., Nature
`285, 335-337, 1980). Both 6,8 thalassemia and HPFH
`are characterized by the production of considerable
`amounts of y chains in adult life; relatively more are
`synthesized in HPFH than in 88 thalassemia. Hence it
`has been suggested that the Alu repeat region may
`be involved in the regulation of y-chain synthesis, and
`that its removal is required for the HPFH phenotype,
`but this notion is based only on a few detailed struc-
`tural analyses, most of which are incomplete at the 3'
`end of the deletions. There is no knowledge of what
`the effects of juxtaposition of normally distal chromo-
`somal sequences may be on the activity of the ,8-gene
`cluster.
`
`The difficulties of interpreting the functional effects
`of these deletions are exemplified further by the dif-
`ferent lesions that result in 936,13 thalassemia. One
`lesion involves an inversion of most of the region
`between the "y— and 8—globin genes together with two
`deletions, a novel rearrangement that may have re-
`sulted from a rare, doub|e—intrachromosomal cross-
`ing-over event (Jones et al., Nature 291, 39-44,
`1981). Two other types of G3/6/3 thalassemia are due
`to deletions involving the 8 and ,8 genes and either
`part or all of the Ay gene (Orkin et al., J. Clin. invest.
`64, 866-869, 1979; Jones et al., NAR 9, 6813-6825,
`1981 ). All three forms have similar phenotypes, yet in
`the first type the Alu repeat region is present, though
`in a reverse orientation, while in the latter two cases
`it has been deleted. It may be, of course, that a single
`remaining Gy locus, even if fully active, cannot com-
`pensate for the lack of ,8-chain production; if it could,
`these disorders might have had an HPFH phenotype.
`Although these findings do not argue conclusively
`for or against there being regions involved in the
`regulation of the changes in globin-gene expression,
`it
`is apparent that most of the major downstream
`deletions are associated with persistent activity of
`upstream loci
`that are normally inactivated during
`development. Further evidence for the interdepen-
`
`dence of the loci within this gene cluster comes from
`studies of one form of 3/8,8 thalassemia that results
`from a long deletion involving both y-globin genes;
`although the ,8 gene is intact,
`,8-globin synthesis is
`markedly reduced or possibly even abolished (van der
`Ploeg et al., Nature 283, 637-642, 1980). This has
`led to the notion that the y and ,8 genes lie in function-
`ally distinct domains, the activities of which are mu-
`tually exclusive (Bernards and Flavell, op. cit.). De-
`spite the lack of evidence for specific regulatory se-
`quences, it does appear that persistent y—chain syn-
`thesis in these conditions is related in some way to
`loss of extensive regions of the y-8-/9 globin-gene
`cluster, and is not simply due to any sort of major
`rearrangement. Thus a duplication of the Gy gene
`resulting in effect in a 5 kb insertion and giving the
`arrangement Gy-Gy-Ay has recently been observed
`(Trent et al., NAR 9, 6723-6733, 1981). The pheno-
`typic effects of this insertion, as for the triplicated 04
`genes, are minimal.
`It
`is becoming clear that the molecular basis for
`most of the thalassemias is a simple cis-acting muta-
`tion within the globin-gene cluster, and that these
`disorders are extraordinarily diverse; often, appar-
`ently homozygous individuals are in fact compound
`heterozygotes for different molecular forms of thalas-
`semia. The coinheritance of one or more deletion or
`nondeletion forms of a thalassemia can significantly
`ameliorate homozygous ,6” or ,8” thalassemia (Weath-
`erall et al., Lancet 1, 527-529, 1981). Other human
`genetic disorders will probably show similar molecular
`diversity; there is no reason to believe that thalassemia
`is unique in this respect.
`Further analysis of some of the splicing defects that
`give rise to the ,8" thalassemias, with more refined in
`vitro transcription systems, will provide useful infor-
`mation about the details of processing of the primary
`transcript. Although no mutations affecting transcrip-
`tion of the globin genes have yet turned up, there is
`still hope; some of the nondeletion or thalassemias and
`the mild ,8* thalassemias are possible candidates.
`With regard to the developmental genetics of hemo-
`globin, we may have concentrated on the wrong con-
`ditions; the forms of pancellular HPFH and 68 thalas-
`semia studied so far are associated with extensive
`
`gene deletions, and may not be the best models for
`analysis of gene regulation during normal develop-
`ment. Conditions such as nondeletion HPFH, although
`less dramatic in their phenotypic effects, may reflect
`more closely the changes that take place during nor-
`mal fetal development.
`It is encouraging that the ge-
`netic determinants for some of these conditions have
`been shown to be linked to restriction enzyme poly-
`morphisms within and flanking the y-8—,8 globin-gene
`cluster, and their locations may soon be pinpointed
`more precisely.
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