IV-b.   Swiss Type of Nondeletional HPFH

The name "Swiss type of HPFH" comes from a study by Marti and coworkers (1-3) who observed, in the Swiss population, apparently normal adults with normal hematology but with elevated levels of Hb F (1-5%). It is also often referred to as heterocellular HPFH because the Hb F is unequally distributed over the red cells (4,5). Swiss HPFH has been found in many populations; its only distinguishing feature is its elevated level of Hb F and elevated number of F-cells. The condition is usually accidentally observed by noticing the elevated Hb F level in starch gel electrophoresis, IEF, or some chromatographic experiment. The quantity of Hb F is usually established by an alkali denaturation procedure (6). None of the above described promoter mutations (^Ggamma or ^Agamma) are considered to belong to this Swiss type of HPFH; the quantities of Hb F in these heterozygotes usually exceed 5% (Fig. 10). An exception is the -110 (A->C; ^Ggamma) mutation. Although not one single variation or abnormality has been discovered which explains the persistent elevation of Hb F (and F-cells) the following observations should be considered:

A.   The C->T mutation at position -158 (^Ggamma);
B.   The A->G mutation at position -161 (^Ggamma);
C.   Sex related influences; the FC locus on Xp 22.2;
D.   The determinant located on chromosome 6q;
E.   Gamma-globin gene rearrangements.

An analysis of cases with the Swiss type of HPFH usually involves the determination of hematological parameters, an exact value for the level of Hb and the number of F-cells, isolation of DNA or RNA, gene mapping analysis, DNA sequencing, and others. Identifying the gamma chain composition by reversed phase HPLC (7) after the Hb F has been isolated, is often complicated by the presence of a common ^Agamma variant, the ^Agamma^T (75 Ile->Thr, ^Agamma) (8,9). Furthermore, chromosomes with this ATA->ACA mutation at ^Agamma75 often carry a 4 bp deletion (-AGCA at positions -222 to -225, ^Agamma) which is associated with a low ^Agamma (and ^Agamma^T) expression in adults (10,11) and may well affect the total Hb F level.

A.   The C->T mutation at position -158 of the ^Ggamma chain

This mutation was first observed by Gilman and Huisman (12) and appears to be associated with elevated level of Hb F because the production of the ^Ggamma chain is increased 3-11 fold. The effect, however, is not consistent and many adult individuals with a homozygosity for T at -158 ^Ggamma have less than 1% Hb F (Table XVIII) (from Ref. 13). Earlier studies (14) involving the population of Macedonia led to a similar conclusion. In a few subjects this haplotype was defined and included 14 restriction sites (HincII 5' to epsilon; XmnI 5' to ^Ggamma; HindIII at ^Ggamma and ^Agamma; PvuI 5' to psibeta; HincII at psibeta and 3' to it; TaqI 5' to delta; HinfI 5' to beta; HgiAI and AvaII at beta; HpaI, HindIII, and BamHI 3' to beta). The observed haplotype [- + + - + + + + + + + + + +] is identical to that seen in patients with sickle cell (SS) anemia, described by Labie et al (15) and Nagel et al (16) as the Senegal type. In SS patients this haplotype is associated with relatively high levels of Hb F and high ^Ggamma values (15,16). Furthermore, the same haplotype is observed in beta-thal major patients who have a milder disease because of an increased production of Hb F. However, many adult subjects with this haplotype have normal levels of Hb F but with an increase in ^Ggamma level which suggests that other factors may accentuate the Hb F production.

• Table XVIII

B.   The A->G mutation at position -161 of the ^Ggamma chain

This mutation is probably rare and has thus far been observed in three Black families (13,17). Homozygotes may have a Hb F level of up to 4.0% (Table XIX from Ref. 13).

• Table XIX

There are definite indications that the increase in Hb F results from an increased activity of the ^Ggamma-globin gene (increased ^Ggamma-mRNA). The mechanism by which this mutation (and the C->T mutation at -158, ^Ggamma) causes the increase in gamma-mRNA and gamma chain production is not known but it could be that both may cause a change in the binding of a regulatory protein (18).

C.   Sex related differences; The FC locus on Xp 22.2

(19,20). There is evidence that the levels of Hb F and the number of F-cells are somewhat higher in normal females than in males. Hb F in females: 0.6±0.4 (n=87), in males: 0.4±0.3 (n=92); and F-cells in females: 3.8±3.0 (n=171), in males 2.7±2.3 (n=121) (20). Extensive linkage analysis using polymorphic restriction sites on the X chromosome mainly in SS families from Jamaica have localized the "F-cell production" locus on Xp 22.2. In male SS patients two phenotypes for F-reticulocytes have been observed: low <12%; high >12%, and three in females: low <12%; high >12 to 24%, and very high >24%.

D.   The determinant located on chromosome 6q

For years Weatherall, Wood, Clegg, Thein, and coworkers have been studying a few large families with heterocellular HPFH or Swiss HPFH (21-24). Their analyses included F-cell counts and levels of Hb F; the pedigrees shown in Ref. 23 are most impressive. Using various segregation analyses, a gene mapping strategy, and complex statistical procedures, it was concluded that, besides the beta-thal allele present in these families and the C->T mutation at position -158 (^Ggamma), a third major determinant for Hb F production can be found on chromosome 6q. This gene could code for a transacting factor which may act as an activator or repressor of transcription through binding to regulatory site(s) of either the beta or the gamma promoters, or even to the LCR 5' to the epsilon gene.

E.   Gamma-globin gene rearrangements

(Reviewed in Ref. 25). During the past several years it has become evident that certain variations in the commonly occurring 5'-epsilon-^Ggamma-^Agamma- psibeta-delta-beta-3' genic arrangement of the beta-globin gene cluster on the short arm of chromosome #11 are responsible for different ^Ggamma and ^Agamma ratios. The most frequently observed variations are:

1. Point mutations or gene conversions resulting in a -^Ggamma-^Ggamma- or -^Agamma-^Agamma- arrangement replacing the normally occurring -^Ggamma-^Agamma-. These anomalies can be readily detected by gene mapping using the enzyme PstI. Restriction sites within and around the two normal gamma-globin genes result in the presence of four fragments (4.9; 4.1; 2.7; 0.8 kb). When -^Ggamma-^Agamma- is replaced by -^Ggamma-^Ggamma- the PstI site within the third exon of the ^Agamma-globin gene is lost , resulting in the occurrence of only three fragments (4.9; 4.1; 3.4 kb). PstI restriction sites are present in both ^Agamma-globin genes of the -^Agamma-^Agamma- arrangement which results in the elimination of the 4.9 kb fragment (-> 4.1 kb and 0.8 kb). Detection of the various fragments requires both gammaIVS-II and inter-gamma probes.

2. The presence of multiple gamma-globin genes. Chromosomes with three, four, and even five gamma-globin genes have been discovered; invariably, the level of ^Ggamma is increased which has led to the assumption that the hybrid gene is an -^AGgamma- gene, i.e. the counterpart of the -^GAgamma- gene found in the gamma-thalassemic condition.

The changes in the ^Ggamma and ^Agamma ratios in these and related conditions (mainly gamma-thal) can best be determined at birth. The ^Ggamma values for normal babies falls between 60 and 70%, and only a limited number of babies have values higher than 70% or lower than 50%. Data for a large number of newborn babies with low ^Ggamma values (53 babies) or with high ^Ggamma values (158 babies) are shown in Fig. 12. Twenty-two of the "low ^Ggamma" babies were identified through gene mapping analyses as having a gamma-thal (listed as -A/GA- that is a simplified terminology for -^GAgamma-/-^Ggamma·^Agamma-). As many as 88 of the 158 newborn babies with high ^Ggamma values were studied in a similar fashion. Twenty-one had the GG/GA (short for -^Ggamma·^Ggamma-/ -^Ggamma·^Agamma-) arrangement, 64 had a GGA/GA triplication (short for -^Ggamma·^Ggamma·^Agamma-/-^Ggamma·^Agamma-), while one each of the remaining three newborns had a homozygosity for the gamma-globin gene triplication (GGA/GGA), a heterozygosity for a gamma-globin gene quadruplication (GGGA/GA) or gamma-globin gene quintuplication (GGGGA/GA). Babies with a gamma-globin gene triplication have slightly lower ^Ggamma values than babies with the -^Ggamma·^Ggamma-/-^Ggamma·^Agamma- (GG/GA) arrangements.

The first of these anomalies observed in adults was the -^Ggamma-^Ggamma-globin gene arrangement (26) and was later found in several Black families (27,28), including in a person with sickle cell anemia (29). The condition is also referred to as the Atlanta type of nondeletional HPFH. Adult heterozygotes have 1.3-9.8% Hb F with ^Ggamma values of 94.6-98.0%, normal levels of Hb A₂ (2.1-2.9%), and normal hematological values (data are from Ref. 27). Gene mapping and sequence analyses (30) have shown that the C->T mutation at position -158 (^Ggamma) occurs at both genes, while no other mutations have been observed. mRNA data have implied that the C->T mutation at -158 (^Ggamma) at both ^Ggamma genes is the major factor responsible for the elevated level of Hb F; it apparently exerts its effect on the transcriptional rate of the gene with which it is associated.

Another anomaly is the gamma-globin gene triplication (^Ggamma·^Ggamma·^Agamma) which in the adult results in elevated Hb F levels (with mainly ^Ggamma chains) provided the C->T mutation at -158 (^Ggamma) is present at both ^Ggamma genes (+^Ggamma·+^Ggamma·^Agamma). Data from Ref. 31 show Hb F levels varying from 3.4-6.4% in 11 such heterozygotes. Other normal adult individuals with a similar gamma gene arrangement but with C at -158 (^Ggamma) usually have lower levels of Hb F and lower ^Ggamma values (Table XX).

FIG. 12. The relative quantities of the ^Ggamma chain in newborn babies with ^Ggamma values <50% and in babies with ^Ggamma values >72%. The types of rearrangements, listed in this figure, are discussed in the text (from Ref. 25).

• Table XX

Studies of adults with gamma-globin gene quadruplication or quintuplication are limited. Members of a Turkish family with a chromosome carrying a specific beta-thal mutation (frameshift at codon 8, -AA) and four gamma-globin genes had mildly elevated levels of Hb F (0.7, 0.9, 1.1, 2.5, 3.1, 3.7, 3.8%) with mainly ^Ggamma (85-95%) and elevated levels of Hb A₂ (4.1-5.1%) (33).

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