Haemochromatosis: genetic and functional studies
Jason Partridge
Royal Free and University College Medical School University College London University of London
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Genetic haemochromatosis (GH) is an autosomal recessive condition common in Northern Europeans. It causes excess absorption of iron, resulting in tissue damage. Two approaches were used to study GH:
Positional cloning: R1 derived artificial chromosomes (FACs), containing large inserts of human genomic DMA, were isolated from around D6S1260, at that time the observed peak of linkage disequilibrium with GH. These clones were used to screen a human small intestine cDNA library. Four of the cDNAs isolated from this library contained C2H2 zinc finger motifs, comprising a single locus. The cDNA and genomic sequence and expression pattern of this locus were determined. The locus has characteristics similar to that of an expressed processed pseudogene, although it retains an open reading frame of 1.2kb. No evidence for a parent locus at another chromosomal site was detected using sequence database screening, somatic cell hybrid analysis and fluorescent in situ hybridisation. The sequence conservation displayed by this zinc finger pseudogene makes it an excellent tool for the identification of zinc finger genes in model organisms.
Assessment of a cellular phenotype: a cellular phenotype for GH would allow functional cloning of the gene, as well as investigation of GH in vitro. The enzyme ferric reductase was previously shown to have increased activity in duodenal biopsies from GH patients compared to controls. This increase paralleled increased uptake of ®^Fe in GH.
GH patients and controls for ferric reductase studies were characterised by investigation of both haplotype and HFE mutations. No significant difference in lymphocyte, monocyte or macrophage ferric reductase activity was observed when GH and control preparations were compared. However, a significant (p<0.05) ten-fold increase in ferric reductase activity accompanied the differentiation of monocytes to macrophages. This increase most likely reflects the co-ordinate upregulation of proteins of iron metabolism during the differentiation of monocyte to macrophage. Contents
Chapter 1. General Introduction: Index 12
1.1. THE HISTORY OF GENETIC HAEMOCHROMATOSIS 13 1.2. CLINICAL FEATURES 14 1.2.1. Diagnosis 15 1.2.1.1. Liver biopsy 16 1.2.1.3. CT and MRI scanning 18 1.2.2. Symptoms 18 1.2.2.1. Cirrhosis 18 1.2.2.2. Skin abnormalities 19 1.2.2.3. Arthropathy 19 1.2.2.4. Diabetes Mellitus 20 1.2.2.5. Other Endocrine Abnormalities 21 1.2.2.6. Cardiac Abnormalities 21 1.2.3. Treatment 22 1.3. GENETICS OF HAEMOCHROMATOSIS 23 1.3.1. Mode of inheritance 23 1.3.2. Human Leukocyte Antigen (HLA) alleles 23 1.3.3. Linkage disequilibrium with telomeric markers 24 1.3.4. Recombinant chromosomes 26 1.3.5. Chromosome rearrangements 26 1.3.6. Candidate Genes 27 1.4. POSITIONAL CLONING OF THE HFE GENE REGION 30 1.5. ISOLATION OF THE HFE GENE 30 1.5.1. The HFE protein and possible function 32 1.5.2. Genetic heterogeneity of haemochromatosis 37 1.6. MOLECULAR ADVANCES IN THE FIELD OF IRON METABOLISM 40 1.6.1. Ferric reductase 40 1.6.2. Divalent Metal Transporter 1 (DMT1 / DCT1 / Nramp2) 40 1.6.4. Hephaestin 44 1.6.5. Hepcidin 44 1.7 PREVIEW OF THESIS 45
Chapter 2. Analysis of GH and Control genotypes: Index. 49
2.1. AIMS OF THE STUDY 50 2.2. INTRODUCTION 51 2.2.1. Polymorphic Markers 51 2.2.1.1. Protein polymorphisms 51 2.2.1.2. Restriction fragment length polymorphisms 51 2.2.1.3. Polymorphism information content 52 2.2.1.4. Minisatellite repeats (VNTRs) 53 2.2.1.5. Microsatellites 54 2.3. METHODS 56 2.3.1. Multiplex fluorescent FOR 56 2.3.1.1. Microsatallite analysis 57 2.3.1.2. Sample preparation 57 2.3.1.3. Setup of the 310 ABI prism autosampler 57 2.3.1.4. Run conditions 58 2.3.1.5. Computer analysis of data 59 2.3.2. HFE mutation analysis 59 2.4. RESULTS 59 2.5. DISCUSSION 63 2.5.1. General problems of microsatellite analysis 63 2.5.2. GH patient microsatellite data versus controls 64
Chapter 3. Isolation and characterisation of genes around D6S1260: index. 67
3.1. INTRODUCTION 68 3.2. METHODS 69 3.2.1 YAC and PAC clones 69 3.2.2. Plating the cDNA library 71 3.2.2.1. The Clontech human small intestine cDNA library HL1133a 71 3.2.2.2. Titering the cDNA library 72 3.2.2.3. Plating a IX coverage of the library 73 3.2.2.4. Preparation of duplicate library filters for screening 73 3.2.3. Screening cDNA library 74 3.2.3.1. PAC insert probe preparation 74 3.2.3 2. Pre-hybridisation of filters with placental DNA competition 75 3.2.3.3. Competition of the radiolabelled probe 76 3.2.3.4. Washing of cDNA filters 77 3.2.3.5. Analysis of results from the 715 / 672 PAC insert probe 77 3.2.3.6. Secondary Screen 78 3.2.3.7. Tertiary screen 78 3.2.4. Subcloning the cDNA clones 79 3.2.4.1. PCR amplification of positive plaques 79 3.2.4.2. EcoR I subclones of cDNA PCR products 79 3.2.4.3. Cloning of PCR products using CloneAmp pAMPI vector 80 3.2.4.4. Amplification of cDNAs for pAMPI cloning 80 3.2.4.5. Ligation of cDNAs into pAMP 1 vector 81 3.2.5. Sequencing of the cDNAs 82 3.2.6. Computer analysis of cDNA sequences 83 3.2.7. Hybridisation analysis of cDNA inserts 84 3.3. RESULTS 84 3.4. DISCUSSION 89 Chapter 4. Characterisation of ZNF204: index. 91
4.1. INTRODUCTION 93 4.2. COMPLETE oDNA SEQUENCE 98 4.2.1. Methods 98 4.2.2. Results 98 4.2.3. Conclusion from sequence investigation of ZNF204. 108 4.3. ORGANISATION OF THE GENOMIC LOCUS 109 4.3.1. Introduction 109 4.3.2. Methods 109 4.3.3. Results and Discussion 110 4.4. NORTHERN BLOT ANALYSIS 112 4.4.1. Introduction 112 4.4.2. Methods 112 4.4.3. Results and Discussion 116 4.5. 5' RAPID AMPLIFICATION OF CDNA ENDS (5’ RACE) 118 4.5.1. Introduction 118 4.5.2. Methods 118 4.5.3 Results 121 4.6. SEARCHING FOR ZNF204 HOMOLOGUES 122 4.6.1. Somatic cell hybrid panel analysis 122 4.6.2 Methods 122 4.6.3. Results and Discussion 123 4.7. HYBRIDISATION SCREENING OF A GENOMIC PAC LIBRARY 125 4.8. FLUORESCENT IN SITU HYBRIDISATION (FISH) 126 4.8.1. Introduction 126 4.8.2. Methods 127 4.8.2.1. cDNA probe 127 4.8.2.2. PAC probes used for FISH 127 4.8.2.3. Method used for FISH 127 4.8.3. Results and Discussion 130 4.9. ZOO BLOT ANALYSIS OF ZNF204 135 4.9.1. Methods 135 4.9.2. Results and Discussion 135 4.10. IN VITRO TRANSCRIPTION AND TRANSLATION OF ZNF204 139 4.10.1. Methods 140 4.10.1.1. TNT reaction conditions 140 4.10.1.2. Denaturing gel analysis of translation products 141 4.10.2. Results and Discussion 141 4.11. ZNF 204 LOCUS DISCUSSION 144 Chapter 5. Ferric reductase functional studies; index. 149
5.1. INTRODUCTION 150 5.2. PATIENTS AND METHODS 153 5.2.1. Patients 153 5.2.2. Mutation analysis 153 5.2.3. Isolation of peripheral blood monocyctes and differentiation to macrophages 154 5.2.4. Ferric reductase assay 158 5.3. RESULTS 159 5.4. DISCUSSION 164
Chapter 6. Conclusions and Perspectives. 166
References. 180
Appendix 1. 220
Appendix 2. 222 Index of Tables
Table 1.1. Chromosomal location of known genes of iron metabolism prior to the isolation of HFE 28 Table 1.2. Genetic Heterogeneity of Haemochromatosis. 39 Table. 2.1. Patient founder haplotype microsatellite and HFE mutation analysis results 61 Table. 3.1. End sequence and hybridisation analysis of eleven cDNA clones isolated by hybridisation of PACs 715 and 672 to a small intestinal cDNA library. 87 Table 3.2. Low resolution YAC / PAC map of the haemochromatosis gene region showing the position of ZNF204. 88 Table. 4.1. Multiple mutations in the zinc finger and linker motifs of ZNF204. 104 Table. 4.2. Predicted binding sequence of ZNF204 of the 10 putative functional 106 Table 5.1. Human ferric reductase activity has the characteristics of a membrane bound enzyme. 162 Table 5.2. Ferric reductase activities. 163 Index of Figures
Figure 1.1. A predicted model of the HFE protein based on sequence homology with classical MHC class I proteins. 34 Figure 1.2. Diagram of iron transport from intestine (enterocyte) to plasma to liver hepatocyte 42 Figure 2.1. GeneScan trace showing relative fluorescent intensity plotted against approximate size (bp). 60 Figure. 4.1. Possible structures of the two histidine two cysteine zinc finger. 95 Figure 4.2. Mapping, isolation and characterization of ZNF204. 92 Figure 4.3. ZNF204 sequence, as deposited in GenBank (Accession No. AF033199) 101 Figure 4.4. Northern analysis of the ZNF204 locus expression on a polyA(+) multi-tissue northern blot. 114 Figure 4.5. Diagrammatic overview of 5’ RACE procedure. 119 Figure 4.6. Fluorescent in situ hybridisation of PAC 715 on normal metaphase chromosomes, showing hybridisation to chromosome 6p21.3. 132 Figure 4.7. Fluorescent in situ hybridisation of cDNA 3AS2 on normal metaphase chromosomes, showing hybridization to chromosome 6p21.3. 133 Figure 4.8. Fluorescent in situ hybridisation of PAC 832 on normal metaphase chromosomes, showing hybridisation to chromosome 14. 134 Figure 4.9. ZNF204 cross hybridizes with multiple zinc finger loci. 138 Figure 4.10. Results of the TNT analysis of ZNF204. 143 Figure 6.1 A model showing the proposed role of Hepcidin in intestinal iron absorption. 175 Acknowledgments
I would like to thank the following people for their kind help and support:
Firstly I would like give my deepest thanks to my supervisor: Dr. Ann Walker for her unfailing support throughout all aspects of this thesis and wise advice through thick and thin.
I would also like to thank my secondary supervisors; Firstly Dr. James Dooley for his support guidance and copious quantities of blood, and secondly, Dr. J Paul Simons for his kind help and reviews of my progress.
Special thanks to Daniel Wallace for help and support throughout my time in the lab and to all the members of the UK Haemochromatosis Consortium for their wise words of advice.
I would especially like to thanks the members of my family for putting up with me writing this thesis; my mother and father Brenda and John, my sister Gemma and my wonderful wife Melanie who makes it all worthwhile.
This work was supported by the Medical Research Council. 10
Dedication
I wish to dedicate my thesis to my daughter Emily Rebecca Partridge who decided to be born 5 and a half weeks early on the same day I was due to take my PhD viva (9^^ of July 2003), Both me and mummy love you very much. Chapter 1 General Introduction Page 11
Chapter 1
General Introduction Chapter 1 General Introduction Page 12
Chapter 1. General introduction: index.
1.1. THE HISTORY OF GENETIC HAEMOCHROMATOSIS...... 13 1.2. CLINICAL FEATURES...... 14 1.2.1. Diagnosis ...... 15 1.2.1.1. Liver biopsy ...... 16 1.2.1.3. CT and MRI scanning ...... 18 1.2.2. Symptoms ...... 18 1.2.2.1. Cirrhosis ...... 18 1.2.2.2. Skin abnormalities ...... 19 1.2.2.3. Arthropathy ...... 19 1.2.2.4. Diabetes Mellitus ...... 20 1.2.2.5. Other Endocrine Abnormalities ...... 21 1.2.2.6. Cardiac Abnormalities ...... 21 1.2.3. Treatment ...... 22 1.3. GENETICS OF HAEMOCHROMATOSIS...... 23 1.3.1. Mode of inheritance ...... 23 1.3.2. Human Leukocyte Antigen (HLA) alleles ...... 23 1.3.3. Linkage disequilibrium with telomeric markers ...... 24 1.3.4. Recombinant chromosomes ...... 26 1.3.5. Chromosome rearrangements ...... 26 1.3.6. Candidate Genes ...... 27 1.4. POSITIONAL CLONING OF THEHFE GENE REGION...... 30 1.5. ISOLATION OF THE HFE GENE...... 30 1.5.1. The HFE protein and possible function ...... 32 1.5.2. Genetic heterogeneity of haemochromatosis ...... 37 1.6. MOLECULAR ADVANCES IN THE FIELD OF IRON METABOLISM ...... 40 1.6.1. Ferric reductase ...... 40 1.6.2. Divalent Metal Transporter 1 (DMT1 / DCT1 / Nramp2) ...... 40 1.6.4. Hephaestin ...... 44 1.6.5. Hepcidin ...... 44 1.7 PREVIEW OF THESIS ...... 45 Chapter 1 General Introduction Page 13
Chapter 1. General introduction:
1.1. The history of genetic haemochromatosis
Genetic haemochromatosis (GH) was first described by Trousseau in
1865 and later Troisier in 1871 in patients with the classical symptoms of diabetes mellitus, cirrhosis and bronzing of the skin. Von Recklinghausen
(1889) was the first to attribute these features to a specific disorder.
Sheldon (1935) was the first to propose that GH was caused by an inborn error of metabolism that led to increased iron accumulation. GH was one of the first genetic disorders to be mapped to a specific chromosome via linkage. This seminal work by Simon and co-workers in 1975 showed an association between GH and the human leukocyte antigen (HLA)-AS.
Linkage was shown with HLA-A, which was later mapped as part of the major histocompatibility complex (MHC) to chromosome 6p21.3 (Simon et a!., 1987; Franke and Pellerino, 1977). The approximate chromosomal position of GH was known for 19 years before the HFE gene was finally cloned. Chapter 1 General Introduction Page 14
1.2. Clinical features
The clinical presentation of GH is dependent on both genetic and
environmental factors. The major genetic component results in increased
iron absorption but this will be affected by environmental factors including the dietary iron intake and blood loss. Patients present between the third and fifth decade of life with a variety of symptoms including arthralgia,
lethargy, upper abdominal pain, abnormal liver function tests, loss of libido or the onset of diabetes mellitus. Late features are skin pigmentation, liver cirrhosis, endocrine abnormalities and cardiac disease. There is often a delay between the first presenting symptoms and diagnosis of GH of approximately 5 years (Adams et al., 1991).
GH is 10 times more common in male than female patients because the onset is delayed in women due to menstrual blood loss and pregnancy
(Finch et a!., 1966). Precirrhotic GH patients have a normal life expectancy and cirrhotic patients may only have a normal life expectancy if iron is removed (Adams et a!., 1991). Those with cirrhosis and diabetes mellitus have reduced survival, due to the development of complications including hepatocellular carcinoma (Niederau et a!., 1985). Since cirrhosis is prevented by the removal of iron by venesection there is a need for early diagnosis. Chapter 1 General Introduction Page 15
1.2.1. Diagnosis
The initial diagnosis of GH is usually based on the phenotypic expression of the disease, assessed by serum iron (normal range 11-36 pmol/l), total iron binding capacity (normal range 53-85 pmol/l), serum ferritin concentration and liver biopsy (normal ranges dependent on age/sex).
From the serum iron concentration and total iron binding capacity (TIBC) the transferrin saturation value can be calculated (serum iron/TIBC). The normal serum iron concentration is between 11 and 36 pmol/l and rises as body iron stores increase.
Patients with iron overload and increased serum iron concentrations have a reduced serum transferrin level, due to a feedback mechanism reducing transferrin synthesis.
Serum ferritin levels generally reflect body iron stores and are increased in GH. Ferritin is an iron storage protein, which is present in all tissues.
The normal serum ferritin concentration ranges for the Royal Free
Hospital laboratory are 10-150 pg/l in females and 20-200 pg/l in males.
Levels are increased in patients with GH; with advanced disease levels can be increased to approximately 10 times the normal level. Chapter 1 General Introduction Page 16
1.2.1.1. Liver biopsy
Liver biopsy is important in the definite diagnosis of GH because it allows
both histological examination and estimation of liver iron. As iron
accumulation occurs in GH it is deposited as both ferritin and
haemosiderin in periportal hepatocytes and in the pericanalicular cells within lysosomes. Later in the disease deposition is found in all areas of the hepatic acinus as well as the bile duct epithelium and connective tissue. Untreated this iron causes perilobular fibrosis and in advanced disease broad fibrous septa and cirrhosis. Areas of preserved
parenchyma surrounded by fibrotic septa lead to a “holly leaf" pattern
(Powell et al., 1981).
The pattern of iron distribution in GH is different to that found in secondary iron overload, for example following transfusion for haemolytic anemia. Kupffer cells (differentiated liver macrophages) do not become iron loaded until late in classical GH. This is in contrast to the iron loaded
Kupffer cells observed in transfusion related siderosis (Yam et al., 1968).
The liver tissue obtained from a needle biopsy is split into two pieces:
(a) One section is fixed in formalin for histological examination to determine the degree of fibrosis and cirrhosis. Haemosiderin iron is Chapter 1 General Introduction Page 17
estimated histologically using Peris (Prussian) stain or Tirmann-
Schmelzer stain.
(b) The remainder is washed to remove any contaminating blood and
then placed in a drying oven to give the dry weight (approximately 30% of
the hepatic wet weight). The iron content is then determined by either
spectrophotometric methods or atomic absorption spectrophotometry.
The iron content is used to determine the hepatic iron index:
Liver iron concentration of iron (pmol/g dry wt)
______= Hepatic iron index
Age of patient in years
A hepatic iron index of above 2.0 is indicative of GH. The index has been valuable in differentiating between GH homozygotes (generally > 2.0) and
heterozygotes and patients with abnormal iron indices caused by other factors such as alcoholic liver damage and hepatitis C (generally < 2.0)
(Basset et al., 1986, Summers et a i, 1990). Chapter 1 General Introduction Page 18
1.2.1.3. CT and MRI scanning
The computed axial tomography (CT) is a non-invasive method, which
may show hepatic iron overload. However its main drawback, apart from
cost, is low sensitivity for mild and moderate iron deposition. Magnetic
resonance imaging (MRI) can detect moderate iron deposition but is not
sensitive enough for detection of early iron deposition. Liver iron
Measurements by Biosusceptometry (SQID) is a far more sensitive
method however, the only two available SQID machines are in North
America. For this reason, liver biopsy with iron measurement may be
used to assess liver iron in the diagnosis of GH.
1.2.2. Symptoms
1.2.2.1. Cirrhosis
Approximately 10 % of GH patients present with cirrhosis (Stremmel et
al., 1995, Adams et al., 1991). Hepatocellular carcinoma is a major
complication and cause of mortality with prevalence more than 200 times
that of the general population (Powell et al., 1994). In cirrhotic patients
hepatocellular carcinoma is still a risk despite the removal of the iron.
Since the advent of genetic testing, the goal is early identification and treatment of haemochromatosis before establishment of cirrhosis. Chapter 1 General Introduction Page 19
1.2.2.2. Skin abnormalities
Untreated GH patients with late disease can have excessive skin
pigmentation. This is caused by increased melanin deposition in the skin.
GH has been called “bronze diabetes” but “bronze” is misleading as most
GH patients display a slate grey discolouration of the skin. The abnormal
melanin deposits are increased within scars, genitalia, flexation creases
and on rare occasions the oral mucosa (Ferrand et al., 1977). Another
cutaneous feature of late GH is lack of facial and pubic hair. This may be
secondary to endocrine abnormalities.
1.2.2.3. Arthropathy
Arthropathy was first described in patients with GH in 1964 by
Schumacher and co-workers. It is now recognized that 25 to 50% of all
GH patients suffer from joint pain with or without signs of arthritis
(Niederau et al., 1985). The prevalence of arthropathy is not related to the degree or duration of the iron overload. It can occur despite venesection and can even occur after iron stores have returned to a
normal level. Arthropathy is often present early in the disease process
preceding diabetes mellitus and cirrhosis (Adams 1991). It is not specific for GH and is observed also in secondary iron overload (Dymock et a!.,
1972). Chapter 1 General introduction Page 20
The most common form of arthropathy observed normally progresses
slowly, although degenerative arthritis is observed. The arthropathy
involves the joints of the hands, proximal interphalangeal joints and
metacarpophalangeal joints (Schumacher 1988). Early radiological
changes consist of subchondrial cyst formation and narrowing of joint
spaces. Knees and hips may also be affected. Chondrocalcinosis occurs
in these joints and is related to the deposition of calcium pyrophosphate,
which is thought to be a direct result of iron deposition. Iron inhibits
pyrophosphatase resulting in the accumulation of pyrophosphates.
1.2.2.4. Diabetes Mellitus
Diabetes mellitus develops in 30 to 60 % of patients with GH (Niederau et
al., 1985). An investigation of 163 GH patients showed 55 % had
diabetes mellitus with another 10 % having glucose intolerance (Niederau
et al., 1985). Insulin dependent diabetes mellitus is associated with late
disease. Complications of diabetes mellitus such as retinopathy,
peripheral neuropathy and nephropathy may also occur. Diabetes is thought to be due to damage to the islet cells (beta cells) of the pancreas
by excess deposition of iron (Stremmel et al., 1988). Chapter 1 General Introduction Page 21
1.2.2.5. Other Endocrine Abnormalities
Loss of libido and testicular atrophy are frequently observed in advanced
GH in men, and may be the first sign of the disorder. The cause of
hypogonadism is pituitary dysfunction due to the selective deposition of
iron within the gonadotrophic cells of the pituitary gland. There is reduced
secretion of lutenizing hormone and follicle stimulating hormone
(Stremmel et ai., 1988), with a subsequent reduction of plasma
testosterone levels (Kley et al., 1985). In young females amenorrhoea
may result (Adams et al., 1990).
1.2.2.6. Cardiac Abnormalities
GH patients may present rarely with cardiomyopathy and congestive
cardiac failure. Cardiomyopathy is normally observed in long-standing
GH patients diagnosed late in the disease but may be the presenting feature in younger adults (Dabestani et al., 1988). The risk of death due to cardiomyopathy is approximately 300 times higher for GH patients
compared with the general population. Although 20 - 35 % of GH patients
have arrhythmias, such as supraventriclular ectopic beats, tachyarrhythmias and atrial flutter and fibrillation, these are normally not
life threatening (Adams etal., 1991). Chapter 1 General Introduction Page 22
1.2.3. Treatment
GH is treated by venesection of 1 unit of blood (approximately 450ml) weekly until the transferrin saturation and ferritin concentration are
normal (Crawford et al., 1991, Bourel and Lenoir 1965). Maintenance
venesection is then continued at 3 to 6 monthly intervals. Each unit of
blood removed represents approximately 250mg of iron. The range of
iron overload in GH patients is 5 to 40g (Bomford and Williams 1976)
equivalent to 20 to 160 venesections. For comparison the normal body
iron content is approximately 5g. Venesection increases the rate of
erythropoiesis thus mobilizing stored iron from parenchymal tissues.
In the rare patient with cardiomyopathy the removal of iron by venesection alone may be too slow, and the iron chelator desferrioxamine is used in combination with venesection.
Desferrioxamine however causes side effects, so treatment must be kept to a minimum and used only in extreme cases. Chapter 1 General Introduction Page 23
1.3. Genetics of haemochromatosis
1.3.1. Mode of inheritance
In 1974 Saddi and co-workers proposed an autosomal recessive mode of
inheritance for GH, which is now accepted. Before this it was thought that
the inheritance of GH was autosomal dominant. The autosomal dominant
mode of inheritance was proposed due the high proportion of children
with an affected parent having GH. However, this was an effect of the
high gene frequency allowing for a high proportion of homozygous /
heterozygous marriages.
1.3.2. Human Leukocyte Antigen (HLA) aiieles
In 1975 Simon and coworkers reported an association of GH with certain
alleles of HLA antigens, in particular the HLA-A3 and B-14 antigens.
These preliminary results were published in French literature in early
1975 (Simon et al., 1975). Full details were later published in English in
1976 (Simon et al., 1976). H LA-A3 and HLA-B14 were found in significantly greater frequencies in GH patients than healthy blood donor controls. HLA-A3 was observed in 78% of GH patients versus 27% of controls, and HLA-B14 in 26% of GH patients compared with only 3% of controls (Simon et al., 1976). HLA-B7 was also shown to be significantly Chapter 1 General Introduction Page 24
increased in GH patients in a later study (Simon et al., 1981). Family
studies also demonstrated linkage of GH to HLA-A (Simon, 1987). The
findings of Simon were a landmark in the history of haemochromatosis
research, and were the first evidence to confirm Sheldon’s hypothesis,
made in 1935, that GH was dependent on genetic factors.
Research in haemochromatosis prior to this date hung on the question whether haemochromatosis was acquired or inherited, with some
authors arguing strongly for GH being an acquired disorder.
Simon’s observations did not rule out environmental factors influencing the extent of iron overload, but did show that the primary defective locus was linked to the major histocompatability complex (MHC), HLA antigen
A, which was subsequently mapped to chromosome 6p21.3 (Franke and
Pellegrino eta!., 1977)
1.3.3. Linkage disequilibrium with telomeric markers
The GH candidate region initially centered on the HLA region but was extended greatly with the investigation of new microsatellite markers.
One crucial CA repeat marker D6S105 was isolated and mapped approximately 2cM telomeric of HLA-A. (Stone et ai., 1994; Raha-
Chowdury et ai., 1995). Allele 8 of this marker has a stronger association with GH than the HLA-A3 allele (Jaswinska et ai., 1993): D6S105 allele 8 Chapter 1 General introduction Page 25
was observed in 93% of GH patients compared to 21% of controls.
Additional CA repeat markers around D6S105 and towards the telomeric marker D6S299 were also investigated. This study revealed that 70% of
GH chromosomes contain the haplotype: D6S306 allele 5, D6S105 allele
8, D6S464 allele 9 and D6S1260 allele 4. This common GH haplotype was only found in 6% of non-GH control chromosomes (Raha-Chowdhury et al., 1995). The linkage of GH to HLA-A and the presence of a common haplotype in the majority of patients suggested that GH has spread by multiplication of a founder mutation, and that the majority of GH chromosomes are derived from this founder individual.
The gene position in the founder haplotype could therefore be mapped by linkage disequilibrium. Linkage disequlibrium is defined as the nonrandom association of alleles at linked loci.
The lambda likelihood method of linkage disequlibrium analysis is powerful as it analyses all recombinations since the original mutation and not just recombination occurring in a single family (Hasbacka et a!.,
1994). Using this method of linkage disequlibrium analysis in the GH critical region Raha-Chowdhury et al. (1995) showed that the marker
D6S1260 was at the peak of linkage disequilibrium with GH in their data set. The peak indicated the most probable region for the gene to be located at this time. The GH gene was thought most likely to lie in the region between D6S105 and D6S1260 (equates to approximately 900 Chapter 1 General Introduction Page 26
kb). This mapping was reproduced by an independent American study
(Seese et al., 1996) using a similar set of markers.
1.3.4. Recombinant chromosomes
Several recombinations involving GH and HLA-A and HLA-B have been proposed but subsequently withdrawn (Edwards et a!., 1986, David et a!.,
1986, Panajotopoulos et a!., 1989; Powell et a/., 1990). However an informative recombination in a GH patient was described by Calandro and co-workers (1995). This positioned the HFE gene telomeric to HLA-
F. This recombinant was therefore consistent with the linkage disequlibrium data (Calandro eta!., 1995).
1.3.5. Chromosome rearrangements
An initial pulsed field gel electrophoresis (PFGE) study by Lord and co workers (1990) showed no GH associated changes. In this study however the most telomeric marker used was HLA-A. More recently, a pericentromeric inversion (6p21.1-6p23) was demonstrated to segregate with disease in a family. However, the breakpoints appeared to flank the predicted site of the HFE gene (Venditti et a!., 1994). Recent studies using PFGE in the region around D6S1260 however showed no rearrangement (Wallace et a!., 1998). The lack of any observable PFGE rearrangements in GH suggested that the common founder mutation Chapter 1 General Introduction Page 27
responsible for GH would not involve large chromosomal rearrangement.
This implied that the GH defect was either a point mutation or a micro deletion or rearrangement, beyond the resolving power of techniques such as PFGE.
1.3.6. Candidate Genes
Known proteins of iron metabolism were excluded as candidates on the basis of their chromosomal location (see Table 1.1.). None of the functional genes of iron metabolism mapped to 6p21.3. Chapter 1 General Introduction Page 28
Table 1.1. Chromosomal location of known genes of iron metabolism prior to the isolation of HFE.
Name Gene Symbol Map Location
Ferritin (H) FTH1 11q3
FTHL 1-4 1p31-p22; 1q32.2-q42; 2q32-q33; 3q21-q23
FTHL7.8, 13q12;Xq26-q28
10-16 5; 8; 9; 14; 17; 6p; 11q13
FTHP1 6p21.3-p12
Ferritin (L) FTL 19q13.3-13.4
FTLL1 20q12-qter
FTLL2 Xp22.3-p21.2
T ransferrin TF 3q21
TFP 3
Transferrin receptor TFRC 3q26.2-qter
Lactoferrin LTF 3q21
Iron requlatory binding protein IRP1 9
Aconitase (soluble) AC01 9q22-q32
Haptoglobin HP 16q22.1
Haemopexin HPX 11q15.5-15.4
Haem Oxygenase -1 HM0X1 22q12
Haem Oxygenase -2 HM0X2 16p13.3
Caeruloplasmin CP 3q21-24
Iron Response Protein 2 IRP2 15
* Adapted from Worwood 1999 Chapter 1 General Introduction Page 29
Pappas and co-workers (1995) described 10 expressed sequence tagged sites (ESTs) mapping around the HFE gene region (Pappas et al., 1995).
ESTs are sequences defined by a pair of primers derived from cDNA.
Pappas and co-workers mapped 10 ESTs to chromosome 6p21-6p23 using a panel of somatic cell hybrids containing different portions of chromosome 6. The fine mapping of these ESTs was not described. The
DNA library used to generate the ESTs however was fetal brain, which was not the most likely tissue for the expression of HFE, unless HFE is ubiquitously expressed.
A zinc finger protein described by Beutler and co-workers was proposed as a candidate for the HFE gene, lying 500kb centromeric of D6S105
(Beutler et a!., 1996). The gene was shown to be polymorphic in 3 out of
55 GH patients and 1 out of 44 control patients, however it was most highly expressed in ovary, testis and prostate, as well as the small intestine. Although zinc finger proteins, as transcriptional regulators, were considered potential candidates for the HFE gene, the expression pattern of the gene and its position made it very unlikely to be the gene causing
GH. Chapter 1 General Introduction Page 30
1.4. Positional cloning of the HFE gene region
Positional cloning is the isolation of a gene by chromosomal map position alone, without the use of antibodies to the protein or prior information on function, structure or sequence. For positional cloning, the candidate region must be refined down to a size that allows efficient use of gene isolation techniques. In GH no recombinations or chromosome rearrangements became apparent that would allow the gene position to be narrowly defined (see section 1.3.4.).
1.5. Isolation of the HFE gene
Mercator Genetics Incorporated used a combined positional cloning approach to isolate the HFE candidate gene (Feder et ai, 1996). They originally and incorrectly named the gene HLA-H (there was already an
HLA-H locus, OMIM 142925). They first generated a Yeast Artificial
Chromosome (YAC) contig stretching from HLA-A to D6S276 and used this to isolate smaller Bacterial Artificial Chromosome (BAC), PI Phage
Artificial Chromosome (PAC) and cosmid clones. Using these clones as a resource they generated 26 short repeat markers across the region, which they used for linkage disequlibrium studies (Feder et ai, 1996) The highest Pexœss obtained (D6S2241) was 0.81, suggesting that at least
81% of disease-bearing chromosomes carried a common mutation. They used this Pexœss data, with the deviation from Hardy-Weinberg equilibrium Chapter 1 General Introduction Page 31
along with ancestral haplotype analysis to narrow the critical region
containing the gene to a region of 600kb. Where family data was
unobtainable a cell line was created containing each individual chromosome 6, to enable haplotype analysis. The precise boundaries for the gene were identified by haplotype analysis of GH chromosomes not
bearing the full extended GH ancestral haplotype, to identify the positions of historic recombination events. This analysis defined the minimal HFE gene region of 250kb between the markers D6S2241 and D6S2238.
Exon trapping and cDNA selection were then used on clones spanning the critical region to identify candidate sequences. The entire genomic region of 250,000bp was also sequenced to ensure complete identification of all possible coding regions. The 250kb region, like the neighboring MHC region, was gene rich, yielding 15 genes: 12 histone genes, a Ro/SSA ribonucleotide protein (an auto antigen that is frequently recognised in patients with Systemic Lupus Erythematosus and Sjogren Syndrome), a sodium phosphate transporter and a HLA-A2 like sequence. To identify the most probable candidate, each gene was individually sequenced, including intron-exon boundaries, in two haemochromatosis patients homozygous for the founder haplotype and 2 control individuals. A single base mutation of a G to A at nucleotide 845 of the open reading frame was found to be homozygous in the HLA-A2 like gene in the patients but not present in the controls. This mutation predicted the substitution of a cysteine at amino acid 282 by tyrosine Chapter 1 General Introduction Page 32
(Cys282Tyr). Sequence homology with other HLA molecules predicted that this mutation would disrupt an intrastrand disulphide bond. This
mutation was detected in 85% of GH chromosomes and only 3% of
control chromosomes. A second variant H63D (His63Asp) was also detected (Feder et al., 1996)
1.5.1. The HFE protein and possible function
Based upon the sequence similarities, the product of the HFE gene was predicted to have similarities with atypical class I MHC proteins (Figure
1.1). HFE is a MHC class I like molecule that interacts with p 2 - microglobulin. HFE was predicted to have with three extracellular domains (ai, a 2 and as with the binding site for p 2-microglobin on as, a transmembrane helix and a short intracellular domain. HFE retains the structural features of MHC class I molecules such as the four cysteine residues that form disulphide bridges within the a 2 and as domains, but unlike classical antigen presenting HLA molecules only two of the normally four critically spaced tyrosine residues that are important in peptide binding are present (Madden et ai., 1992). This two tyrosine structure will destroy the peptide binding groove as is observed with nonclassical members of the MHC Class I family, such as the Fc receptor that do not bind peptides (Ravetch and Marguiles, 1994; Story et ai.,
1994). In the Fc receptor the peptide binding groove is effectively closed by a proline in the a2 domain; in HFE there is an equivalent proline at Chapter 1 General Introduction Page 33
codon 188 (Feder et al., 1996) It seemed unlikely that HFE is involved in presentation of peptides, but how it could be involved in regulation of iron metabolism was unclear. Chapter 1 General Introduction Page 34
Figure 1.1. A predicted model of the HFE protein based on sequence homology with classical MHC class I proteins.
H63D Mutation
a Heavy Chain
NH2| NH2
Extracellular COOH C282Y Mutation
Plasma membrane ,
Intracellular COOH
The model shows the three extracellular domains a^, «2 , as, the binding site for p 2-microglobin on as, the transmembrane helix and intracellular domain, as well as the sites of the C282Y and H63D mutations (adapted from Feder et al., 1996;
Bacon et a!., 1999). Chapter 1 General Introduction Page 35
The Cys282Tyr mutation disrupts a critical disulphide bridge in the a3 loop of the
HFE protein (Figure 1.0.) This affects the protein's ability to interact with p2- microglobulin, retarding its presentation on the surface of the cell and binding to the transferrin receptor protein (TfR). The His63Asp mutation does not affect the association of HFE with >52-microglobulin, and in vitro this form is presented on the cell surface (Waheed etal., 1997; Feder ef a/., 1997).
Northern blot analysis, showed a single major transcript present in most tissues of about 4kb. Highest levels were observed in small intestine, the site of iron absorption and the liver, the major site of iron deposition in haemochromatosis
(Feder et a/., 1996).
Immunohistochemical studies showed staining for the HFE protein in some epithelial cells in every segment of the alimentary canal. The most intense staining was seen in the deep crypts of the small intestine (Parkkila at a/., 1997).
The crypt cells do not absorb iron; this occurs as they migrate up the villus before they are sloughed off. This was an unexpected finding as it was assumed the staining would be more intense on the mature villus entrocytes, the site of iron absorption. Chapter 1 General Introduction Page 36
A major breakthrough was the demonstration of binding of HFE to the transferrin receptor (Waheed etal., 1999, Parkkila etal., 1997b and Feder et al., 1999).
Although the mechanism of action of HFE is not fully understood, it appears to modulate the classical transferrin receptor - mediated endocytosis pathway of iron entry into cells.
In the small intestine, the transferrin receptor is expressed on the basolateral surface of the enterocyte. It remains controversial, but it may be that the HFE - transferrin receptor complex in the basolaterial crypts may “sense” body iron stores, and determine the iron absorption of the mature enterocyte. Chapter 1 General Introduction Page 37
1.5.2. Genetic heterogeneity o f haemochromatosis
Since the discovery of the HFE gene, several other causes of haemochromatosis have been found (Table 1.2.).
Juvenile haemochromatosis is a rare autosomal recessive disorder causing accelerated iron overload presenting in the second and third decades of life and affects men and women equally. The gene has been mapped by linkage analysis to chromosome 1q, but is as yet undiscovered (Roetto et al. 1999)
Transferrin receptor 2-related haemochromatosis is an autosomal recessive disorder caused by mutations in the gene TFR2, which encodes a transferrin receptor isoform (Camaschella et al., 2000, Roetto et ai., 2002)
Ferroportin 1 related iron overload is an autosomal dominant disorder caused by mutation of the gene for the ferroportin iron transporter molecule (also known as iron-regulated transcript 1, IREG1; metal transporter protein 1, MTP1,
SLC11A3). It causes iron overload, though markedly in cells of the reticulo endothelial system (eg. Kupffer cells). It is difficult to manage clinically using venesection as this may cause anaemia (Njajou et a!., 2001; Montosi et al.,
2001; Devalia ef a/./2002; Wallace etal., 2002) Chapter 1 General Introduction Page 38
H-Ferritin related iron overload is an autosomal dominant iron overload that has
(so far) only been observed in a single Japanese family. It is caused by a mutation in 5' iron responsive element (IRE) in the gene that encodes the H- subunit of the iron storage molecule ferritin (Kato et a i, 2001 ). Table 1.2. Genetic Heterogeneity of Haemochromatosis.
Disease OMIM* Gene Locus Mode of Mutation(s) Treatment
(onset) (year identified) inheritance
HFE-related haemochromatosis 235300 HFE 6p31.3 Autosomal C282Y, H63D, Venesection
(adult) (1996) recessive S65C, IVS3+1G->T
Juvenile haemochromatosis 602390 Not yet cloned 1q21 Autosomal Chelation e.g. desferrioxamine
(Juvenile / early adult) recessive (Venesection - care)
Transferrin receptor 2-related 604720, TFR2** 7q22 Autosomal Y250X, M172K, Venesection haemochromatosis 604250 (2000) recessive 84-88lnsC (disease appears to be similar to HFE-
(adult) related haemochromatosis)
Ferroportin 1 - related Iron overload 604653, FPN1 2q32 Autosomal N144H, A77D, Not yet defined. Some patients become
(Adult) 606069 (also called IREG1 dominant V162del anaemic if venesected.
or SLC11A3 or
MTP1)
(2000)
H-ferrltIn IRE*** - related Iron 134770 H-ferritln 11q12-q13 Autosomal A49U Not yet defined overload (Adult) (mutation dominant
identified 2001)
* edited OMIM, Online Mendelian Inheritance In Man:httD://www.ncbi.nim.nih.Qov/Omim/ , ** TFR2, transferrin receptor 2, IRE, Iron responsive element
8 Chapter 1 General Introduction Page 40
1.6. Molecular advances in the field of iron metabolism
1.6.1. Ferric reductase
The ferric reductase gene was cloned by a subtractive cloning strategy designed to identify intestinal genes involved in iron absorption. The cDNA sequence predicted a plasma membrane di-haem protein, named duodenal cytochrome B
(DcytB). Immunohistochemical staining for the protein localised to the duodenal brush border membrane (McKie et a!., 2000). Ferric reductase reduces Felll to
Fell in preparation for its uptake by Divalent Metal Transporter 1 (DMT1) (Figure
1.2 .).
1.6.2. Divalent Metal Transporter 1 (DMT1 / DCT1 / Nramp2)
DMT1 was identified by cDNA expression cloning in Xenopus oocytes (Gunshin et a!., 1997). It is a plasma membrane glycoprotein with 12 membrane spanning domains. It has been demonstrated to be a metal ion transporter with a broad substrate range that includes Fell, Znll, Coll, Gdll, Cull, Nill and Pbll. DMT1 is found on luminal side of the upper gastrointestinal tract and on the plasma membrane of most cells. It is also present in endosomal vesicles of macrophages and other cells, where it functions to transport iron from within the endosome into the cytoplasm (Flemming et a/., 1998; Gruenheid et a/., 1999).
On the small intestinal brush border, it is the site of iron uptake by the cell after Chapter 1 General Introduction Page 41
its reduction by ferric reductase (Figure 1.2.). Expression of DMT1 (possibly ferroportin 1 also) has been reported to be increased (or inappropriately high in relation to body iron stores) in haemochromatosis patients, but as yet the mode of this regulation is not yet fully understood (Zoller et al., 2001 ) Chapter 1 General Introduction Page 42
Figure 1.2. Diagram of iron transport from intestine (enterocyte) to plasma to liver hepatocyte (Fleming and Sly, 2001).
enterocyte
Fp1/lreg1
Hepnaestin femreductase
Kupffer cell
hepatocyte Chapter 1 General Introduction Page 43
1.6.3. Ferroportin (Iregl)
Ferroportin was identified using three separate methodologies: subtractive cloning (McKie et a/., 2000); positional cloning of the gene responsible for hypochromic anemia in zebrafish (Donovan at al., 2000) and by using an affinity column to bind messenger RNAs containing Iron Response Elements (IREs)
(Abboud at a!., 2000). It is an iron export protein localised to the enterocycte basolateral membrane (connecting the enterocycte to the portal vein circulation; see Figure 1.2.). Ferroportin (Fpl) is also found on Kupffer cells and on placental syncytiotrophoblast cells (Donovan at a!., 2000). An IRE has been reported in the
5’ untranslated region (UTR), but its function is not yet understood. Expression of ferroportin is increased in conditions that increase iron absorption, for instance iron deficiency. This is in contrast to the effect of the classical 5'-IRE in H-ferritin, where under low iron conditions, ferritin expression is decreased (Klausner at a!.,
1993). Ferroportin has been shown to be mutated in some forms of autosomal dominant haemochromatosis (Montosi at a/., 2001; Njajou at a!., 2001; Devalia at a!., 2002). Basolateral transport has been proposed as the rate limiting step of iron absorption and this step is probably mediated by ferroportin 1 (Fpl)
(McLaren at a!., 1991) Chapter 1 General Introduction Page 44
1.6.4. Hephaestin
The Hephaestin gene (HEPH) was cloned as a result of looking for the gene defect in the sex linked anemia (s/a) mouse (Vuipe et al., 1999). It was shown to be a caeruloplasmin homologue with predominantly perinuclear localization within intestinal enterocytes. This multicopper protein seems to act as the ferroxidase, necessary for the exit of the iron from the intestinal enterocyte into the systemic circulation (see Figure 1.2. above). However the exact function of hephaestin has yet to be determined.
1.6.5. Hepcidin
Knock out mice for the USF2 gene (upstream of the hepcidin gene) that also prevented expression of the hepcidin gene showed increased circulatory iron and hepatic iron (Nicolas at a!., 2001). The iron loading pattern was similar to that displayed by the HFE knockout mouse. Hepcidin has sequence homology with anti microbial peptides and is synthesized in the liver. It is suggested to be part of the signaling between the body iron stores in the liver and iron absorption in the intestine (Fleming and Sly 2001 ). Chapter 1 General Introduction Page 45
1.7 Preview of thesis
Chapter 2 describes the genetic characterisation of the Royal Free cohort of haemochromatosis patients and a control group. The genotype for four microsatellite markers flanking the haemochromatosis gene region was determined. Following the identification of the HFE gene, the patients were also investigated for the Cys282Tyr and His63Asp mutations. This was done to identify a genetically homogeneous group of Cys282Tyr homozygous patients for later ferric reductase studies. It also confirmed absence of disease related genotypes in the control group, and highlighted possible non HFE-related haemochromatosis patients.
Chapter 3 describes the isolation and characterisation of genes from around the microsatellite marker D6S1260. At the time of study this was the apparent peak of linkage disequilibrium within haemochromatosis chromosomes. The study was done in an attempt to identify candidate genes for haemochromatosis. A small intestinal cDNA library was screened with two PAG clones known to contain
D6S1260. Eleven cDNA clones were identified via hybridisation, four of which were identified to have single copy inserts that showed sequence homology to zinc finger loci. It was decided to further investigate these four clones. Chapter 1 General Introduction Page 46
Chapter 4 describes the complete sequencing and characterisation of the four cDNA clones, which were all derived from a novel zinc finger locus on chromosome 6p21.3, designated ZNF204 (deposited as GenBank accession number AF033199). Genomic and RNA analyses suggested that ZNF204 was an expressed processed pseudogene and is unrelated to haemochromatosis.
However, no parent locus could be identified using somatic cell hybrid panels or fluorescent in situ hybridisation (FISH). No product could be identified following coupled transcription and translation of ZNF204. This locus is probably an expressed processed pseudogene. The sequence is however highly conserved across species and may prove valuable in identifying other zinc finger genes in model organisms.
Chapter 5 describes the first analysis of ferric reductase activity in white blood cells from haemochromatosis patients and control individuals. Previous studies had shown increased activities in haemochromatosis compared with control duodenal biopsy ferric reductase activity. Macrophages, like duodenal enterocytes, remain paradoxically iron poor in haemochromatosis and were therefore considered likely to express the same abnormality in ferric reductase activity as duodenum. This study was conducted in an attempt to identify an in vitro index of the disease and to investigate the possibility of functional complementation by candidate haemochromatosis genes. No significant difference was observed in either monocytes or macrophages from Chapter 1 General Introduction Page 47
haemochromatotis patients versus controls, ruling out a functional complementation approach. However, a ten fold increase in ferric reductase activity was observed as monocytes matured into macrophages, possibly reflecting the putative upregulation of gene of iron metabolism in differentiation. Chapter 2 Analysis of GH and control genotypes Page 48
Chapter 2 Analysis of GH and control genotypes Chapter 2 Analysis of GH and control genotypes Page 49
Chapter 2. Analysis of GH and Control genotypes: Index.
2.1. AIMS OF THE STUDY...... 50 2.2. INTRODUCTION...... 51 2.2.1. Polymorphic Markers ...... 51 2.2.1.1. Protein polymorphisms ...... 51 2.2.1.2. Restriction fragment length polymorphisms ...... 51 2.2.1.3. Polymorphism information content ...... 52 2.2.1.4. Minisatellite repeats (VNTRs) ...... 53 2.2.1.5. Microsatellites ...... 54 2.3. METHODS...... 56 2.3.1. Multiplex fluorescent PGR ...... 56 2.3.1.1. Microsatallite analysis ...... 57 2.3.1.2. Sample preparation ...... 57 2.3.1.3. Setup of the 310 ABI prism autosampler ...... 57 2.3.1.4. Run conditions ...... 58 2.3.1.5. Computer analysis of data ...... 59 2.3.2. HFE mutation analysis ...... 59 2.4. RESULTS...... 59 2.5. DISCUSSION...... 63 2.5.1. General problems of microsatellite analysis ...... 63 2.5.2. GH patient microsatellite data versus controls ...... 64 Chapter 2 Analysis of GH and control genotypes Page 50
Chapter 2. Analysis of GH and control genotypes.
2.1. Aims of the study
Four polymorphic (CA)n repeat markers from the HFE gene region were chosen for analysis of GH patient and control genotypes. D6S265,
D6S105 and D6S1260 have been shown to form part of the founder haplotype in GH using linkage disequilibrium studies (Jazwinska et al.,
1993, Raha-Chowdury et al., 1995, Seese et al., 1996). A marker towards the telomeric extent of the GH gene region, D6S1621, was also tested. Towards the end of this study, the HFE gene was identified
(Feder et al., 1996), and so the C282Y and H63D mutations of the HFE gene were also determined. These studies were performed:
(1) To determine microsatellite markers of the HFE gene region, and the
C282Y and H63D mutations of HFE, in order to identify patients with a secure diagnosis of HFE-related haemochromatosis, and to compare GH and control patients to be investigated by ferric reductase assay (Chapter
5).
(2) To screen the Royal Free Hospital cohort of GH patients, and control patients, to identify appropriate individuals for a pulsed field gel electrophoresis approach to mapping the GH gene (Wallace et al., 1996). Chapter 2 Analysis of GH and control genotypes Page 51
2.2. Introduction
2.2.1. Polymorphic Markers
2.2.1.1. Protein polymorphisms
The first genetic linkage studies in humans relied on protein polymorphisms of blood to define linkage (Clark et al., 1956). Linkage is defined as the occurrence of two loci sufficiently close together on a chromosome so that their assortment is non independent. The first linkage to be shown in GH was with HLA-A (Simon et al., 1977).
2.2.1.2. Restriction fragment length polymorphisms
Due to the limitations of protein analysis investigations, research moved towards recombinant DMA technology to investigate linkage in GH.
Restriction fragment length polymorphism (RFLP) occurs because sites recognized by restriction endonucleases are polymorphic. Single base- pair changes are frequent in the human genome (estimated to be as high as 1 in lOOObp. The nucleotide sequence difference can create or destroy enzyme recognition sites. Individual chromosomes can be traced down generations using these polymorphisms. There are however two main drawbacks to using RFLPs for haplotype analysis: (1) the level of polymorphism is normally low and limited by the number of alleles, and Chapter 2 Analysis of GH and control genotypes Page 52
(2) RFLP analysis requires restriction enzyme digestion, electrophoresis, blotting and hybridization. This is time consuming and not well suited to automation.
2.2.1.3. Polymorphism information content
The degree of polymorphism of a marker is expressed as the polymorphism information content (PIC). PIC values are calculated using the formula: -
n-1 n
PIC= 1 -(Zpi^ )-Z £2pi^pj^
/ = 1 /■ = 1 y = / + 1
where p\ and pj are the frequency of the /th and /th alleles.
High PIC values indicate a greater likelihood that a locus will be informative for linkage analysis. RFLP analysis on the human genome, due to its large size and low rates of polymorphism, is limited in application. Therefore more informative polymorphic markers were sought. Chapter 2 Analysis of GH and control genotypes Page 53
2.2.1 A. Minisatellite repeats (VNTRs)
Minisatellites are highly polymorphic regions of the genome. They are composed of tandem repeats of 8-90bp with variation deriving from the number of tandem repeats. The length of the total array is variable from
1-30 kb giving rise to a large number of alleles, each of which may be rare in the population. Over 70% of minisatellites are highly polymorphic
(Litt et al., 1989). The overall size of the minisatellite is related to its degree of polymorphism.
Small minisatellites (2 kb or smaller) are frequently associated with monomorphic or only minimally variable loci. Large minisatellites (bigger than 4kb) are nearly always derived from hyper-variable loci.
Minisatellites are found throughout the genome, although there is clustering in human centromeric and telomeric regions. Uninterrupted minisatellite repeats are termed perfect repeats; interrupted minisatellites are termed imperfect repeats, and if fragmented into several blocks termed compound repeats (Weber et al., 1989). They are inherited in a
Mendelian fashion with a low mutation rate. Their uses include genetic fingerprinting and linkage analysis although this is hampered by the clustering. Chapter 2 Analysis of GH and control genotypes Page 54
2.2.1.5. Microsatellites
Microsatellites consist of around 10-50 copies of a 1-6 bp motif; therefore they can be highly polymorphic. They were originally analyzed and detected by radiolabelled PCR and denaturing polyacrylamide gel electrophoresis (Weber et al., 1989). Microsatellites therefore became a frequently used marker in genetic analysis. The di-nucleotide repeat CA is most commonly used for genetic analysis. The abundance of the CA repeat has been investigated (Tautz at al 1989, Litt et al., 1989, Weber et a/., 1989) showing that there are approximately 0.5 X 10^ to 1 X 10® copies in the human genome with an average separation of between 30-
65 kb.
This high frequency throughout the genome and high degree of polymorphism are advantageous for genetic analysis. Examples of the usefulness of microsatellites include the Genethon human genetic linkage map (Gypay et al., 1994) and the Genome Directory (Venter et al., 1995).
The Genethon (1994) map is a second generation genetic linkage map of the human genome containing over 2000 CA repeats, about 56% of these at a distance of less than IcM from another of its markers. The
Genome Directory described the use of CA repeat markers to create a first order YAC contig map across the entire human genome, and to Chapter 2 Analysis of GH and control genotypes Page 55
obtain high resolution second generation maps of chromosomes 3,12,6 and 22. These maps are particularly useful in mapping both monogenic diseases and multifactorial traits. The recent publication of the draft human genome sequence built upon these successes, and marked a culmination of the efforts of the Human Genome Project (International
Genome Sequencing Consortium, 2001)
The mechanism for the creation of variation in the length of microsatellite repeats is thought to be that of replication slippage (polymerase stutter) randomly occurring in internally repetitive stretches. Another possibility causing variation in repeat length is unequal mitotic exchange. This variation in length however occurs at a very slow pace estimated to be
6 .2 X 10'"^ mutations per locus per gamete for the longest of repeats
(Weissenbach et al., 1996). This slow mutation rate enables alleles to be traced successfully through families. CA repeats are highly conserved as a repetitive element in mammalian genomes. CA repeats are effectively single locus probes, and exploited in genomic mapping. Once mapped, their highly polymorphic nature and Mendelian inheritance allows them to be used effectively in linkage and linkage disequilibrium analysis to map disease genes. In this Chapter, microsatellite analysis of the founder haplotype and HFE mutation analysis were used to identify patients with a secure diagnosis of HFE-related haemochromatosis. Chapter 2 Analysis of GH and control genotypes Page 56
2.3. Methods
2.3.1. Multiplex fluorescent PCR
The following work was performed at the kind invitation of Professor Mark
Worwood within the Department of Haematology, University of Wales
College of Medicine, Cardiff. Multiplex fluorescent PCR amplification of
CA repeats was performed on genomic DMA from both GH and control patients using a Hybaid Omnigene PCR machine. Four primer pairs were used in the multiplex PCR, and the forward primer of each pair was fluorescently labeled as indicated: D6S105 (HEX), D6S265 (RAM),
D6S1260 (TET) and D6S1621 (TET). It was possible to use the fluorescent marker TET for both D6S1260 and D6S1621 primers as these microsatelites give PCR products differing in size by approximately
200bp, which can be readily differentiated from each other. The reaction profile was: 94.2°C for 40 seconds; 55°C for 40 seconds; 72°C for 40 seconds (30 cycles) followed by a final extension at 72°C for 5mins then a 26°C soak. PCR reactions contained; 1X Bioline PCR buffer; 1mM
MgCl2 ; lOOpM each of dGTP, dCTP, dATP, dTTP; 1pM each of forward and reverse primers; 1U of Bioline Tag polymerase, in a total volume of
15pl. Chapter 2 Analysis of GH and control genotypes Page 57
2.3.1.1. Microsatallite analysis
The allele sizes of amplified CA repeats were analyzed on a Perkin Elmer
ABI Prism 310 GeneScanner using the GeneScan application kit. The
310 was run in high resolution mode containing a 6.6M urea denaturing gel. A 3 percent polymer was used for this resolution giving a 50-250bp read range. The capillary tube containing the polymer was set to 40cm gel length, with sample injection set at 10 seconds to optimize resolution.
Internal size standards were included with each sample to permit accurate sizing.
2.3.1.2. Sample preparation
PCR reactions (Ipl) were mixed with 15pl deionised formamide and O.SpI
GS 500 size standards. Samples were denatured 2mins at 95°C, snap cooled on ice, and loaded into the 310.
2.3.1.3. Setup of the 310 ABI prism autosampler
Buffers and polymer for the electrophoresis were made according to the
Perkin Elmer operational manual. For each sample the 310 will automatically form a new matrix within the capillary tube. After sample analysis the capillary is cleaned and neutralized by 0.3M NaOH and 1M
HCI washes respectively and washed with deionised water. The next Chapter 2 Analysis of GH and control genotypes Page 58
aliquot of polymer is then drawn up into the capillary and analysis of the next sample starts. Tubes containing buffers and polymers were placed into the 310 in the following positions: 1, GeneScan polymer solution; 2, dH2 Ü; 3, dH20; 4, 0.3M NaOH; 5, 1M HCI; 6, left empty for emergency back flush. The anode buffer consisted of 1g genetic analyzer buffer
(lOx) and 2.8g GeneScan polymer (7% w/w) made up to 10ml. The cathode buffer consisted of 0.5g genetic analyzer buffer 10X, 2.2g
Genescan polymer (7% w/w), 2.0g urea plus 800ul of deionized water, giving a 3% Gene scan polymer solution containing 6.6M urea. After dissolving the urea the solution was passed through a 0.45pm filter before use.
2.3.1 A. Run conditions
The ABI 310 was set to the following run and injection parameters:
Analyzer range 3250 - 5500; size call range 75 - 500bp; split peak detection off; data smooth on; peak detection 70
RED/BLUE/GREEN/YELLOW; fit to line "global Southern”; run module denatured; GS XT long denatured; injection 10 sec; Kv 15.00; run 15.00
Kv; temperature of block 42°C; runtime for each sample 20 mins; matrix file dye set 0 and autoanalysis default on. Chapter 2 Analysis of GH and control genotypes Page 59
2.3.1.5. Computer analysis of data
Raw data were analyzed using Perkin Elmer Gene Scan Analysis software version 1 alpha on an Apple Macintosh™. An example of the data obtained is shown in Figure. 2.1.
2.3.2. HFE mutation analysis
DNA was prepared from peripheral blood from GH patients and controls using the QIAamp Blood kit (Qiagen). Cys282Tyr and His63Asp mutations were analyzed by PCR and restriction enzyme digestion
(Worwood et ai., 1997).
2.4. Results
An example result of the output from the ABI 310 GeneScanner is shown in Figure. 2.1., below. The results from the analysis of 14 GH patients and 7 controls are summarised in Table. 2.1. Chapter 2 Analysis of GH and control genotypes Page 60
Figure 2.1. GeneScan trace showing relative fluorescent intensity
plotted against approximate size (bp).
Apparent size (bp)
96 100 104 109 112 116 120 124 128 132 136 140 144 148 152 156 160 16 * tn c S c 3200 c 800 (Ü I This Genescan trace above shows homozygosity for one marker (D6S1260-4/4, Green), also showing heterozygosity for two markers (D6S105-8/6, Black, D6S265-1/3, Blue), peaks for size standards are shown in red. The conversion between allele size and allele number is shown in Appendix 1. Chapter 2 Analysis of GH and control genotypes Page 61 Table. 2.1. Patient founder haplotype microsatellite and HFE mutation analysis results Individual Age Sex Genotype HFE D6S1621 D6S1260 D6S105 D6S265 C282Y H63D GH1 5-5 4-4 6-8 1-5 ++ GH2' m 5-6 6-8 1-3 GH3 m 5-10 6-8 1-6 ++ GH4 74 5-10 . 8- 8= 1-3 ++ GH5 5-7 3-3 6-8 3-3 GH6 5-10 6-8 1-6 ++ GH7 5-10 4-4 8-8 1-6 ++ GH8 5-10 % 4 8-8 ++ GH9' 5-7 4-4 5-8 1-6 ++ GH105-5 3-4 6-8 1-3 ++ GH11 m 7-10 5-6 3-6 GH1 NT NT NT NT ++ GH13 5-7 4-5 2-6 6-6 + - GH102' 48 5-10 5-8 3-4 + + C l 48 10-10 4-4 4-5 3-6 + - 0 2 3-4 2-8 3-3 + - 03 4-4 3-6 5-5 04 m 10-10 4-4 6-7 3-3 05' 60 4-11 4-5 6-12 5-5 + - 06 6-10 5-6 3-54-5 07 m 5-10 4-4 3-8 3-5 + - Legend on next page. Chapter 2 Analysis of GH and control genotypes Page 62 Results are shown for GH and control (C) patients. Homozygosity for alleles of the founder haplotype (D6S1621-5; D6S1260-4; D6S105-8; D6S265-1) is dark shaded. Regions of possible heterozygosity for the founder haplotype are lightly shaded. NT, not tested; C282Y, Cys282Tyr; H63D, His63Asp; + and -, presence and absence of mutation; ® patients investigated in ferric reductase assays (Chapter 5); ‘^juvenile haemochromatosis; ^ homozygous for HLA- A3 and HLA-B7. The position of HFE is between the markers D6S1621 and D6S1260. Chapter 2 Analysis of GH and control genotypes Page 63 2.5. Discussion 2.5.7. General problems of microsatellite analysis Microsatellites were originally observed on standard DNA sequencing gels. The microsatellites were frequently amplified using Taq polymerase and [ a^^P] dCTP, to allow the polymorphic bands to be detected by autoradiograpy. This manual method is prone to the occurrence of three main artifacts: (1) The differing mobility of the two strands of DNA may give rise to multiple bands. This is because the CA and GT strands of the amplified DNA migrate with differing mobilities under denaturing conditions. It may be remedied by using a single radiolabelled primer which will label only one strand, rather than incorporating a radiolabelled deoxynucleotide, which will label both strands. (2 Polymerase slippage (by mispairing during the PCR) may give rise to extra bands differing by 2n in size. Changing the PCR reaction conditions however does not lead to any significant improvement of resolution. (3) Non complementary base addition at the 3’ end of PCR products (Clark 1988). This is treatable by treating the PCR end products with 3’-5’ exonuclease. Chapter 2 Analysis of GH and control genotypes Page 64 The ABI 310 is not prone to mobility shift problems as only one of the primers is fluorescently labeled. Polymerase stutter may occur, but by using the GeneScan software it is possible to reduce any ladder to background, leaving clear peaks. Non-complementary base addition may be remedied by 3'-exonuclease treatment as for conventional micosatellite analysis. Although a drawback to using the 310 is its initial cost, the data produced are of high quality. 2.5.2. GH patient microsatellite data versus controls The numbers of patients analysed are too small for independent linkage disequlibrium analysis. Even in the limited data set, however, there is evidence for the conservation of the founder haplotype (D6S265-1; D6S105-8; D6S1260-4; D6S1621-5; Raha-Chowdhury ef a/., 1995) in GH patients from the Royal Free Hospital. Two patients (GH2 and GH11) had early onset of iron overload with cardiac involvement, and were diagnosed as having juvenile haemochromatosis. Of note was the observation that these patients did not have the founder haplotype, or either the C282Y or H63D mutation of HFE. Linkage of juvenile haemochromatosis to HFE was later excluded, and this disease has subsequently been recognized as a distinct Chapter 2 Analysis of GH and control genotypes Page 65 disorder, and linked to another locus on chromosome 1q (Roetto et al., 1999). The genotype analysis also allowed selection of appropriate controls and GH patients for physical mapping of patient chromosomes by pulsed-field gel electrophoresis (Wallace eta!., 1998). These studies allowed the comparison of genotypes between the GH and control patients studied for ferric reductase analysis (Chapter 5). Following mutation analysis it was also possible to select patients with a secure diagnosis of HFE-related haemochromatosis who were homozygous for C282Y, and controls who were negative for C282Y, for ferric reductase analysis. Chapter 3 Isolation and characterisation of genes from around D6S1260 Page 66 Chapter 3 Isolation and characterisation of genes around D6S1260 Chapter 3 Isolation and characterisation of genes from around D6S1260 Page 67 Chapter 3. Isolation and characterisation of genes around D6S1260: Index. 3.1. INTRODUCTION...... 68 3.2. METHODS...... 69 3.2.1 YAC and PAC clones ...... 69 3.2.2. Plating the cDNA library ...... 71 3.2.2.1. The Clontech human small intestine cDNA library HL1133a .... 71 3.2.2.2. Titering the cDNA library ...... 72 3.2 2.3. Plating a IX coverage of the library ...... 73 3.2.2.4. Preparation of duplicate library filters for screening ...... 73 3.2.3. Screening cDNA library ...... 74 3.2.3.1. PAC insert probe preparation...... 74 3.2.3.2. Pre-hybridisation of filters with placental DNA competition 75 3.2.3.3. Competition of the radiolabelled probe ...... 76 3.2.3.4. Washing of cDNA filters ...... 77 3.2.3.5. Analysis of results from the 715 / 672 PAC insert probe 77 3.2.3.6. Secondary Screen ...... 78 3.2.3.7. Tertiary screen ...... 78 3.2.4. Subcloning the cDNA clones ...... 79 3.2.4.1. PCR amplification of positive plaques ...... 79 3.2.4.2. EcoR I subclones of cDNA PCR products ...... 79 3.2.4.3. Cloning of PCR products using CloneAmp pAMPI vector 80 3.2.4.4. Amplification of cDNAs for pAMPI cloning ...... 80 3.2.4.5. Ligation of cDNAs into pAMP 1 vector ...... 81 3.2.5. Sequencing of the cDNAs ...... 82 3.2.6. Computer analysis of cDNA sequences ...... 83 3.2.7. Hybridisation analysis of cDNA inserts ...... 84 3.3. RESULTS...... 84 3.4. DISCUSSION...... 91 Chapter 3 Isolation and characterisation of genes from around D6S1260 Page 68 Chapter 3. Isolation and characterisation of genes from around D6S1260. 3.1. Introduction This work was undertaken in spring 1996, before the cloning of the haemochromatosis candidate gene (HFE) by Feder and co-workers (1996). As described in Section 1.3 (Genetics of haemochromatosis), at this time the apparent peak of linkage disequilibrium was the chromosome 6 polymorphic CA repeat marker D6S1260 (Raha- Chowdury et al., 1995). We therefore concentrated on this region to isolate and characterise new genes hoping to identify potential candidate genes for haemochromatosis as well as other novel gene sequences. There are several methods, which can be used to isolate genes from cloned regions of genomic DNA. The method used was the direct hybridisation of genomic clones to a cDNA library. This approach requires the use of cDNA from a defined tissue. This is potentially useful in reducing the number of transcripts to be screened, but involves the presumption that the gene or genes are expressed in the particular tissue. The major advantage of this method, however, is that it is simple and rapid. Chapter 3 Isolation and characterisation of genes from around D6S1260 Page 69 Two overlapping PACs 11g1pCYPAC2N (715) and 302f12pCYPAC2N (672), spanning 148kb of genomic DNA, and containing D6S1260, were used to screen a cDNA library of human small intestine. The choice of cDNA reflected the probability that this tissue expresses the gene for haemochromatosis, since the small intestine is the site of iron absorption from the diet. 3.2. Methods 3.2.1 YAC and PAG clones Yeast artificial chromosome (YAC) clones from the HFE gene candidate region were obtained from the CEPH library (Albertsen et a!., 1990; Chumakov eta!., 1995). PI-derived artificial chromosome (PAC) clones from the Roswell Park Park Cancer Institute (RPCI-1) PAC library were obtained from the Human Genome Mapping Project Resource Centre (HGMP-RC). PACs from the HFE gene region were identified by several methods: direct request of previously published PACs; hybridization of whole YACs onto gridded PAC library filters (Baxendale et al., 1991); PCR amplification from primary, secondary and tertiary PAC library pools; and by PAC ‘walking’ using T7 and SP6 end specific riboprobes on gridded PAC library filters (Stratagene SuperCos protocol). Chapter 3 isolation and characterisation of genes from around D6S1260 Page 70 YAC clones were grown at 30°C for two days in selective media deficient in uracil and tryptophan. YAC DNA was prepared in agarose blocks according to the lithium dodecyl sulphate (LIDS) method of the Wellcome Trust Summer School - Human genome analysis: from genome to function (http://www.wellcome.ac.uk/summerschools). Cells were immobilized in agarose blocks, and DNA isolated via treating with zymolase and yeast lysis solution containing lithium dodecyl sulphate. PAC clones were grown at 37°C overnight in Luria-Bertani (LB) media containing the selective antibiotic kanamycin. PAC DNA was prepared using standard alkaline lysis mini-preparation methods (Sambrook et al., 1989) or by using QIAfilter plasmid midi kits (Qiagen). The alkaline lysis procedure is based on the following principle: Cells are first pelleted and lysed by treating them with sodium dodecyl sulphate and NaOH. The SDS solubilizes the cell membranes causing the cells to lyse thus releasing their contents. NaOH causes the DNA and proteins to denature. Subsequent addition of acetate buffer neutralizes the lysate and allows the DNA to renature. The smaller size of the plasmid DNA and the proximity of the two interlocked strands means that the plasmid DNA renatures quickly and remains in solution. The chromosomal DNA however remains single stranded, and together with the denatured proteins and other cellular debris becomes trapped in the insoluble salt and detergent complexes. The Oiagen plasmid purification procedure uses an anion exchange column, which binds double stranded DNA. Chapter 3 Isolation and characterisation of genes from around D6S1260 Page 71 Contaminating RNA, proteins and other impurities can then be removed in a low salt wash and DNA eluted in a high salt wash. The purified plasmid can then be precipitated using either ethanol or isopropanol and plasmid DNA resuspended in TE buffer ( 10mM Tris-HCI, 1mM EDTA, PH8). Pulsed-field gel electrophoresis analysis, Southern blotting and hybridization with 32P-radio-labeled total human DNA determined the size of YAC clones. The size of the PAC inserts was determined by Not I digestion and PFGE analysis. YAC and PAC clones were digested with Hind III, fragments separated by agarose gel electrophoresis and Southern blotted to Hybond N+ membranes. Probes and polymorphic markers across the HFE region were localized on the YACs and PACs by hybridization to Southern blots and by PCR analysis. 3.2.2. Plating the cDNA library 3.2.2.1. The Clontech human small intestine cDNA library HL1133a A cDNA or complementary DNA is synthesized as a single-stranded copy of an RNA template, using an RNA-dependent DNA polymerase (reverse transcriptase). The first strand cDNA is copied using a DNA-dependent DNA polymerase, giving double stranded cDNA, which is cloned into a suitable vector. A cDNA library is a collection of cDNAs that ideally represents all the RNA molecules expressed by a specific tissue. The Chapter 3 Isolation and characterisation of genes from around D6S1260 Page 72 cDNA library chosen for screening was a Clontech human small intestine 5’-Stretch cDNA library (HL1133a). This is a commercial library prepared from the duodenal tissue of a 15 year old Caucasian female. The mRNA was primed with both oligo (dT) and random primers for reverse transcription, and then EcoR I cloned into the vector XGT10. 3.2.2.2. Titering the cDNA library The following methodology is modified from Sambrook et al., (1994). The Escherichia coil host strain Y1090r- was revived from glycerol stock held at -70°C and streaked onto an LB agar 1.5% (w/v) Petri dish containing 50pg/ml ampicillin and grown at 37°C overnight. A single colony was used to inoculate 10ml of LB broth containing lOmM MgS 0 4 and 0.2% (w/v) maltose without added antibiotic. The broth was incubated at 37°C in a shaker at 200rpm overnight until the ODeoo reached approximately 2.0. The culture was centrifuged at 650g for lOmins and the pellet re suspended in the same volume of lOmM MgS 0 4 (“plating cells”) and stored at 4 °C. Titering dilutions of 1/500, 1/5,000 and 1/50,000 were prepared from the library using IX lambda dilution buffer (0.1 M NaCI, lOmM MgS 0 4 .7 H2 0 , 33mM Tris-HCI (pH7.5) and 0.01% (w/v) gelatine). The titering dilutions (lOOpI) and control (lOOpI IX dilution buffer) were added to 200pl of the Y1090r- plating cells, and incubated at 37°C for 15mins. Melted (45°C) LB top agarose 0.7% (w/v) containing lOmM MgS 0 4 was mixed with each dilution thoroughly and poured onto pre Chapter 3 Isolation and characterisation of genes from around D6S1260 Page 73 warmed (37°C) LB plus 10mM MgS 0 4 agar plates, ensuring even spreading across the plate. The plates were allowed to solidify and then incubated at 37°C inverted for approximately 7 hours, until plaques were clearly visible. Plaques were counted and plaque forming units (pfu) per ml of stock calculated. 3.22.3. Plating a 1X coverage of the library Using the titering information from above, dilutions were prepared from stock to give 250,000 pfu on each of eight 22 X 22cm LB plus 10mM MgS 0 4 agar plates. The Clontech HL1133a small intestine library is estimated to contain 1.6 X 10® independent cDNA clones. Inoculating 250,000 pfu on eight 22 x 22cm agar plates thus gave a single coverage of all the cDNAs present. The plates were poured using the same method as for titering, except that each 22 x 22cm plate contained 200ml of LB plus lOmM MgS 0 4 agar and 50ml LB top agarose plus lOmM MgS 0 4 . The plates were inverted and incubated at 37°C for approximately 7 hours until the plaques were clearly visible, and not touching each other. 3.2.2A. Preparation of duplicate library filters for screening When plaques reached the optimum size, the plates were removed from the 37°C incubator and chilled at 4°C to harden the top agarose. Sterile Hybond™ N+ (Amersham) 22X22cm nylon membrane filters (2 filters per plate) were marked with the corresponding plate numbers. These were Chapter 3 Isolation and characterisation of genes from around D6S1260 Page 74 placed using sterile forceps carefully on top of the top agarose for 60 seconds. Care was taken not to smudge the plate, introduce bubbles or disturb the pattern of plaques. The orientation of the nylon filters was marked by piercing each one with an 18 gauge needle to give an asymmetric pattern. The first filter was removed, and the duplicate filter placed on the same plate for 60 seconds, and marked with the same needle holes. The filters were incubated twice plaque-side up on Whatman 3MM filter paper saturated with DNA denaturing solution (1.5M NaCI, 0.5M NaOH) for 60 seconds. The filters were then placed on Whatman 3MM saturated with neutralizing solution (1.5M NaCI, 0.5M Tris-HCI (pHB.O) twice for 60 seconds. The filters were briefly washed in 4 X SSC, placed on damp Whatman 3MM and UV cross linked at 1200J using a Stratagene StrataLinker, and allowed to dry prior to hybridization. 3.2.3. Screening cDNA library The human genomic inserts of PACs 715 and 672 were used to screen the small intestine cDNA library. 3.2.3.7. PAC insert probe preparation PACs 715 and 672 were digested using the restriction enzyme Not I to release the human insert. The digest was separated by pulsed field gel Chapter 3 Isolation and characterisation of genes from around D6S1260 Page 75 electrophoresis (PFGE) using a BioRad CHEF DRIII system (Ho & Monaco 1992). A 0.8% (w/v) agarose gel was pulsed in three blocks of 24 hours with switch times of 12sec (96°, 2V/cm), 15sec (100°, 2V/cm), 18 sec (96°, 2V/cm). The DMA was visualised by ethidium bromide staining. The human DNA insert bands (PAC 715, lOkb and 115kb; PAC 672, 33kb and 49kb) were excised from the gel under long wave UV light. The excised human DNA inserts from each PAC were pooled, p-agarase-digested and phenol chloroform extracted. The DNA was resuspended in 30|xl TE pH8.0. The DNA concentration of the inserts was estimated by absorbance at A 26 0 . 25ng of the human insert DNA was radiolabelled by random hexanucleotide priming (Feinberg & Vogelstein, 1983) using a^^P dCTP (Amersham). 3.2.3.2. Pre-hybridisation of filters with placental DNA competition The duplicate cDNA library filters were pre-wet using 4X SSC, sandwiched between hybridisation meshes and placed in an AppliGene hybridisation bottle. 20ml of formamide pre-hybridisation solution containing 10Opg/ml denatured, sheared human placental DNA was added in order to quench repetitive DNA sequences. The bottle was placed in a rotating oven at 42°C overnight. Chapter 3 Isolation and characterisation of genes from around D6S1260 Page 76 3.2.3.3. Competition of the radiolabelied probe The radiolabelied probe was pre-annealed with a mixture of sheared human placental DNA, Alu / Line repeats and ribosomal probes to quench repeat sequences contained within human genomic DNA. Approximately 500,000 to 1,000,000 copies of the Alu repeat element and 100,000 - 800,000 copies of the Line repeat have been estimated to occur in the human genome (Boeke, 1997). To compete the probe, the following reagents were added to give a final concentration as indicated: human placental DNA (2.5mg/ml); Bluescript DNA (50pg/ml); Alu repeat DNA (5pg/ml); LINE repeat (5pg/ml); ribosomal DNA (R7.3 and R5.8 probes; 5pg/ml each). The probe mixture was denatured at 100°C for lOmin and snap-cooled on ice. The probe was briefly centrifuged to collect evaporation, cold 20X SSC was added to a final concentration of 5X, and the mixture was placed at 65°C for 90mins to allow the repeat DNA to anneal to the probe. The prehybridisation solution was then removed from the filters and replaced with 20ml fresh hybridisation solution. The competed probe was added to give 1 X 10®cpm/ml and the filters were hybridised overnight at 42°C. Chapter 3 Isolation and characterisation of genes from around D6S1260 Page 77 3.2.3.4. Washing of cDNA filters After hybridisation the hybridisation solution was discarded and the filters washed twice in 4X SSC at room temperature. The filters were washed twice in 0.2X SSC, 0.1% SDS at 60°C for ISmins, rinsed in 4X SSC and placed between plastic wrap. The filters were placed together on X- OMAT XAR-5 x-ray film for 2 days (Kodak) at -70°C using an intensifying screen to enhance the signal. 3.2.3.5. Analysis of results from the 715 / 672 PAC insert probe After developing, the x-ray films were marked at the 3 asymmetrical needle holes. The duplicate filter films were aligned and a total of 24 potentially positive plaques were found to be reproduced on both filters. Due to the high density of the plaques on the 22 X 22cm agar plates it was not possible to pick out individual positive plaques for PCR amplification at this stage. A 5mm diameter agar plug was pulled from the plates containing the positive plaques and placed in 1 ml of sterile lambda dilution buffer and left to elute overnight. A second round of screening was then undertaken. Chapter 3 Isolation and characterisation of genes from around D6S1260 Page 78 3.23.6. Secondary Screen A ^[i\ aliquot from each of the 24 eluted phage was added to 100pl of the Y1090r- plating cells. The mixture was incubated for lOmins at 37°C then added to melted (45°C) LB top agarose 0.7% (w/v) containing 1QmM MgS 0 4 , and poured onto pre-warmed (37°C) LB plus 1GmM MgS 0 4 15cm diameter agar plates. The plates were incubated inverted for 12-14 hours until individual plaques could be seen. Sterile Hybond™ N+ (Amersham) 15cm circular nylon membrane filters were used to copy the plates as described previously. Again the orientation of the nylon filters was marked by piercing asymmetrically 5 times with an 18 gauge needle. The filters were probed by hybridisation as described in screening cDNA library. 3.2.3.7. Tertiary screen From this second round of screening 11 positive plaques were identified. Due to the high density of plaques on the second round plates there was the possibility of cross contamination of clones picked for PCR. As a precaution a third round of screening was undertaken to reduce the density of plaques and possibility of cross contamination while picking. The 11 positives were pulled as plugs and re-screened using the above method. This third round of screening yielded single positive plaques that were distinct enough to be picked for PCR. Chapter 3 Isolation and characterisation of genes from around D6S1260 Page 79 3.2.4. Subcloning the cDNA clones 3.2.4.1. PCR amplification of positive plaques The 11 positive plaques identified by hybridisation were picked using sterile pipette tips and innoculated into the following PCR reaction mixture to act as DNA template: IX GibcoBRL PCR buffer; 1.5mM MgCl 2 ; 200pM each of dGTP, dCTP, dATP, dlTP; 500ng kGTIO forward and reverse primers in a total volume of 100|il. A no DNA negative control was also included. The reaction profile was that of a hot start: 94°C for 2mins then hold and add 2.5U (0.5pl) GibcoBRL Tag polymerase, then 94°C for 45 seconds; 72°C for 45 seconds; 55°C for 2mins increasing by 1 second per cycle for 35 cycles followed by a 10°C soak. The PCR products were separated by electrophoresis on a 1 % (w/v) agarose gel and visualised by ethidium bromide staining. From the lOOpI total PCR reaction, 60pl was purified using QIAquickspin (Qiagen) purification columns and eluted with 50pl lOmM Tris HCI (pH8.0). 3.2.4.2. EcoR I subciones of cDNA PCR products lOpI of purified DNA was digested with EcoR I, examined on an agarose gel and subcloned into the vector pBluescript (Stratagene). Before ligation, EcoR I was heat inactivated for 20mins at 6 8 °C. The ligation Chapter 3 Isolation and characterisation of genes from around D6S1260 Page 80 reaction contained: 100-200ng EcoR l-digested PCR product (3.0pl): 200ng EcoR l-digested and calf intestinal alkaline phosphatase-treated Bluescript; 6.25pl dH 2 0 ; O.SpI 10X T4 ligation buffer (NEB); 0.25pl T4 DNA ligase (2 X10® U/ml (NEB) at 11°C overnight. XL-1 Blue (Stratagene) competent cells were transformed using 5pi of this ligation mixture. Transformants were picked for plasmid DNA preparation by alkaline lysis “mini-preps” (Sambrook et al., 1994), and for preparation of glycerol stocks. 3.2.4.3. Cloning of PCR products using CloneAmp pAMP1 vector CloneAmp is a system developed by GibcoBRL to facilitate the high efficiency cloning of PCR amplification products. Sequence specific PCR primers are designed so that they incorporate dUMP residues in their 5’ ends (forward 5’ CAD CAU CAD CAD 3', reverse 5’ CUA CUA CUA C U A 3’). After PCR, the amplified product is treated with uracil DNA glycosylase (UDG), resulting in the dUMP residues becoming abasic and unable to base pair. This exposes a 12 base pair 3’ overhang which can be annealed to the complementary sequence in the linearised vector pAMP 1. 3.2.4.4. Amplification ofcDNAs forpAM PI cloning PCR was performed on the QIAquickspin-purified primary amplification products of the cDNAs. The primers used were adaptations of kGTIO Chapter 3 Isolation and characterisation of genes from around D6S1260 Page 81 primers, kGT 10F-CAU and 1GT10R-CUA. The PCR reaction contained; 0.3pl of QiaQuick (Qiagen) cDNA amplification product, or sterile dH 2 0 negative control; 1X GibcoBRL PCR buffer; 1.5mM MgC^; 200pM each of dGTP, dCTP, dATP, dTTP; 500ng ^uGTI0F-CAU/1GT1OR-CUA forward and reverse primers in a total volume of 100pl. The reaction profile was that of a hot start: 94°C for 2mins, then hold and add 2.5U (0.5pl) GibcoBRL Tag polymerase; then 94°C for 45 seconds, 55°C for 45 seconds, 72°C for 3mins, increasing by 1 second per cycle for 20 cycles; followed by a 10°C soak. The PCR products were separated on a 1 % (w/v) agarose gel and then visualised by ethidium bromide staining. 3.2.4.5. Ligation of cDNAs into pAMP 1 vector The dUMP-containing PCR product (1pl; 50-1 GOng) was added to a reaction mixture containing: IX annealing buffer (20mM Tris-HCI (pH8.4), 50mM KCI, 1.5mM MgCb) (Gibco BRL); 0.5pl pAMP 1 vector DNA (12.5ng); 0.25|liI (10U/uI) uracil DNA glycosylase in a total volume of 5pi. The mixture was incubated at 37°C for 30mins and placed on ice. 1 pi of the reaction mixture was used to transform competent XL-1 blue E. coii (Stratagene). Transformants were picked for plasmid DNA preparation by alkaline lysis “mini-preps”, and for preparation of glycerol stocks. Chapter 3 Isolation and characterisation of genes from around D6S1260 Page 82 3.2.5. Sequencing of the cDNAs A single colony from each of the transformed cDNAs was used to inoculate 100ml of LB broth containing 50)ig/ml ampicillin. The cultures were grown overnight at 37°C in a shaking incubator at SOOrpm. When the culture reached approximately 1 optical density unit (ODD) at Aeoo. the culture was harvested and centrifuged at SOOOrpm to obtain a pellet. This pellet was then used for alkaline lysis maxi-preparation of plasmid DNA using QIAfilters (Qiagen). The maxi-preparations of plasmid DNA were sequenced by the Sanger dideoxy-chain termination method using the Sequenase Version 2.0 sequencing kit (United States Biochemical). The 5’ and 3’ ends of the cDNAs were sequenced with the >10 forward and reverse PCR primers. Sequencing products were subjected to electrophoresis under denaturing conditions for 5 hours at 60V in a BioRad Sequi-Gen sequencing tank containing 6 % acrylamide : bisacrylamide (> 2 0 0 bp range) SequaGEL (National Diagnostics). The sequencing gel was vacuum dried at 80°C for 2 hours and exposed to BioMAX MR film (Kodak). Chapter 3 Isolation and characterisation of genes from around D6S1260 Page 83 3.2.6. Computer analysis of cDNA sequences The DNA sequences obtained from the ends of the cDNAs were screened for regions of homology to known sequences using the BLAST and FASTA (Altschul et a!., 1994, Pearson & Lipman 1988) citations. These programs search for regions of similarity between the test sequence and sequences held within specified databases. Two types of BLAST search were performed: BLASTN (DNA test sequence searching for homology to DNA sequences held on the database) and BLASTX (DNA test sequence with six frame translation searching for homology to predicted protein sequence held on the database). The nucleic acid databases searched with BLASTN were: G, GenBank Library; E; EMBL Library, R; Alu-like sequences, C; cDNA sequence library, P; Eukaryotic Promoter Data, J; Fugu Project sequences, 2; Sanger CpG Island library, 3; EMBL minus EST/STS/GSSs. The following are sections of EMBL that were used for more focussed searches: x; ESTs , h; Human, I; Invertebrates, o; Organelles, a; Other Mammals, v; Other Vertebrates, t; Patent Sequences, d; Rodents, s; STSs, u; Unclassified, q; New EMBL sequences. Protein databases searched with BLASTX were: S; Swissprot Protein Library, M; TREMBL (translation of EMBL), N; NBRF (PIR) Protein Library. The databases were also searched using the command nr for non-redundant database. Chapter 3 Isolation and characterisation of genes from around D6S1260 Page 84 FASTA searches were made using the same combination of databases as BLASTN and BLASTX. Results from the homology search (BLASTN, BLASTX) are returned with a homology score and a probability score. The homology score shows local alignment as a percentage identity (nucleotide against nucleotide and predicted protein against protein) and as a percentage similarity (predicted protein against protein only). The probability gives an indication of the strength of the similarity. The data obtained must however be taken as whole when considering possible homologies. 3.2.7. Hybridisation analysis of cDNA inserts cDNA insert probes were prepared by EcoR I digestion of PCR products and gel purification, radiolabelied by the random hexamer method (Feinberg and Vogelstein 1983) and hybridised to Southern blots of the 11 cDNA inserts; Hind Ill-digested PAG clones from the region; and Hind Ill-digested human genomic DNA. 3.3. Results The results from screening the cDNA library and analysis of the positive clones are summarised in Table. 3.1. Of the 11 cDNAs isolated, only 4 clearly mapped back to one of the parent PACs originally used to isolate the cDNAs. The cDNAs 1,2,3 and 4 all hybridised to PAC 715 Chapter 3 Isolation and characterisation of genes from around D6S1260 Page 85 (11g1pCYPAC2N) as well as to YACs 81 and 119, (CEPHy900E03878 and CEPHy904727B2). These four cDNAs all cross-hybridised to each other. All four had BLAST and FASTA sequence homologies to zinc finger transcription factors. cDNA3 was shown to contain the expressed sequence tag STS-9945. Of these 4 cDNAs, two gave single copy bands when hybridised to Hind Ill-digested human genomic DNA, the remaining two zinc finger cDNAs, however, gave a smear on hybridisation. The ends of these two zinc finger clones when sequenced were found to be chimeric. The cDNA 3 (2CS) contained 110bp of the p-amyloid gene, from amino acid 172 to 226 (100% nucleotide identity) adjoining the pBluescript cloning vectors SK primer site. The p-amyloid gene maps to chromosome 21 with no pseudogenes reported on chromosome 6p21.3. The cDNA 4 (4CS) contains approximately 3kb of the epidermal growth factor sequence (100% nucleotide identity), adjoining the XGT10 forward primer in the cloning vector pBluescript. The gene for epidermal growth factor has been mapped to chromosome 19 with no reports of any pseudogenes in chromosome 6p21.3. Chimeric clones such as these are frequently observed in cDNA libraries, and are a consequence of the method by which the libraries are created. Of the other 7 cDNAs, sequence from cDNA 5 (4BS) did not have significant homology to any sequences held in the BLAST database. The sequence of cDNA 6 (1AL) had 92% nucleotide identity to various Chapter 3 Isolation and characterisation of genes from around D6S1260 Page 86 cytochrome oxidase subunits. The cDNA 7 (2AL) sequence showed a 100% amino acid identity to bacterial rRNA sequence. The cDNAs 8 (2BS), 9 (4AS), 10 (5AS) and 11 (5AS2) when sequenced all showed high homology to repeat sequences held in the BLAST databases: cDNAs 8 and 9 contained Alu sequence; cDNA 10 had high homology to the Alu class of retrotransposons and cDNA 11 had high homology to the LINE class of repeat elements, and also contained a CA repeat of unknown origin. On hybridisation cDNAs 5 to 11 did not give a signal on any of the PACs originally used to screen the cDNA library, and gave a smear on Hind III digested genomic DNA. Table. 3.1. End sequence and hybridisation analysis of eleven cDNA clones isolated by hybridisation of PACs 715 and 672 to a small intestinal cDNA library. YACs 81 and 119 correspond to clones CEPHy900_e_03878 and CEPHy904727_b_2, respectively: PACs 715 and 672 correspond to clones 11g1pCYPAC2N and 302f12pCYPAC2N, respectively. cDNA No. Name Size (kb) Homology by BLAST Hybridisation Comments analysis cDNA PAC YAC genomic 1 5FL 0.9 zinc finger protein 1.2.3.4 715 81.119 single copy contained within cDNA 2 2 3AS2 2.7 zinc finger protein 1.2.3.4 715 81.119 single copy contains cDNA 1 3 2CS 1.6 zinc finger protein 1.2.3.4 715 81.119 multiple bands chimeric: poly-A tall; STSG-9945 4 4CS 5.5 zinc finger protein 1,2.3.4 715 81.119 multiple bands Repetitive; chimeric 5 4BS 5.5 None 5 -ve -ve smear Repetitive 6 1AL 1.7 Cytochrome oxidase 6 -ve -ve smear Repetitive 7 2AL 1.2 bacteriai rRNA 7 -ve 81.119 smear Repetitive 8 2BS 0.9 Alu 8.9 -ve -ve smear Repetitive 9 4AS 2.9 Alu 8.9 -ve -ve smear Repetitive 10 5AS 1.2 retro-transposon 10.11 -ve -ve smear Repetitive 11 5DS2 1.5 LINE and CA repeat 11 -ve -ve smear Repetitive N00 Chapter 3 Isolation and Characterisation of genes from around D6S1260 Page 88 Table 3.2. Low resolution YAC / PAG map of the haemochromatosis gene region showing the position of ZNF204. o CD CD M 00 CM O YAC Size [kb] T- (/) CO CM (/) (/) o Li. O U) CD 5 o Z > CM CM o i Q. QQ E CL o N N Q. 0 0 0 0 878 0 3 950 h 11 727 b 2 899_g_1 779 d 7 947 f 6 792_g_12 790_g_7 755 g 10 97 c 8 97 d 16 313 I 6 302 f 12 The position of ZNF204 is shown in red; blue indicates the presence of markers in YACs; green indicates presence of markers in PACs; light blue indicates YAC deletions; NT indicates not tested. Yeast artificial chromosome (YAC) clones were obtained from the CEPH library (Albertsen et al., 1990; Chumakov et al., 1995). The position of HFE was subsequently shown to be between probe p44T and butyrophilin (BTN) (Federe/a/., 1996). Chapter 3 Isolation and Characterisation of genes from around D6S1260 Page 89 3.4. Discussion At the time of isolation before the cloning of the haemochromatosis candidate gene (Feder et aL, 1996), genes isolated from around D6S1260 (at that time the apparent peak of linkage disequilibrium) were potential candidates for the haemochromatosis gene. Haemochromatosis is a disease where the normal regulation of iron absorption is lost leading to increasing iron overload. Zinc finger proteins regulate the expression of other genes and loss of function due to mutation may therefore lead to loss of regulation. The 4 cDNAs containing the zinc finger homology were chosen to be fully sequenced, because they were considered potential haemochromatosis candidates on the basis of the available mapping data and their possible functional role, and because they represented a potentially novel zinc finger locus which had been isolated by positional cloning from the 6p21.3 region. The other 7 cDNAs were excluded from further analysis at this time because of their cross-homology to known repeat sequences, repetitive hybridisation pattern on genomic Hind III digested DNA and the fact that they did not map back to either of the PACs used to original isolate the cDNAs (PAC 715 or 672). The identification of these cDNAs was most likely due to cross hybridisation of repeat sequences contained within the PACs, despite the use of competing DNA in the hybridisation. Chapter 3 Isolation and Characterisation of genes from around D6S1260 Page 90 Repeat sequences are found throughout the genome, although there is evidence of clustering in centromeric and telomeric regions in humans. There are two classes of dispersed repetitive DNA: SINEs (short interspersed elements) and LINEs (long interspersed elements). SINEs are shorter than 500bp and occur 10^-10® times in the human genome. An example of which is the Alu family of repeats, classically around 300bp in length. LINEs are longer (average size 5kb) and occur approximately 10"^ times in the human genome. Retrotransposons comprise part of this family of repeats. This identification of false positives due to cross hybridisation with SINEs and LINEs is one drawback to screening a cDNA library by hybridisation with genomic clones. Other methods such as exon trapping (Buckler et a/., 1991) and genomic sequencing are unaffected by the problem of cross hybridisation, although these methods are more time consuming and / or large scale procedures. The characterisation of the novel zinc finger locus encoded by cDNAs 1-4 is described in Chapter 4: Characterisation of ZNF204. Chapter 4 Characterisation of ZNF204 Page 91 Chapter 4 Characterisation of ZNF204 Chapter 4 Characterisation of ZNF204 Page 92 Chapter 4. Characterisation of ZNF204: Index. 4.1.INTR0DUCTI0N...... 93 4.2. COMPLETE CDNA SEQUENCE...... 98 4.2.1. Methods ...... 98 4.2.2. Results ...... 98 4.2.3. Conclusion from sequence investigation of ZNF204 ...... 108 4.3. ORGANISATION OF THE GENOMIC LOCUS...... 109 4.3.1. Introduction ...... 109 4.3.2. Methods ...... 109 4.3.3. Results and Discussion ...... 110 4.4. NORTHERN BLOT ANALYSIS...... 112 4.4.1. Introduction ...... 112 4.4.2. Methods ...... 112 4.4.3. Results and Discussion ...... 116 4.5. 5’ RAPID AMPLIFICATION OF CDNA ENDS (S' RACE)...... 118 4.5.1. Introduction ...... 118 4.5.2. Methods ...... 118 4.5.3 Results ...... 121 4.6. SEARCHING FOR ZNF204 HOMOLOGUES...... 122 4.6.1. Somatic cell hybrid panel analysis ...... 122 4.6.2 Methods...... 122 4.6.3. Results and Discussion ...... 123 4.7. HYBRIDISATION SCREENING OF A GENOMIC PAC LIBRARY 125 4.8. FLUORESCENT IN SITU HYBRIDISATION (FISH)...... 126 4.8.1. Introduction ...... 126 4.8.2. Methods ...... 127 4.8.2.1. cDNA probe ...... 127 4.8.2.2. PAC probes used for FISH ...... 127 4.8.2.3. Method used for FISH ...... 127 4.8.3. Results and Discussion ...... 130 4.9. ZOO BLOT ANALYSIS OF ZNF204...... 135 4.9.1. Methods ...... 135 4.9.2. Results and Discussion ...... 135 4.10. IN VITRO TRANSCRIPTION AND TRANSLATION OF ZNF204 139 4.10.1. Methods ...... 140 4.10.1.1. TNT reaction conditions ...... 140 4.10.1.2. Denaturing gel analysis of translation products ...... 141 4.10.2. Results and Discussion ...... 141 4.11. ZNF 204 LOCUS DISCUSSION...... 144 Chapter 4 Characterisation of ZNF204 Page 93 Chapter 4. Characterisation of ZNF204. 4.1. Introduction Zinc finger (ZNF) proteins are transcription factors, which regulate the transcription of other genes. One of the first gene regulators to be characterized was TFIIIA, a zinc finger protein from Xenopus that regulates the transcription of ribosomal 5S RNA (Miller et al., 1985). The zinc finger transcription factors take their name from their structure, where four key residues are arranged in a tetrahedral formation, and together co-ordinate a zinc atom. The zinc finger domain is composed of 25 to 30 amino acid residues, with two cysteine and two histidine residues arranged at the extremities of the domain (the so called C2H2 domain). It is these residues that co-ordinately bind the zinc atom. Multiple zinc finger domains may be found tandemly arranged in transcription factors. The protein sequence of the full -30 amino acid zinc finger domain is highly conserved: Tyr/Phe-X“Cys-X-Cys-X2,4"Cys-X3-Phe-X5-Leu-X2"Hls-X2,5“HiS“X5 Chapter 4 Characterisation of ZNF204 Page 94 where X is any amino acid. The 30 amino acid repeating unit is referred to as the zinc finger domain on the basis of its tertiary structure forming a loop of 12 amino acids. This loop contains the conserved leucine and phenylalanine residues as well as being rich in basic residues. It is these basic residues such as arginine and lysine that interact with the acidic DNA (see Figure. 4.1). Chapter 4 Characterisation of ZNF204 Page 95 Figure. 4.1. Possible structures of the two histidine two cysteine zinc finger. / Zn (a) (b) Adapted from Latchman1995. Showing the loop of 12 amino acids and (b) showing the proposed structure of an antiparallel beta-pleated sheet and an alpha-helix around the co-ordination sites of the zinc ion. This binding of the zinc atom has been directly confirmed by Extended X-ray absorption fine structure Spectroscopy (EXAFS) (Diakun, et al., 1986). Chapter 4 Characterisation of ZNF204 Page 96 The zinc fingers interact with the major groove of the DNA helix. The finger interacts with five bases of the DNA sequence (or half a helical turn). Successive fingers bind on opposite sides of the helix (Klug and Rhodes 1993). Although first shown in TFIIIA, an RNA polymerase Ill- associated transcription factor, C2H2 fingers have subsequently been identified mainly as transcription factors of RNA polymerase II (Struhl 1989). The association between the presence of a zinc finger motif and transcription factors is so strong that it is possible to probe with the zinc finger domain to isolate new transcription factors. An example of this was the isolation of a zinc finger ZNF141, the gene responsible for Wolf-Hirschhorn syndrome on chromosome 4p. The gene was isolated using a degenerate oligonucleotide of the C2H2 consensus sequence (Tommerup et al., 1993). It is estimated that the human genome contains several hundred zinc finger genes containing the highly conserved C2H2 domain (Bray et a/., 1991). Screening of cosmid and cDNA libraries with zinc finger sequences has led to the identification of over 130 novel zinc finger sequences (Hoovers et a/., 1992). Another example of a disease caused by mutation of a C2H2 zinc finger gene is Greig cephalopolysydactyly syndrome Engelkamp and Heyningen (1996). Wilm’s tumor is also associated with the presence of a defective C2H2 zinc finger on chromosome 11p13. This defective zinc finger causes both Wilm’s tumor (Gessler et a/., 1990), and congenital malformations of the urogenital tract (Pritchard-Jones et a!., 1991). Chapter 4 Characterisation of ZNF204 Page 97 Zinc finger proteins therefore have been shown to play a key role in regulation of gene transcription. Several human diseases result from mutation of a zinc finger protein. The complete characterization of the novel zinc finger locus, which was isolated from chromosome 6p21.3, is described in this Chapter. To characterise the novel ZNF cDNA the complete sequence was first determined and analysed. The sequence obtained was screened for sequence homologies to known gene sequences, to determine motifs and potential open reading frames. The genomic locus was also sequenced, to search for introns. Expression was investigated using northern blot analysis to determine sites of expression. 5’ rapid amplification of cDNA ends was used to search for additional 5’ transcribed sequences. The possibility of translation from the ZNF was investigated by coupled transcription translation analysis. Four approaches were used to search for loci homologous to the ZNF: Computer based database homology search; hybridisation screening of a genomic PAC library; single chromosomal hybrid panel analysis and fluorescent in situ hybridisation. Chapter 4 Characterisation of ZNF204 Page 98 4.2. Complete cDNA sequence 4.2.1. Methods The complete cDNA sequences of the 4 zinc finger-containing cDNAs, 2CS, 3AS2, 5FL and 4CS were determined by primer walking. Each primer walk was sequenced by the Sanger dideoxy-chain termination method, using the Sequenase Version 2.0 sequencing kit (United States Biochemical). Individual sequences were aligned by eye and using the BESTFIT and PILEUP programs of the GOG suite available via telnet at menu.hgmp.mrc.ac.uk. 4.2.2. Results The sequence analysis revealed that the four cDNAs were all from the same locus (Genome nomenclature approved name ZNF204, GenBank accession number AF033199). Two of the four cDNAs were found to be chimeric. Figure. 4.2. summarises the mapping and positional cloning of the cDNAs encoding the ZNF204 locus. The ZNF204 sequence, as deposited in GenBank, is given in Figure. 4.3. Figure 4.2. Mapping, isolation and characterization of ZNF204. (See following page for legend). Z N F 2 0 4 ii TEL CEN Genomic -► pCYPAC2N302f1 PACs pCYPAC2N11g1 10kb EST449 STSG-9945 I 1 cDMA 1 CDNA2 CDNA3 cDNA CDNA4 200bp PCR prob« y I 1 M Consensus p A S' ------— L- 3' PCR products CO CO (a) Fine resolution genomic map around D6S1260 indicating marker positions, with ZNF204 shown in bold. Below, PAC clones used to screen the small intestinal cDNA library; dots indicate the presence of markers (Raha-Chowdhury et al., 1995; Pappas et al., 1995; Volz et a/., 1997; Wallace ef a/., 1998) as determined by PGR or hybridization, (b) Overlapping cDNA clones showing the position of EST449 and STSG-9945; a double slash indicates the junction of ZNF204 cDNA clones with chimeric sequence, (c) Schematic representation of ZNF204 showing the 1.1 kb ORF consensus sequence and the positions of its start ATG (arrow) and polyadenylation consensus sequence (pA). Blocks indicate the positions of individual zinc finger motifs with * representing termination codons and lightning indicating the location of the frame shift mutation. The position of the PGR hybridization probe is indicated above the consensus. Below the consensus, the 3 bars represent the 3 overlapping PGR products amplified from genomic DNA using cDNA primers; cDNA and genomic DNA products were of identical size. Chapter 4 Characterisation of ZNF204 Page 101 K Figure 4.3. ZNF204 sequence, as deposited in GenBank (Accession No. AF033199) LOCUS AF033199 2630 bp mRNA PRI 25-JUN-1998 DEFINITION Homo sapiens C2H2 zinc finger protein pseudogene, mRNA sequence. ACCESSION AF033199 NID g3252864 KEYWORDS SOURCE human. ORGANISM Homo sapiens Eukaryota; Metazoa; Chordata; Vertebrata; Mammalia; Eutheria; Primates; Catarrhini; Hominidae; Homo. REFERENCE 1 (bases 1 to 2630) AUTHORS Partridge,J., Wallace,D.F., Robertson,A., Fox,M.F ., Simons,J.P., Dooley,J.S. and Walker,A.P. TITLE Cloning and molecular characterization of a cross-homologous zinc finger locus ZNF2 04 JOURNAL Genomics 50 (1), 116-118 (1998) MEDLINE 98292504 REFERENCE 2 (bases 1 to 2630) AUTHORS Partridge,J., Wallace,D.F., Robertson,A., Dooley,J.S. and Walker,A.P. TITLE Direct Submission JOURNAL Submitted (06-NOV-1997) Academic Medicine, Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF, UK FEATURES Location/Qualifiers source 1..2630 /organism="Homo sapiens" /db_xref= "taxon: 9606" /chromosome="6" /map="6p21.3 ; between D6S1260 and D6S105" /tissue_type="small intestine" gene 1..2630 /note="expressed processed pseudogene" /gene="C2H2 zinc finger protein" /pseudo Chapter 4 Characterisation of ZNF204 Page 102 BASE COUNT 943 a 474 C 534 g 679 t ORIGIN 1 cggggcatgc tgcttccctt caccttccac catgattgta agtttcctga ggcGtcGGGa 61 ggtgtgcttc tgtacagcct gtggaatgtt accaaagacg ttggaagagg tggctatggg 121 acatcacctg ggagaagtgg aagcaaatgg acactgttca gaagtccata tacagaaaca 181 tacttggaaa aatatagaaa cctggttttg ctagatggga agcttgcagc tggggccaag 241 acatcaagag tagagcagca ggacatttca aaagaagatt aactcaaaga ttagagatgg 301 aagaacttgc aaagagaaag tctgtaccgg aagaaatctg gaaatctaga ggccagttta 361 agaatcagca gctaaacaag gagaataatc tagggcaaga gatagctacc tgcacaaaaa 421 ttcctaccag aaaaagagac atagaatcta atgaatttgt gaaaaatttt actgtaagat 481 caatacttgt tgcagaacag atagatccta tggaagagaa ttgtcataaa tatggtacat 541 gttgaaagat gctcaaacaa aactcagatt taattataca aagaaagtat gatggaaaaa 601 aaaaaacctt gtaaatatag tgaatgtggg agaaccttca gaggccacat cactGttgtt 661 cagcatcaaa taactcattg tggagagaga ccctgtaaat gtactgagtg tagaaaggga 721 tttaatcaga gttcccactt aagaaataat cagagaaaaa ctctttcagg agaaaagcGG 781 tacaaatgca gtgagtgtgg gaaggccttc agttattgct tagttcttaa tcaacacGag 841 agaattcaca gtggagagaa accttatgag ggtactgaat gtggcaagac attcattcag 901 tcgtacatac cttactcagc atcaaagaat tcacacactg gtgagaagcc Gtatacatgt 961 cttgaatgtg gaaggctttt tagtcagaac acacatctta ctctacatca gagaatGcat 1021 actggagaga aaccttatga atgcaatgaa tgtggtaggt cctttagtca gactgcacat 1081 cttactcaac atcaaagaat gtatacagga gaaaaactct atgaatgtaa tgaatgtgag 1141 aaagccttcc atgatcactc agctcttatt caacatcata ttgtccatac tgcagagaaa 1201 ccctatgata tcatgactgg gaaaactttc agttactgtt cagacctcat tcaacatcag 1261 agaatgcaca G tggagagaa accatacaaa tgcaatgaat gtgggaatgc Gtttagtgat 1321 tgttcatccc ttattcagca tcaaagaact cacactggag aagagcctta tgaatgtaag 1381 caatgtggaa aagcctttag cagaagcaca taccttactc aacatcagag aagtcacgca 1441 ggagagaaac agtataaatg caatgaatgt gagaaaactt tcagcctgag ttcattGGtt 1501 acacagcata tgagggttca gactggagaa aaaccctaca aatataatga atatggaaaa 1561 gcttttagtg actgctcagg acattttcag agaactcaca Gtggagagaa gcGGtgtgaa 1621 tgtaatgact gtgggaaacc tttcagtttc tgttcagccc taattcaaca taagagaatt 1681 cataccagaa agaagccctg actgtacctt cataccagta aatgcactga Gtgtggaaaa 1741 gccttcagtg attggttagc acttgttcaa catcagataa Gtcaacactg gagaaaaacG 1801 gtataaatgt actgaatgtg gaaaagcctt cagttggagt acagacGtca aaaatcacca 1861 gaaaactcat actagtgaaa aatcctataa atgtaatgaa tgtagaaagg GGtttagtta 1921 ctgctctggt cttattcaat gtcaggtcat tcatactata gaaaaacGtt atgaatacgg 1981 taaatgtggc aaagccttta ggcagaggac agaccttaaa aaacatcaga aaatgcatac 2041 cgaagagaaa ccctatgaat gtaatgaatg tgggaaagcc tttagccaga gcacatatct 2101 tacaaaacac caaaaaattc atagtgaaga gaaatcaaat atacatactg agtgtgggga 2161 aaccattaga caaaactctt ctttttacaa caataaaacc tcacactgga gagttctctg 2221 aatgccttaa gaatttggtt aatatggaga cccttcccag ggaaacagaa ggaggatcgt 2281 gaaaaccgtt gactacttga atgatcacat ggtttagtgg agagagcatg attctgggtt 2341 ttaaaagtca tggatctcaa tctcagctcc tattactaac tagatctttt actttggggt 2401 aagtcacttc atatctttag gccttaattt cctcatctga aaactggaag gcGtgacttg 2461 acttgttgag cttaagatcc tcaattatta tatttactag gaattcaagt ttctatagat 2521 gtggttcaga attgtgactt atttattgta catcaggtgt gattcacaag tgagcttgta 2581 gtagttatta aggagtcaat aaagatatga tataaaaaaa aaaaaaaaaa Chapter 4 Characterisation of ZNF204 Page 103 The ZNF204 locus contained the primer sequences for two expressed sequence tags (ESTs), STSG-9945 (Whitehead Institute) and EST449 (Pappas et al., 1995), (Figure. 4.2.). As expected, STSG-9945 was located within the 3’ untranslated region, originating from oligo dT priming of the polyA tail. However EST449 was derived from an internal polyA region, and was located close to the 5’ end of the transcript. Neither of the EST sequences contained a zinc finger motif, thus it is their placement within the transcript which allowed them to be classified as part of a zinc finger locus. The ZNF204 transcript contained a total of 18 two cysteine-two histidine (C2H2) zinc finger motifs, the complete sequence of the ZNF204 cDNAs revealed the presence of a premature stop codon upstream of zinc finger motifs 14-18 and a frame shift mutation within zinc finger motif 17 (Figure. 4.2.). Sequence analysis of both genomic and cDNA clones revealed that only 10 of the 18 zinc finger motifs conformed fully with the core consensus (C-X 2 .4 -C-X 3 -F-X5 -L-X2 -H-X2 ,5 H; Klug and Rhodes, 1987). The remaining motifs contain mutations which predict amino acid substitutions at conserved cysteine and histidine residues (see Table. 4.1.). In Drosophila and yeast, mutations of these conserved residues have been shown to eliminate the function of the Kruppel and ARD1 zinc finger proteins, respectively (Blumberg et a!., 1987; Redemann eta!., 1988). Chapter 4 Characterisation of ZNF204 Page 104 Table. 4.1. Multiple mutations in the zinc finger and linker motifs of ZNF204. The consensus motif for the zinc finger loop (Choo and Klug 1994) is shown along with the 13 ZNF204 motifs before the first stop codon. The most highly conserved residues of the motif are highlighted in bold red; mutations are shown in blue (ZNFs 3,12,13). Zinc finger Consensus Sequence; No. CX 2-4 CXXXFXXXXXLXXHX 2-5 H 1 CKYSECGRTFRGHITLVQHQITH 2 CSECGKAFSVCLVLNQHQRIH 3 CLECQRLFSQNTHLTLMQRIH 4 CNECEKAFHDHSALIQHHIVH 5 CNECGNAFSDCSSLIQHQRTH 6 CKQCGKAFSRSTYLTQHQRSH 7 CNDCGIPFSFCSALYQHKRIH 8 CNDCRKPFSRCSALIQHIRIH 9 CT DCGKAFS DWLALVQHQITQH 10 CNECGKAFSQSTYLVKHQKIH 11 CTECGRAFSWSTDLKNHQKTH 12 CNECRKAFSYCSGLYQCQVIH 13 YGKCGKAFRQRT DLKKHQKMH Chapter 4 Characterisation of ZNF204 Page 105 A possible DNA binding sequence for the 10 putative functional zinc finger motifs is postulated in Table. 4.2. It is possible to postulate a DNA binding sequence as the zinc finger binds to the DNA double helix by inserting the a-helical part of the finger into the major groove. The positioning of the three amino acids at positions -1, 3 and 6 of the ZNF consensus control the binding specificity (Choo and Klug 1994; and Nardelli, etal., 1991). It was hoped that the predicted DNA binding sequence of the functional zinc finger motifs could be used to determine the gene this zinc finger regulated, however, the predicted sequence., nCnnT/cnnGA/TnGA/TnTCnTcCnnGnnnnT nnnnnTcnnn is too non specific to enable further electronic or PGR investigations (Table 4.2.). Chapter 4 Characterisation of ZNF204 Page 106 Table. 4.2. Predicted binding sequence of ZNF204 of the 10 putative functional zinc finger motifs (legend on next page). Zinc Finger No. Zinc finger motif amino acid sequence Predicted DNA binding 1 CKYSECGRTFRGHITLVQHQITH n C n 2 CSECGKAFSVCLVLNQHQRIH n Tc n 4 CNECEKAFHDHSALIQHHIVH n G A/T 5 CNECGNAFSDCSSLIQHQRTH n G A/T 6 CKQCGKAFSRSTYLTQHQRSH n T C 7 CNDCGIPFSFCSALYQHKRIH n Tc C 8 CNDCRKPFSRCSALIQHIRIH n n G 9 CTDCGKAFSDWLALVQHQITQH n n n 10 CNECGKAFSQSTYLVKHQKIH n T n 11 CTECGRAFSWSTDLKNHQKTH n Tc n Chapter 4 Characterisation of ZNF204 Page 107 Only the ten zinc fingers upstream of the first stop which conform to the consensus are shown. The amino acids highlighted in blue determine the interaction of that finger with DNA, and allow some prediction of the bound DNA sequence, as shown (Choo and Klug 1994 and Nardelli etal., 1991). Where both capital and lower case letters are used to show the predicted nucleotides bound, capitals indicate the statistically more likely nucleotide. Chapter 4 Characterisation of ZNF204 Page 108 4.2.3. Conclusion from sequence investigation ofZNF204. The detection of multiple mutations of conserved zinc finger residues lead to the conclusion that although ZNF204 was discovered via a cDNA library, thus indicating ZNF204 RNA was produced, it may well be a pseudogene. To investigate this possibility the genomic locus was investigated to look for introns. Chapter 4 Characterisation of ZNF204 Page 109 4.3. Organisation of the genomic locus 4.3.1. Introduction PCR primer pairs were designed to span the ZNF204 cDNA consensus sequence. These were used to investigate genomic DNA to define intronic boundaries. Seven overlapping sets of primers were used to amplify cDNA and genomic DNA templates. Intron containing PCR products would give rise to larger PCR products detectable by agarose gel electrophoresis. Three key overlapping PCR reactions were used to screen ZNF204 (Figure 4.2.). 4.3.2. Methods The PCR reaction was as follows: IX GibcoBRL PCR buffer; 1.5mM MgCb; lOOpM each of dGTP, dCTP, dATP, dTTP; 500ng of each primer set and template DNA or sterile dH20 negative control in a total volume of 25pl. The reaction profile was that of a hot start: 94°C for 2mins then hold and add 2.5U (0.5pl) GibcoBRL Tag polymerase then 94°C for 45 seconds; 55°C for 45 seconds; 72°C for 2mins for 35 cycles followed by a 10°C soak. The PCR products were separated by electrophoresis on a 1% (w/v) agarose gel and visualised by ethidium bromide staining. Chapter 4 Characterisation of ZNF204 Page 110 The genomic sequence was determined by analysis of Hind III subclones of PAC pCYPAC2N11g1 in pBluescript phagemid. Sequence homology analysis was performed using the BLAST program (Altschul etal., 1994). 4.3.3. Results and Discussion Amplification of the genomic ZNF204 locus in 7 overlapping segments using cDNA primers revealed that the 2.7kb ZNF204 is colinear with the genomic locus at the level of resolution of electrophoresis of PCR products. Lack of introns is suggestive evidence for ZNF204 being a pseudogene. However, functional genes can exist without introns an example of which is the histone family. The possibility that a small exon was not detected by amplification was investigated by sequence analysis of the genomic locus. This analysis was performed on Hind III subclones of PAC pCYPAC2N11g1 in pBluescript phagemid. The genomic sequence of the 2.7 kb ZNF204 locus was identical with the cDNA sequence, confirming the lack of introns within this region. The lack of introns, multiple mutations in functional zinc finger motifs, and apparent truncation of the 5’ region together indicate that the locus is may be an expressed processed pseudogene. Chapter 4 Characterisation of ZNF204 Page 111 To search for a “parent” locus and investigate the similarity of ZNF204 to published sequences, homology analysis was performed using the BLAST program (Altschul et al., 1994) on a non-redundant database. BLASTN revealed the highest homology to ZNF135: 66% nucleic acid identity over 117 nucleotides. However reanalysis of the highest 10 homologies using the BESTFIT (GCG) program showed that ZNF184 (second highest homology with BLASTN) had the closest homology to ZNF204 with 68% nucleic acid identity over 1963 nucleotides. ZNF184 is also from the 6p21.3 region and within 500kb of ZNF204 (Goldwurm at a!., 1997; Wallace at a/., 1998). When only the putative 5’ UTR of ZNF204 was searched for homology, ZNF184 was again detected. This homology was however to the predicted coding “spacer” region of ZNF 184 suggesting that the 5’ region of ZNF204 may have been derived from coding sequence. It seems likely that the putative “parent” locus is in 6p21.3, and possibly related to ZNF184. Chapter 4 Characterisation of ZNF204 Page 112 4.4. Northern blot analysis 4.4.1. Introduction To investigate the size of the mRNA transcript(s) homologous with the ZNF204 locus, 2 cDNAs 1 and 2 (spanning the ZNF204 locus) and a short PCR product (STSG-9945) were used as hybridisation probes on a multi tissue northern blo t. By analysing hybridisation on a northern blot it may be possible to ascertain if the overlapping DNA sequence for cDNAs 1,2,3 and 4 represent the entire cDNA sequence of the ZNF204 locus, or only part of it. Northern analysis also enables the simultaneous analysis of mRNA expression and transcript size in different tissues. The multi-tissue northern blot analysed contained human polyA(+) mRNA from spleen, thymus, prostate, testis, ovary, small intestine, colon (non-mucosa) and peripheral blood leukocyte (Clontech Human II #7759-1). 4.4.2. Methods cDNAs 1,2 and 3 (25ng) were radiolabelled by random hexanucleotide priming (Feinberg and Vogelstein, 1983) using dCTP (Amersham). PCR products from STSG-9945 (cDNAI as a template) and from ZNF184 (Goldwurm at a!., 1997), genomic DNA as a template were also radiolabelled. The northern blot Chapter 4 Characterisation of ZNF204 Page 113 and suitable positive and negative control Southern blots were pre-hybridised. The prehybridisation solution was removed from the filters and replaced with 20ml fresh hybridisation solution. The probe was competed as previously described (Chapter 3) and added to give 1 X 10®cpm/ml. The filters were hybridised overnight at 42°C. The blots were briefly washed using 4X 880 then washed twice in 0.5X 880 0.1% 8D8 at 55°0 for 15mins. The filters were rinsed in 4X 880 and placed on BioMAX M8 x-ray film (Kodak) at -70°0 using an intensifying screen to enhance the signal. Chapter 4 Characterisation of ZNF204 Page 114 Figure 4.4. Northern analysis of the ZNF204 locus expression on a polyA(+) multi-tissue northern blot. O CO o 8.5kb 3.2kb a) P-actin b) Legend on next page. Chapter 4 Characterisation of ZNF204 Page 115 a) The hybridisation illustrated is for STSG-9945, it is however representative of all the ZNF204 hybridisations performed (cDNA 1 and 2 and STSG-9945 PCR products). A 3.2kb transcript was visible in all tissues tested: spleen, thymus, prostate, testis, ovary, small intestine, colon and peripheral blood leukocyte. An additional 8.5kb band was also seen in prostate, ovary and testis. The lane loading observed using )9-actin would indicate however, the 8.5kb band could be observable in other tissues if loading was more comparable. b) Hybridisation of the probe yff-actin on the multi-tissue northern blot to check lane loading. The hybridisation pattern suggests near equal lane loading. Chapter 4 Characterisation of ZNF204 Page 116 4.4.4. Results and Discussion ZNF204 hybridization was detected in spleen, thymus, prostate, testis, ovary, small intestine, colon and peripheral blood leukocyte (Figure 4.4.). The polyA(+) mRNA transcript size for ZNF204 is 3.2kb. The total overlapping cDNA sequence obtained for ZNF204 is however 2.7kb. Therefore approximately 500bp of sequence is missing from the overlapping cDNAs sequences. The observation that both ZNF204 and ZNF184 hybridise to a 3.2kb transcript on northern analysis is consistent with the BLAST analysis, suggestive of a common ancestry. The BLAST analysis also suggests that the 5’ region of ZNF204 may be truncated, consistent with the northern analysis. In an attempt to clone this missing 5’ sequence the 5’ Rapid Amplification of cDNA Ends approach was used. On three tissues, prostate, ovary and testis an additional 8.5kb transcript was observed. There are several possible explanations for this larger transcript. An alternative downstream polyadenylation sequence could be used, giving rise to a larger 3' untranslated region. An alternative splicing event could give rise to a larger transcript; however, no evidence has been found for introns in the genomic locus of ZNF204. The 8.5kb band could correspond to mRNA from a homologous locus. Finally, the “polyA(+) mRNA” used for the northern blot has previously been shown to contain nuclear RNA precursors (Clontech personal Chapter 4 Characterisation of ZNF204 Page 117 communication of unpublished data). This therefore seems a likely explanation for the additional 8.5kb bands. Chapter 4 Characterisation of ZNF204 Page 118 4.5. 5’ Rapid Amplification of cDNA Ends (5’ RACE) 4.5.1. Introduction The creation of a cDNA library is often based on the priming of reverse transcriptase with oligo[dT]. This method ensures the production of 3' sequence, however, depending on length and secondary structure there is often loss of the 5’ terminal sequence. ZNF204 has a 3.2kb mRNA transcript on northern analysis. The overlapping cDNA sequence for ZNF204 however only gives a total of 2.7kb. There is therefore potentially 0.5kb of missing 5’ sequence to ZNF204. In an attempt to clone this missing 5’ end of ZNF204, the 5’ rapid amplification of cDNA ends method was used (5’ RACE). 4.5.2. Methods The method of 5’ RACE used was a modification of that of Frohman (1993). 5' RACE or "anchored" PCR allows the isolation of 5’ terminal sequence from mRNA via the utilisation of an anchored PCR primer. Chapter 4 Characterisation of ZNF204 Page 119 Figure 4.5. Diagrammatic overview of 5’ RACE procedure. m RNA (A), A nneal first strand primer, GSP1, to mRNA G SP1 (A ) Copy m R N A into cD N A 3 M- with Si»>Ef,ScRir^'” il RT Degrade RNA with 3-4- R N ase Mix Purify cD N A with G lass MAX Spin Cartridge Tail purified cDNA with 3 C C C C - dCTP and TdT Abridged Anchor Primer PCR amplify dC tailed cDNA SCZZT-JGI ■ IGC> using the Abridged Anchor Pnmer 3 'C C C C ------and nested GSP2 G S P 2 UAP AUAP Ream plify primary P C R prcnJuct □ G I I G - 3 C 3 C C C C - using AUAP. or UAP, and nested G S P nested GSP Chapter 4 Characterisation of ZNF204 Page 120 Poly(A)+ lymphocyte mRNA was used for the first round cDNA synthesis. Total RNA can be used for this procedure but, with the low level of ZNF204 mRNA expected from the northern blot analysis, poly(A)+ was used to enrich for rare cDNA sequences (Figure 4.7). GSP1 was designed 730bp downstream of the 5’ end of the ZNF204 cDNA sequence: (a) to allow hybridisation screening using ZNF204 5’ probes on the 5’ RACE products and (b) to give a PCR product over 500bp, for better recovery using the GlassMAX spin columns. The cDNA product was purified to remove un-incorporated dNTPs and GSP1. The purified product was then incubated with the recombinant enzyme terminal deoxynucleotidyl transferase (rTdT) and dCTP. This enzyme produces a homopolymeric tail on the 3’ termini of the cDNAs. A PCR reaction was then used to amplify the cDNA of interest using an “abridged-anchored” primer to the 3’ homopolymeric tail and a nested PCR primer 3’ to the reverse transcription primer GSP1, GSP2 (GSP2 was designed to have an annealing temperature close to that of the “abridged-anchor” primer, 660bp from the ZNF204 cDNAs 5' termini). A second round of PCR was used to increase the concentration of product produced for cloning using a 3' nested primer to GSP2, GSP3. The primers used for this final stage of amplification were designed to allow the use of the CloneAmp system (GibcoBRL) of PCR cloning by the incorporation of a CAU-tail to the upstream “anchored” primer and a CUA-tail to GSP3 (630bp from ZNF204 cDNA sequence 5’ terminus). Chapter 4 Characterisation of ZNF204 Page 121 4.5.3 Results The PCR amplification of cDNA using GSP2 gave a smear of bands on the ethidium bromide stained agarose gel (all negative controls were negative). PCR amplification using the nested primer GSP3 however failed to give any bands (all negative controls were also negative). No bands were observed after hybridization of these Southern blots, indicating no specific 5’ RACE product had been amplified. The GSP2 and GSP3 primer positioning 660bp and 630bp from the current ZNF204 cDNA 5’ terminus means products of 1.66Kb and 1.63Kb respectively should be amplified from ZNF204 RNA if the transcript was 3.2kb and not 2.7kb as indicated by the northern analysis. The lack of any products therefore probably reflects the technical limitation of this procedure. Chapter 4 Characterisation of ZNF204 Page 122 4.6. Searching for ZNF204 homologues 4.6.1. Somatic cell hybrid panel analysis A somatic cell hybrid panel is made by the fusion of a mouse or hamster cell line with a human cell line. As the fused cells divide, the human chromosomes are systematically lost. The process of human chromosome retention is random, creating chromosomally distinct cell lines some eventually containing a single human chromosome. These “human mono-chromosomal" cells are then isolated from the cell population and cultured into a single human chromosome cell line (Warburton etal., 1990) 4.6.2 Methods As described previously the 2.7kb ZNF204 cDNA is colinear with genomic DNA. Three pairs of PCR primers spanning the ZNF204 locus (Figure 4.2.) were used to screen the human DNA contained within a monochromosomal somatic cell hybrid panel of Povey and co-workers (UK HGMP Resource Centre), in order to search for a putative “parent" locus. Each of the three PCR reactions contained 50ng of each chromosome somatic cell hybrid DNA, a sterile dH20 control and parental rodent cell line controls (50ng human, mouse or hamster genomic DNA). Chapter 4 Characterisation of ZNF204 Page 123 Mouse and hamster genomic DNA templates were included with each primer set because the somatic cell hybrid DNA also contains the genomic DNA from the host cell line. This “host” DNA could potentially also be amplified with the ZNF204 primers. 4.6.3. Results and Discussion All three primer sets only gave the expected size bands in the genomic positive control PCR reaction. The lack of PCR amplification from the chromosome 6 monochromosomal hybrid cell line appears to be due to the cell line used for chromosome 6, MCP6BRA. This cell line (MCP6BRA) has a breakpoint in the short arm of chromosome 6 and is missing the tip of this arm including the region 6p21.3. Hybridisation of PCR reactions with the radiolabeled cDNA 3AS2 was consistent with this interpretation, only showing a positive signal with the genomic control. The lack of an apparent parent locus detected by the monochromosomal somatic cell hybrid panel PCR analysis may be due to several reasons. The primers chosen to screen the panel may have flanked large introns in the “parent” locus, the PCR conditions being optimised for the smaller ZNF204 intron-less copy of the gene. The “parent” locus may be contained in a “gap” in the library panel's chromosomal coverage. The parent may be close to ZNF204, hence missing in the chromosome 6 cell line MCP6BRA. In an attempt to resolve Chapter 4 Characterisation of ZNF204 Page 124 these possibilities, homologues of ZNF204 were sought by genomic library screening and fluorescent in situ hybridisation. Chapter 4 Characterisation of ZNF204 Page 125 4.7. Hybridisation screening of a genomic PAC library The PGR product of STSG-9945 was used as a hybridisation probe to screen human PAG library filter grids (UK HGMP Resource Gentre) in order to search for homologues of ZNF204. The sequence of STSG-9945 is contained within the 3’ UTR of ZNF204 and as a consequence contained within PAG 715 (pGYPAG2N11g1). Three PAGs were found to be positive with STSG-9945: PAG 715 (as expected) as well as 832 (pGYPAG2N 145g14), and 833 (pGYPAG2N102h8). Further PGR analysis of these PAGs showed that PAG 833 contained PY117, a marker approximately llOkb centromeric to D6S1260. When 833 was analysed by PGR using primers spanning ZNF204, it gave the same size PGR products as 715, indicating it was most likely also from chromosome 6p21,3, and probably overlaps the same locus as 715. PAG 832 however did not contain PY117, and failed to give PGR products from the set of primers spanning ZNF204. PAG 832 therefore might contain a possible homologue and/or “parental” locus of ZNF204, and as such was included as a probe in fluorescent in situ hybridisation. Chapter 4 Characterisation of ZNF204 Page 126 4.8. Fluorescentin situ hybridisation (FISH) 4.8.1. Introduction Fluorescent in situ hybridisation (FISH) is a technique that allows the detection of specific nucleic acid sequences on morphologically preserved chromosomes by the use of a fluorescent probe. The position of the fluorescent probe on the metaphase chromosomes gives mapping data on the probe used. In situ hybridisation was originally developed by Pardue and Gall (1969). This was before the advent of molecular cloning and utilised biochemically isolated DMA (e.g. satellite DMA, ribosomal RNA) that was radiolabeled as the probe. There are now non-radioactive methods for labeling DNA for in situ hybridisation such as fluorescence. FISH may use either direct or indirect reporting. In direct reporting, the detectable molecule (reporter) is directly bound to the nucleic acid probe. In the indirect method, the probe contains a reporter molecule, but detection of the probe requires additional incubation with an affinity partner to produce the signal. The presence of the premature stop and frame shift mutations and lack of introns in ZNF204 makes it most likely an expressed processed pseudogene. By using FISH it was possible to screen human metaphase spread chromosomes for a “parent” locus or identify the position of other copies of this locus. Chapter 4 Characterisation of ZNF204 Page 127 4.8.2. Methods 4.8.2.1. cDNA probe The cDNA used as a probe for a “parent” locus was cDNA2 as it was not only the largest of the four cDNAs (2.7kb) it did not contain any chimeric sequence. The use of a 2.7kb cDNA sequence in FISH is close to the limit of resolution (approximately 1.5kb of probe sequence). For this reason an indirect method of labelling was adopted, as it allows the enhancement of signal strength. 4.8.2.2. PAC probes used for FISH PAC 832 was tested as a possible parental locus of ZNF204. PAG 715, which contains cDNA2, was included as a positive control probe for FISH. 4.8.2.3. Method used for FISH FISH was performed using a modification of the method described by Pinkel and co-workers (1986) (Gillet et al., 1993, Banfi et al., 1996). DNA from PAC and cDNA clones was labeled with biotin-14-dATP using a GibcoBRL (Life Technologies) Bionick Kit. The labeled probe was mixed with two competitor DMAs: Cot-1 DNA (GibcoBRL) and polydeoxyadenylic acid-deoxyathymidylic Chapter 4 Characterisation of ZNF204 Page 128 acid (poiy[dA]-[dT] (Sigma). Herring sperm DNA was also added as a blocking agent. The DNA was precipitated using sodium acetate and ethanol and re suspended in hybridisation buffer containing 50% formamide (v/v), 10% dextran sulphate (w/v) and 2X SSPE at pH 7.0. Slides of human metaphase chromosomes were prepared from normal male lymphocyte culture. The normal male lymphocyte culture was synchronised by the addition of thymidine to block DNA synthesis. The block was then removed and 5 bromo-deoxyuridine was allowed to incorporate prior to harvest for use in background chromatin staining. Standard cytogenetic methods were used in the colcemid arrest and hypotonic treatment of the cultured cells prior to their fixing using methanol/acetic acid onto glass slides (Banfi et al., 1996). The slides of metaphase chromosomes can be stored at -70°C for up to 6 months. Metaphase spreads were kindly provided by Dr M. Fox, Galton Laboratory, UCL. The metaphase slides used were prepared for FISH by pre-treating with RNAse (10pg/ml) and proteinase K (0.035|ig/ml) and prefixed with 1% (v/v) formaldehyde (in PBS containing 5% MgCL w/v). The slides were dehydrated in an ethanol series (70% / 90% /100%) before denaturing in 70% formamide, 2 X SSC for 5min at 75°C. The slides were dehydrated again in a series of 4°C ethanol washes (70% / 90% /100%). The probe was then denatured at 75°C for 5mins, added to the slides and allowed to anneal overnight at 37°C. Chapter 4 Characterisation of ZNF204 Page 129 The annealed slides were washed by immersion in 50% (v/v) formamide in 2X SSC at 42°C (three times for Smins), and in 0.1 X SSC, (two times for 2.5min at 42°C). Slides were blocked using 5% (w/v) Marvel non fat milk in 4 X SSC for 20min, and washed in 0.05% (v/v) Tween 20 / 4 X SSC. The fluorescent signal was produced by incubation in a 5pg/ml solution of fluorescein isothiocyanate- avidin conjugate (FITC-avidin; Vector Laboratories) in 5% (w/v) Marvel, 4X SSC, for 20mins. This signal was amplified by adding biotinylated rabbit anti-avidin (5pg/ml in Marvel, 4X SSC; Vector laboratories) for 20mins, followed by a second round of FITC-avidin incubation again for 20mins. Chromosomes were identified by banding made visible by using the fluorescent counter stain diamidino phylindole (DAPI) and propidium iodide which were included in the cover slip mounting medium Vectashield ( Vector laboratories). The slides were investigated using a Nikon Optiphot fluorescence microscope connected to a confocal laser microscopy (MRC600) image capturing system. Digital images were obtained using a cooled CCD (Charge Coupled Device) camera (Photometries) attached to a Zeiss Axiophot fluorescent microscope. The digital image obtained was analysed and stored on an Apple Macintosh using the SmartCapture software from Digital Scientifiic. Chapter 4 Characterisation of ZNF204 Page 130 4.8.3. Results and Discussion Numerous individual chromosomal metaphase spreads were examined using fluorescent microscopy and digital imaging enhancement software. PAG 715 gave a strong signal only on chromosome 6p21.3 indicating that it was not chimeric and confirming the original PGR mapping data in (Figure 4.6.). The ZNF204 probe, cDNA2, had high background fluorescence due to the small size of the probe and the length of exposure time required to observe a signal (Figure 4.7.) cDNA2 did however give small specific signals only on chromosome 6p21.3. To confirm this signal 11 cells were imaged in detail. Of these 64% of the chromatids contained a clear signal on chromosome 6p21.3. A small number of other chromosomes contained a signal. These were however not reproduced in either sister chromatids or other cells examined, making them most likely to be non-specific. However, the resolution of standard FISH is approximately 1Mb. A second “parent” locus within about 1Mb of ZNF204 on chromosome 6p21.3 therefore cannot be ruled out. PAG 832 gave a strong clear band on chromosome 14q in the immunoglobulin region (Figure 4.8.). Further analysis of the insert of PAG 832 using STSG-9945, 3AS2, 2GS, and 5FL as hybridisation probes however failed to give any positive a signals. The non-reproducibility of the original positive identification of PAG Chapter 4 Characterisation of ZNF204 Page 131 832, and the lack of FISH or human monochromosomal somatic cell hybrid panel PCR evidence for a second locus for ZNF204 on chromosome 14, leads to the conclusion that PAC 832 may have been identified originally by cross hybridisation and not by sequence specificity. Chapter 4 Characterisation of ZNF204 Page 132 Figure 4.6. Fluorescent in situ hybridisation of PAC 715 on normal metaphase chromosomes, showing hybridisation to chromosome 6p21.3. Chromosomes were counter stained with 2,6’-diamidino-phenylindole (DAPI) and the signal detected with FITC. Images were captured by cooled CCD (Photometries) from a Zeiss Axioskop fluorescence microscope using Smartcapture software (Digital Scienific). Chapter 4 Characterisation of ZNF204 Page 133 Figure 4.7. Fluorescent in situ hybridisation of oDNA 3AS2 on normal metaphase chromosomes, showing hybridisation to chromosome 6p21.3. Chromosomes were counter stained with 2,6’-diamidino-phenylindole (DAPI) and the signal detected with FITC. Images were captured by cooled CCD (Photometries) from a Zeiss Axioskop fluorescence microscope using Smartcapture software (Digital Scienific). Chapter 4 Characterisation of ZNF204 Page 134 Figure 4.8. Fluorescent in situ hybridisation of PAC 832 on normal metaphase chromosomes, showing hybridisation to chromosome 14. 'Z: s I Chromosomes were counter stained with 2,6’-diamidino-phenylindole (DAPI) and the signal detected with FITC. Images were captured by cooled CCD (Photometries) from a Zeiss Axioskop fluorescence microscope using Smartcaputre software (Digital Scienific). Chapter 4 Characterisation of ZNF204 Page 135 4.9. Zoo blot analysis of ZNF204 To analyse the evolutionary conservation of ZNF204 zinc finger locus, zoo Southern blots of Hind Ill-digested and Tag l-digested DNA were screened with a PCR fragment from ZNF204. The zoo Southern blots contained restriction enzyme digested DNA from human, pig, cow, dog and hamster. 4.9.1. Methods A 0.5kb region of the cDNA2 was amplified and radiolabelled. The PCR probe contained the cDNA sequence of the zinc finger motifs 1, 2, 3, 4 and 5. The hybridisation of the probe was performed at 42°C. The blots were washed at low stringency: once with 4X SSC at room temperature, twice for 20mins in 0.5X SSC, 0.1% SDS at 42°C and once for 20mins in 0.5X SSC, 0.1% SDS at 65°C. 4.9.2. Results and Discussion The ZNF204 probe was shown to be conserved across species. The Hind III digested zoo blot displayed a ladder of hybridising bands increasing uniformly in size by approximately 80bp in all species tested (human, cow, pig, and hamster; Figure 4.9.). This ladder of bands was absent from the Tag I digested zoo blot where only a smear was observed (data not shown). Chapter 4 Characterisation of ZNF204 Page 136 The step-wise increase in the size of the Hind III fragments corresponds to the distance between the phenylalanine residues of adjoining zinc finger domains of 84bp. The DNA-encoding sequence Lys-Ala-Phe occurs commonly in zinc fingers (where phenylalanine is part of the conserved zinc finger consensus), and gives rise in some cases to a Hind III site. A computer analysis of the 10 zinc finger genes with highest homology to ZNF204 also revealed that 15 out of the 20 Hind III sites present were positioned at the phenylalanine residue within the conserved sequence. Two zinc finger genes contained 5 Hind III sites, all at the phenylalanine residue of the consensus, giving rise to a ladder of Hind III fragments as detected by hybridisation in Figure 4.9. The conservation of such zinc finger probes has been previously reported, with one or several cross hybridizing bands detected on Southern blots of human DNA, and on zoo blots (Shannon et al., 1996; Villa et al., 1996; Chowdhury et al., 1987). This experiment demonstrated a remarkable regular ZNF204 hybridization pattern conserved across all species tested, which may be explained in terms of the periodicy of Hind III sites within the consensus sequence of multiple zinc finger proteins. At least 35 individual bands can be resolved in human and bovine DNA, suggesting that the zinc finger genes, and therefore proteins, of these species can in theory contain at least 35 motifs. Indeed Xfin protein of Xenopus, contains 37 C2H2 zinc finger motifs, consistent with these results (Ruiz-i-Altaba et al., 1987). The cross-hybridization of ZNF204 Chapter 4 Characterisation of ZNF204 Page 137 to multiple zinc finger loci across species may provide a tool for the analysis of C2H2 zinc fingers in different species throughout evolution. Chapter 4 Characterisation of ZNF204 Page 138 Figure 4.9. ZNF204 cross hybridizes with multiple zinc finger loci. 0) (0 CL Ü 3.0 1.6 1.0 _05_ 0.34 Southern blot of a Hind III digested DNA from different species probed with a 0.5kb fragment of ZNF204, with size in kb shown on right. A ladder of bands increasing uniformly by approximately 80bp was seen in all lanes reflecting cross-hybridization of ZNF204 to multiple zinc finger loci across species. Chapter 4 Characterisation of ZNF204 Page 139 4.10. In vitro transcription and translation of ZNF204 As described earlier the sequence investigation of cDNA ZNF204 showed ATG and inframe termination codons upstream of the main ZNF open reading frame, as well as a “premature” stop codon upstream of zinc finger motifs 14-18, and a frame shift mutation within zinc finger motif 14. To investigate whether the ZNF open reading frame may be translated, and whether these mutations may result in a truncated protein, cDNA2 was analysed using a cDNA-directed protein synthesis reaction, utilising a coupled eukaryotic in vitro transcription-translation system. The 2.7kb of cDNA2 contains an ATG start codon that conforms to the Kozak consensus (Kozak 1997), followed by all 18 zinc finger motifs which comprise the 1.1 kb open reading frame, the 3’ untranslated region, a polyadenylation sequence and a polyA tail, making it the most suitable of all the cDNA to use in this analysis. The coupled rabbit reticulocyte lysate system; used for the in vitro transcription was an adaptation of the plasmid cDNA-directed protein synthesis coupled transcription-translation system described by Jackson and co-workers (1992) namely the TNT (Transcription ‘n’ Translation) coupled reticulocyte lysate system (Promega). This is a single tube system that enables the translcription of gene sequences cloned downstream of SP6, T3 or T7 RNA polymerase promoters, and translation by using rabbit reticulocyte lysate. Chapter 4 Characterisation of ZNF204 Page 140 4.10.1. Methods Firstly PCR of cDNA2 plasmid was used to determine which of the vector (pBluescript) RNA polymerase promoters (T3, SP6) was suitable for in vitro transcription. The PCR results showed that the promoter T3 was upstream of the cDNA2 ATG start codon. The RNA polymerase T3 was therefore used to transcribe cDNA2. Two reactions were performed: (a) using the standard conditions and, (b) with the addition of 500)iM ZnS 0 4 . Zinc is structurally important in the zinc finger protein, and its addition has been shown to be beneficial in the in vitro translation of zinc finger proteins (Choo and Klug, 1994) A T3 luciferase positive control plasmid (Promega) and a dH20 negative control were also tested. A second group of locally available controls was also included (kindly provided by Dr. P. DeZoysa). These plasmids use the RNA polymerase T7: pKAL and pGAL (deZoysa at a!., 1998). 4.10.1.1. TNT reaction conditions Stock TNT rabbit lysate stored at -70°C was quickly thawed on ice and immediately aliquoted (5pl each) into reaction tubes containing: IX TNT reaction buffer, 1,000 Ci/mmol ^^S-methionine, ImM amino acid mixture (minus methionine), ID RNA polymerase (T3 or T7) and 1U RNase Guard (GibcoBRL) Chapter 4 Characterisation of ZNF204 Page 141 in a total volume of 4|il. 1|il (approximately 200ng) QiaQuick column-purified plasmid DNA was added to the TNT rabbit lysate mixture to start the reaction. The tube was incubated for 2 hours at 30°C then placed on ice. 4.10.1.2. Denaturing gel analysis of translation products 2pi aliquots of the TNT reactions and 2pi protein molecular weight markers ([methyl-^"^C] methylated NEC-811 (NEN)) were separated on two 12% SDS- polyacrylamide gels containing a 4% acrylamide stacking gel at 60V for 5 hours. The two identically loaded polyacrylamide gels were washed and vacuum dried at 80°C for 2 hours, and exposed to Kodak BioMAX XR x-ray film at -70°C overnight, and developed. 4.10.2. Results and Discussion The results of the TNT analysis are presented in Figure. 4.10. The T3 luciferase positive control gave a strong signal at the correct position 61kDa. The two negative dH20 controls gave no products. However no protein bands were observed in either the standard or zinc addition lanes of cDNA2. The predicted protein pi and size for cDNA2 using the e-mail SwissProt program was 8.83 pi / 42 kDa. Chapter 4 Characterisation of ZNF204 Page 142 The lack of observable translation could be due to a weak signal from the protein product, as the predicted protein sequence for cDNA2 contains only 4 methionine residues, which could be labelled by The T7 pKAL control however was detected using the same protocol in later experiments, the protein product of this plasmid also contains 4 methionine residues. Another possible reason for the lack of signal is the need for optimisation in the addition of ZnS 0 4 . The concentration of ZnS 0 4 was previously optimized by Choo and Klug 1994 in in vitro transcription. A weaker signal was observed from the non ZnS 0 4 supplemented in vitro translations. However, the presence of start and inframe stop codons up stream of the putative start ATG of the 1.1 kb open reading frame may be the major factor preventing the in vitro translation of ZNF204 (Kozak, 1996). Chapter 4 Characterisation of ZNF204 Page 143 Figure 4.10. Results of the TNT analysis of ZNF204. LANE 1 2 3 4 5 6 97kDa 69kDa 61 kDa 53kDa 22kDa 12kDa Lane 1, cDNA2 / T3; Lane 2, cDNA2 / T3 plus 500pM ZnS 0 4 : Lane 3 methylated molecular weight marker NEC811; Lane 4, Positive control luciferase / T3; Lane 5, Empty; Lane 6, Negative control dhl20 / T3. Although translation of the positive control was demonstrated, no signal was detected for cDNA2 with or without ZnS 0 4 supplementation. Chapter 4 Characterisation of ZNF204 Page 144 4.11. ZNF 204 locus discussion The identification of 4 different ZNF204 cDNAs from a small intestinal cDNA library confirms that the ZNF204 locus is transcriptionally active. Analysis of the complete cDNA sequence for ZNF204 shows homology to the C2H2 class of zinc finger transcription factors. The amino acid structural motif of zinc finger transcription factors is C-X 2 ,4 -C-X3 -F-X5-L-X2 -FI-X2 ,5 -H; (Klug and Rhodes, 1987) and is highly conserved as is this intervening “linker” sequence TGEKPY. The complete sequence of the ZNF204 consensus cDNA reveals the presence of a premature stop codon upstream of zinc finger motifs 14-18 and a frameshift mutation within zinc finger motif 17 (Figure. 4.2). Sequence analysis of both genomic and cDNA clones reveals that 10 of the 18 zinc finger motifs conform fully to the highly conserved zinc finger consensus Klug and Rhodes, 1987). The remaining 8 motifs however contain mutations, which predict amino acid substitutions at the conserved cysteine and histidine residues that bind the zinc atom (Table 4.1). In drosophila and yeast, mutations at these conserved residues have been shown to eliminate the function of the Kruppel and ARD1 zinc finger proteins, respectively (Blumberg et a i, 1987; Redemann et a i, 1988). The amplification of the genomic ZNF204 locus in 3 overlapping segments using cDNA primers revealed that the 2.7kb of the ZNF204 locus contains no intronic sequence. Chapter 4 Characterisation of ZNF204 Page 145 Together, these data suggest that this locus is most likely an expressed (known to make RNA as isolated from a cDNA library), processed (no introns detected), pseudogene. Such loci are thought to arise by integration into chromosomes of a natural complementary DNA sequence produced by reverse transcriptase from an RNA transcript. The processed appearance of this pseudogene is consistent with a reverse transcribed mRNA intermediate rather than a gene duplication event. Consistent with this cDNA being non functional is the presence of potential ATG start codons and in frame termination codons within the putative 5’ UTR, often seen in non functional loci (Kozak, 1996). Processed pseudogenes, by the nature of their retrotransposition, typically have the remnants of their polyA tails represented in genomic DNA at their 3’ termini. The first polyadenylation signal in the 3' genomic region of ZNF204 has no detectable remnant of a polyA tail. However, there is a second putative polyadenylation signal 181 bp down stream in genomic DNA that appears to have the remains of a polyA tail. The second polyadenylation signal is not the classical AATAAA but ATAAAA. This non classical polyadenylation signal sequence has been shown to retain a reduced functionality in vitro (Sheets et al., 1990). The parent mRNA of ZNF204 may have therefore been alternatively polyadenlylated and 176bp longer than the longest ZNF204 cDNA detected. The wide band observed on the northern blot at 3.2kb could be consistent with two transcripts 176bp apart. Chapter 4 Characterisation of ZNF204 Page 146 Fluorescent in situ hybridisation and monochromosomal somatic cell hybrid panel analysis only detected ZNF204 on chromosome 6p21.3. A possible explanation for this apparent lack of a “parent” locus could be the technical limitations of these methods in the detection of an intron-containing homologue, However, several other explanations are possible. The original locus may no longer be present in the human genome. The resolution of these methods (approximately 1Mb for FISH) is such that the “parent” locus may also be in 6p21.3. This chromosomal band does indeed appear to contain a cluster of zinc finger genes, six having been isolated from the region 6p21.3: SRE-BP, ZNF165, ZNF193, ZNF192, ZNF184, (Goldwurm et a i, 1997; Lee et al., 1997; Tirosvoutis et al., 1995). The results of the homology analysis performed using the BLASTN program (Altschul et al., 1994) revealed the highest homology to ZNF135: 66% nucleic acid identity over 117 nucleotides. However reanalysis of the highest 10 homologies using the BESTFIT (GCG) program showed that ZNF184 (second highest homology with BLASTN) had the closest homology to ZNF204 with 68% nucleic acid identity over 1963 nucleotides. The zinc finger protein ZNF184 is also from the 6p21.3 region, and has been mapped to within SOOkb of ZNF204 (Goldwurm et al., 1997; Wallace et al 1998). When only the putative 5' UTR of ZNF204 was searched for homology, ZNF184 was again detected. This homology was however to the predicted coding “spacer” domain of ZNF184 suggesting that the 5’ region of ZNF204 may have been derived from coding Chapter 4 Characterisation of ZNF204 Page 147 sequence. Taking the mapping and homology analysis together, it seems likely that the putative "parent" locus is in 6p21.3, and possibly related to ZNF184. Indeed, ZNF204 and ZNF184 both hybridise to a 3.2kb transcript on northern analysis, which is consistent if they shared their ancestry. In conclusion, the ZNF204 locus on chromosome 6p21.3 is most likely an expressed processed pseudogene that arose by a local retrotransposition of a gene within the 6p21.3 ZNF184 cluster. Its conservation across species suggests that it could be used to isolate novel ZNF genes from other species. Chapter 5 Ferric reductase functional studies Page 148 Chapter 5 Ferric reductase functional studies Chapter 5 Ferric reductase functional studies Page 149 Chapter 5. Ferric reductase functional studies: index. 5.1. INTRODUCTION...... 150 5.2. PATIENTS AND METHODS...... 153 5.2.1. Patients ...... 153 5.2.2. Mutation analysis ...... 153 5.2.3. Isolation of peripheral blood monocyctes and differentiation to macrophages ...... 154 5.2.4. Ferric reductase assay ...... 158 5.3. RESULTS...... 159 5.4. DISCUSSION...... 164 Chapter 5 Ferric reductase functional studies Page 150 Chapter 5. Ferric Reductase Functional Studies 5.1. Introduction Iron is of vital importance in all living organisms, being an essential co factor of enzymes involved in respiration, cell replication and electron shuttling. The homeostasis of iron is maintained primarily by its absorption in the duodenum and the proximal region of the jejunum (McCance and Widdowson, 1937). Normally the daily loss of iron (1-2mg) is matched by absorption from the diet. This fine balance between iron excretion and absorption is lost in GH. This autosomal recessive disease leads to increased absorption of iron from the diet despite increasing body iron stores. Iron is deposited in the parenchymal tissue of the liver, pancreas, heart and other tissues; if untreated, the iron overload eventually leads to tissue damage. The prevalence of GH in Europeans is approximately 1 in 300, which is higher than the combined prevalences of cystic fibrosis, phenylketonuria and Duchenne muscular dystrophy (Bothwell et al., 1995; Dooley et al., 1997; Witte et al., 1996). Abnormalities of iron metabolism in the intestine, liver and macrophages continue to be implicated in the pathogenesis of GH (Powell et al., 1990; Cox 1990; Olynyk 1995). In the haemochromatotic macrophage, despite the elevation of serum iron and transferrin saturation, concentrations of iron are minimal until late in the disease (Yam et al., 1968). This is in Chapter 5 Ferric reductase functional studies Page 151 direct contrast to transfusional siderosis where there is progressive accumulation of iron by the macrophage. This difference exists despite similar increases in serum ferritin levels (Saab et ai, 1986). Cultured GH monocytes also have elevated levels of ferritin release, and abnormal kinetics of iron release, compared to control monocytes (Flanagan et ai, 1989; Fillet et ai, 1989; Moura et ai, 1998). The uptake of “free” iron provided in the form of ferric citrate by mononuclear cells has been established (Parmley et ai, 1981; Bonkowsky et ai, 1979). Both “free” iron uptake and ferric reductase activity have been shown to be increased in duodenal biopsies from haemochromatotics as compared to controls (Cox and Peters, 1978; Raja et ai, 1996). More recent work has confirmed these findings in Hfe knockout mice (Simpson et ai, 2003(a); Simpson et ai, 2003(b)). Ferric reductase activity has been described in both prokaryotic and eukaryotic cells, including E.coli, S.cerevisiae, mouse, rat, rabbit and human duodenum, rat liver endosomes, rabbit reticulocytes and various cultured cell lines. Its conservation across species indicates a functional role. The S. cerevisiae gene for ferric reductase {FRE1) has been cloned and sequenced (Dancis et ai, 1994; Dancis et ai, 1992) . The ferric iron uptake mechanism in the yeast, S. cerevisiae, utilises the ferric reductase enzyme to reduce Fe(lll) before absorption. The ferrous Fe(ll) iron is Chapters Ferric reductase functional studies Page 152 transported through the membrane, and is thought to be re-oxidised back to Fe(lll) as it passes through the membrane. The human homologue has now been cloned. Partial purification of human ferric reductase from both human Hutu 80 duodenal adenocarcinoma cells and human duodenal microvillus membranes has shown that the activity is NADH-dependent and the protein is membrane bound (Riedel etal., 1995). In this chapter, ferric reductase activity is demonstrated in the K562 human erythroleukaemic cell line and primary cultures of human leukocytes, monocytes and macrophages. Ferric reductase activity is investigated in cells from GH patients compared to controls, under conditions of high “free” iron and during monocyte-macrophage differentiation. Chapter 5 Ferric reductase functional studies Page 153 5.2. PATIENTS AND METHODS 5.2.7. Patients Ten male patients were investigated: five GH and five control patients with no family history of iron disorders. The diagnosis of GH was made from clinical and biochemical data and confirmed by a hepatic iron index >1.9 and / or removal of >5g iron during initial phlebotomies (Dooley et a!., 1997; Won/vood et a!., 1997; Summers et a!., 1990). The groups were age matched: GH, 49+17.4 y, control, 55±5.3 y, (meanlS.D.). 5.2.2. Mutation analysis The Cys282Tyr and His63Asp mutations of the haemochromatosis gene HFE were determined by PCR and restriction enzyme digestion (Worwood et al., 1997). All haemochromatosis patients studied were homozygous for the Cys282Tyr mutation and negative for His63Asp. Two controls carried a single copy of His63Asp; all controls were negative for Cys282Tyr. Chapter 5 Ferric reductase functional studies Page 154 5.2.3. Isolation of peripheral blood monocytes and differentiation to macrophages Peripheral blood was collected into 4X 10ml heparinized tubes. White blood cells were isolated from peripheral blood as mononuclear cells by density gradient centrifugation using Lymphoprep™ (Nycomed) medium according to the manufacturers protocol. Monocytes were then separated from white blood cell mass using their adherence to plastic surfaces. This was achieved by incubating the cells in plastic tissue culture dish for 1 hour. After washing the dish, adherent monocytes were released by scraping with 4°C PBS. Monocytes were then counted, and dispensed into wells of a 96-well microtitre plate. Each well contained 0.2 x 10® cells in a volume of 200pl. The cells were incubated at 37°C, 5% CO 2 for 14 days to fully differentiate into macrophages. Growth medium containing 20% human AB negative serum (Sigma), 100 units/ml penicillin and lOOpg/ml streptomycin, 2mM L-glutamine in RPMI 1640 (ICN) was replaced on day 7 (Tormey et al., 1997) Cell numbers were determined both using a haemocytometer and by fluorescence activated cell sorter (FACs) analysis (Coulter EpicXL). Cell viability was confirmed by trypan blue exclusion (>95%). The purity of the primary cell cultures was assessed by both morphological criteria (Leishmanns stain) and CD14 FACs analysis; an example can be seen in Figure 5.1. It shows before and after separation via plastic adherence. Chapter 5 Ferric reductase functional studies Page 155 Typical purities were 80% for monocytes and 9 5 % for macrophages the other 5-20 % being contamination with neutrophils, eosinophils, basophils and lymphocytes. The clonal erythroleukaemic cell line K 562 was included in all experiments as an internal control. Post experimental macrophage numbers were calculated from DNA concentration using the assumption that each cell contains 6pg of DNA. DNA concentrations were measured spectrophotometrically at 600nm using the Burton assay methodology (Burton 1956). A standard curve of calf thymus DNA (Sigma) was run in parallel to all macrophage DNA. The cell number was then used to calculate ferric reductase activity as nmol Fe/min/IO^cells. Cells were cultured at 37°C either in conditions of “normalised iron” (24pM iron, transferrin saturation 30%), or in normalised medium supplemented with “lOOpM ferric citrate”, to reflect the elevated in vivo concentrations of serum iron and “free” iron of a heavily iron loaded haemochromatotic (Batey et al., 1980; Gutteridge et al., 1985). The “lOOpM ferric citrate” solution was freshly prepared as a mixture of lOOpM ferric chloride to 150|ilVI sodium citrate, as previously described (Neumannova et al., 1995). Cell cultures supplemented with ferric citrate were protected from light; fresh medium was substituted after 7 days. In addition to the protection from light and chelation by citrate, stability of iron (III) was also conferred by the RPMI 1640 culture medium itself, which contains many other chelating agents at high concentration. These include amino acids, sugars, vitamins, bicarbonate and phosphate, which Chapter 5 Ferric reductase functional studies Page 156 also prevent the hydrolytic polymerization of iron (Richardson and Baker, 1991). The amino acids and glucose alone provide an additional 150-fold molar excess of chelating agent to iron (Richardson and Baker, 1991). Chapter 5 Ferric reductase functional studies Page 157 Figure 5.1. CD14 FACS analysis showing macrophage purity before and after plastic adherence purification. i a) Before plastic adherence purification 1 1000 FS LOG b) After L 3 - plastic adherence purification . 1 1000 FS LOG FS (forward scatter) log is proportional to the size and S3 (side scatter) log is proportional to granularity, a) White blood cells before plastic adherence purification. The monocytes (circled) are contaminated with vesicles, debris and other cell types, which are largely removed after plastic adherence, b). Chapter 5 Ferric reductase functional studies Page 158 5.2.4. Ferric reductase assay I Cells were harvested, rinsed, then incubated at a concentration of 2 X10® I cells/ml in 200|il oxygenated physiological buffer (125mM NaCI, 3.5mM ! 1 KOI, 1mM CaCl2 , 10mM MgS0 4 , 10mM D-glucose in 16mM Hepes/NaOH, pH 7.4) in 96-well tissue culture plates at 37°C. The I reaction was started by the addition of lOOpM Fe(lll), as a ferric chelate of nitrilotriacetate (NTA) in a 1:2 ratio (FeiNTA), and ferrozine (ImM). The reaction was performed at 37°C. The rate of reaction was measured spectrophotometrically at 562nm to detect the reduction of the yellow Fe(lll) to the purple coloured stable Fe(ll)-ferrozine complex (Raja et a!., 1996). All controls including analysis of the K562 cell line were performed in parallel with every primary cell culture monocyte and macrophage assay to confirm uniformity of results. Chapter 5 Ferric reductase functional studies Page 159 5.3. RESULTS Preliminary experiments investigated the characteristics of ferric reductase activity of the clonal control K562 erythroleukaemic cell line. In each experiment the rate of ferric reductase activity was determined by linear regression analysis of at least three time courses; each individual time point was performed in duplicate. All time courses were linear for at least 60 minutes (Figures 5.2. and 5.3.). After mild trypsinisation, ferric reductase activity was reduced to 38.7% of control (Table 5.1.). Heating the cells to 65°C for 5min prior to assay reduced activity to 26.7% of control. When the assay was performed at 10°C, 17% of control activity was observed. These observations are consistent with assay of a membrane bound protein. When K562 and macrophage cell-free supernatant was taken for reductase activity at the end of the incubation period, only background levels of reductase activity were detected (Table 5.1.). This indicated that the activity was not due to a soluble, released factor. These observations indicate that ferric reductase is a membrane bound enzyme. Investigation of primary cultures of lymphocytes, monocytes and macrophages demonstrated ferric reductase activity in these blood- Chapter 5 Ferric reductase functional studies Page 160 derived cells (Table 5.2.). No significant difference (p>0.05) was observed between GH (Cys282Tyr homozygous) and control (Cys282Tyr negative) in either lymphocyte, monocyte or macrophage preparations. Figure 5.2. Monocyte ferric reductase activity: GH versus controls. 0.040 0.035 - 0.030 - I 0.025 - CN Î8 m 0.020 - § I 0.015 I < 0.010 - 0.005 0.000 H I “ T ~ 10 20 30 40 50 60 70 Time in Mins Linear regression analysis of ferric reductase data ( • = GH, ■= control). The error bar = 1 S.D. There was no significant difference between GH and control groups (n=5; p>0.05 unpaired student t-test). Chapter 5 Ferric reductase functional studies Page 161 Figure 5.3. Macrophage ferric reductase activity: GH versus controls. 0.10 0.08 - 0.06 - Î8 < 0.02 - 0.00 - 0 10 20 30 40 50 60 70 Time in Mins Linear regression analysis of ferric reductase data ( • = GH, ■= control), The error bar = 1 S.D. There was no significant difference GH and control groups (n=5; p>0.05 unpaired student t-test). Chapter 5 Ferric reductase functional studies Page 162 Table 5.1. Human ferric reductase activity has the characteristics of a membrane bound enzyme. Cell type Treatment Ferric reductase activity (%)^ K562 none 100 K562 0.1 mg/ml trypsin, 37'^C, 60min 38.7 + 1.5** K562 65*^0, 5min 26.7 ± 1.5** K562 assay performed at 10*^0 17.0 + 1.7** K562 supernatant activity^ 0.33 ± 0.58** macrophage supernatant activity^ 0.02 ± 0.02** The cells were subjected to various treatments and then ferric reductase activity was measured and expressed as a percentage of K562 control activity at 37°C (mean ± standard deviation; n = 3). For the control K562 cells, the ferric reductase activity was 6.93nmol Fe/min/10® cells, where 1 pM ferric iron gives an absorbance of -0.03 at 562nm. 2, the reaction was performed in the absence of ferrozine, cells were removed by centrifugation, and supernatant activity determined after the addition of ferrozine. Activities are compared with control: ** p < 0.01 (Student’s t- test). Chapter 5 Ferric reductase functional studies Page 163 Table 5.2. Ferric reductase activities. Activities are shown for lymphocytes, monocytes and macrophages from classical C282Y homozygous GH patients and Cys282Tyr negative control patients. Ferric reductase activity (nmol Fe/min/10®cells) Cell type Fe Mean S.D. n P lymphocytes GH N 4.37 3.13 3 >0.05 lymphocytes control N 3.98 2.51 3 monocytes GH N 4.38 1.71 3 >0.05 monocyte control N 3.75 0.71 3 macrophages GH N 43.3 5.13 4 >0.05 macrophages control N 31.3 8.67 4 cell line K562 N 6.93 0.88 3 >0.05 cell line K562 100pM 7.87 0.77 3 macrophages GH 100pM 10.0 4.87 5 >0.05 macrophages control 100pM 9.2 9.60 5 Ferric reductase activities for GH and control cultured in N (normalised iron medium); 100pM, iron loaded (additional 100pM ferric citrate in normalized iron medium, p = (Students t-test). Chapter 5 Ferric reductase functional studies Page 164 5.4. DISCUSSION The ferric reductase activity of human K562 cells was demonstrated to have the characteristics of a membrane bound enzyme and was not attributable to the secretion of reducing factors. There was no significant difference in the reductase activity between GH and controls for leukocyte, monocyte or macrophage preparations. This was observed under both normalised iron conditions and in the presence of 100pM ferric citrate. Ferric reductase activity of differentiated macrophages was -900% that of monocyte activity (p<0.05). This increase most likely reflects the co-ordinated upregulation of proteins of iron metabolism during the transition into macrophages (Cottrell and Jones, 1979). Macrophage ferric reductase activity was decreased when cultured in lOOpM ferric citrate compared to that in normalised iron. From these results it would appear that macrophage ferric reductase activity may be determined by external “free” iron concentrations during macrophage differentiation. Macrophage ferric reductase activity was down regulated by increased “free” iron concentration. This control of ferric reductase activity could contribute to the low levels of iron observed in the haemochromatotic macrophage. As haemochromatotic macrophages differentiate in high “free” iron conditions in vivo, their ferric reductase activities may be decreased according to the amount of free iron present. Future studies could Chapter 5 Ferric reductase functional studies Page 165 address this issue by isolation of differentiated macrophages from GH and control human subjects, to determine the activity of the in vivo differentiated cells. The clonal reference cell line K562 did not show modulation of ferric reductase activity by iron. The K562 cell line originates from an erythroid cell clone derived from a patient with myeloid leukaemia in acute blast crisis (Andersson and Gahmberg 1981). The lack of modulation of K562 ferric reductase activity by iron may reflect either the erythroid lineage or changes in the clonal expansion of this cell line. The identification of the human ferric reductase gene will ultimately establish whether it is transcriptionally repressed by iron, as in S. cerevisiae. Future studies will elucidate whether ferric reductase plays a role in the apparent iron deficiency of macrophages. Chapter 6 Conclusions and Perspectives Page 166 Chapter 6 Conclusions and Perspectives Chapter 6 Conclusions and Perspectives Page 167 Chapter 6. Conclusions and Perspectives This thesis reports two approaches initially directed towards the mapping and identification of the chromosome 6-linked haemochromatosis gene. At the beginning of this thesis, twenty years had elapsed since initial observations of association of haemochromatosis with particular HLA alleles. A positional cloning approach was used to identify potential candidate genes from the chromosome 6 gene region. A functional cloning approach was investigated by analysis of the first reported potential index of disease in vitro. Before the discovery of the HFE gene, chromosome 6-linked haemochromatosis was investigated first by analyses of HLA serotypes and then later by analysis of DNA markers from the HLA region. The availability of highly polymorphic DNA microsatellite markers was an important step in the mapping and the ultimate identification of HFE. Four polymorphic microsatellite markers spanning the 6p21.3 'founder haplotype' were originally used in this thesis to characterise the patient and control groups. Some of the patients were homozygous for several of these markers, providing strong indirect evidence for chromosome 6- linked haemochromatosis. Later HFE mutation analysis confirmed that most of the patients were homozygous for the Cys282Tyr mutation and that none of the control group had disease related genotypes. Because the link between HFE and iron metabolism was not apparent Chapter 6 Conclusions and Perspectives Page 168 immediately, it remained a 'candidate' gene for some time. However, HFE mutation analysis is now widely used for confirmation of the diagnosis of haemochromatosis and in family screening and has reduced the need for liver biopsy. Due to the pressure of commercial gene hunters also searching for the haemochromatosis gene, the mapping information and clones available at that time were used in an attempt to identify candidate genes from around the apparent peak of linkage disequilibrium (D6S1260). This was done by direct hybridisation of genomic PAC clones to a human intestinal cDNA library. This methodology proved effective by the identification of four single copy cDNAs for the zinc finger locus ZNF204. The gene locus was shown to be intronless and no 5' end could be identified by genomic analysis or by 5'-RACE. Sequence analysis indicated historic mutations that would have inactivated individual zinc finger protein fingers. In vitro coupled transcription and translation studies were unable to demonstrate translation of ZNF204. The locus was therefore considered likely to be an expressed processed pseudogene and unrelated to haemochromatosis. If the parent locus could be discovered, then the point at which this pseudogene was created could be estimated by looking at the mutation rate compared to the original sequence. However, as yet computer Chapter 6 Conclusions and Perspectives Page 169 database screening, somatic cell hybrid panel analysis and FISH has not alluded to a parent locus for ZNF204. Zinc finger proteins have a potential future role in the manipulation of gene expression via targeted promoter méthylation. This targeting can be achieved by creating zinc finger proteins that bind to gene-specific promoter DNA sequences (Latchman, 1995). The fusion of these site- specific zinc finger proteins to methyltransferase results in the targeted méthylation of CpG islands upstream of the zinc finger binding site (Xu and Bestor,1997). An advantage of using this methodology for the selective silencing of genes is that even after short exposure, the cells own methylating pathways will maintain this new pattern of méthylation. The action of other regulatory methods such as antisense nucleic acids, ribozymes and other analogous agents all dissipate after time and require constant exposure. The future therapeutic role of zinc finger proteins is still waiting to be fully exploited. The study of ferric reductase activity in macrophages was conducted in an attempt to identify an in vitro index of the disease. This would allow investigation of potential candidate haemochromatosis genes by functional complementation. Previous studies on duodenal biopsies had shown increased ferric reductase activity in GH compared with controls (Raja et al., 1996). In GH, several lines of evidence indicate that macrophages (like duodenal enterocytes) remain paradoxically iron poor Chapter 6 Conclusions and Perspectives Page 170 despite iron overload (Powell et al., 1990; Cox 1990; Olynyk 1995). Macrophages were therefore considered likely to express any altered functional Index. Ferric reductase activity of K562 cells was demonstrated to have properties consistent with a membrane bound protein. In contrast to earlier criticisms, the activity was not attributable to a secreted reducing factor. Ferric reductase activity was demonstrated for the first time In white blood cells (leukocyte, monocyte and macrophage preparations), but there was no significant difference between GH and control cultures. Macrophage ferric reductase activity was decreased following culture under Iron loaded conditions, although the mechanism of this Iron response was not known. The gene Dcytb has been cloned since these studies were completed and confirmed as the mammalian plasma membrane ferric reductase that catalyzes the reduction of ferric to ferrous Iron as part of the process of Iron absorption (McKle et a/., 2000). The cloning was via a subtractive cloning strategy designed to Identify Intestinal genes Involved In Iron absorption. One of the cDNAs obtained by this strategy had a sequence homology to a plasma membrane dl-haem protein, and was named duodenal cytochrome b (Dcytb) and shared 40-50% homology to cytochrome b561. Immunohlstochemlcal staining for the protein localized It to the duodenal brush border membrane (McKle et a/., 2000), although expression was also found In other tissues. Expression of Dcytb In Xenopus oocytes and cultured mammalian cells led to Induction of ferric Chapter 6 Conclusions and Perspectives Page 171 reductase activity, providing confirmation of function. The level of Dcytb mRNA and protein expression in duodenum was shown to be highly upregulated by three conditions that stimulate iron absorption: chronic anaemia (hypotransferrinaemic mouse), iron deficiency and hypoxia, consistent with the proposed role of ferric reductase in the pathway of iron absorption. It was postulated that DcytB may utilize cytoplasmic ascorbate as an electron donor for the transmembrane reduction of iron, prior to transport into the cell by DMT1 (McKie et al., 2000). This proposal was supported by the demonstration that, in cells transfected with Dcytb, iron and iron chelators modulate the cellular ascorbate levels (Latunde- Dada et a!., 2002). Ascorbate-stimulated reduction of both iron(lll), and interestingly copper(ll), was demonstrated by a putative rabbit homologue of Dcytb, purified from duodenal brush border membranes (Knopfel and Solioz, 2002). If there is a relationship between Dcytb and copper, this is not clear at present. Han and Wessling-Resnick (2002) were not able to identify changes in the expression of Dcytb mRNA in Caco-2 cells in response to copper supplementation. However, the measurement of apical ferric reductase activity did show modulation via copper status. It was proposed that multiple ferric reductases other than Dcytb may exist, which may have different specificities and distribution (Han and Wessling-Resnick, 2002). Chapter 6 Conclusions and Perspectives Page 172 Unlike some proteins of iron metabolism, Dcytb has no identifiable iron responsive elements for the direct modification of expression in response to iron. Therefore, the sensitivity of macrophage ferric reductase activity to iron loading, reported in this thesis, cannot be attributed to an iron response element-mediated mechanism, and the basis of this regulation remains to be resolved. For example, it may be that the ferric reductase gene contains novel sequences which confer regulation by iron; alternatively, the response to iron may be secondary and be mediated via the products of other genes. It has been proposed, by analogy with the regulation of yeast ferric reductase enzymes FRE1 and FRE2 by the iron- inducible transcriptional regulator, Aft1p, that mammalian ferric reductase activity may be regulated by an as yet unidentified, iron-responsive transcription factor (Latune-Dada et al., 2002). Dcytb expression has been demonstrated in several cell types other than the duodenal enterocyte, including spleen, liver, brain and cultured cell lines. The finding of high levels in the neutrophil suggested the possibility of a role in host defence. Ferrous iron, from Dcytb activity, may react with superoxide and H 2 O2 (produced by the respiratory burst oxidase) via Fenton chemistry, to produce a highly reactive hydroxyl radical. The potential role of Dcytb in host defence remains to be explored (McKie et a!., 2002). Chapter 6 Conclusions and Perspectives Page 173 Ferric reductase has been considered as a potential genetic "modifier" of the rate of iron absorption, in mice, significant differences have been observed between different strains of mice in their predisposition towards iron loading, with DBA/2 mice being more susceptible to iron loading than C57BL/6. Recent quantitative RT-PCR studies have shown that knockout mice on the DBA/2 background have increased levels of duodenal ferric reductase, DMT1 and Ireg1 mRNA, as compared to wild type controls. In comparison, the HFE knockout mice on the C57BL/6 background had no such detectable increase in expression of these genes (Dupic et al., 2002a). Recent studies of duodenal ferric reductase activity in HFE knockout mice (mixed background stain "129yOla - C57Bly6") confirmed a modest increase in nitroblue tétrazolium staining in comparison to wild type mice, in agreement with the earlier study (Simpson et a!., 2003). A more detailed analysis of four different mouse strains, C57BL/6, DBA/2, CBA and 129/Sv, showed strain to strain differences not only in serum transferrin saturation and hepatic iron stores, but also in the duodenal mRNA expression of several key genes of iron metabolism: Dcytb, DMT1, Ireg1, and transferrin receptor 1. This study supports some degree of genetic control of the mRNA levels of these molecules (Dupic et a/., 2002b) These mouse studies clearly suggested that functional polymorphisms of duodenal ferric reductase, DMT1 and Ireg1 may be responsible for individual variations in the clinical penetrance of human Chapter 6 Conclusions and Perspectives Page 174 haemochromatosis. Lee et al, (2002) investigated a total of 26 potential modifier genes, including Dcytb, DMT1 and Iregl, by sequencing the coding regions, exon-intron junctions and promoters in DNA from 20 subjects with different HFE genotypes (homozygous C282Y or wild type) and different degrees of clinical penetrance / iron load. However, only one transferrin mutation showed a relationship with iron deficiency anaemia. There was no evidence that polymorphisms of ferric reductase, or any of the other candidate modifier genes, influence the clinical penetrance of haemochromatosis. The putative genes influencing the expression of HFE mutations remain unclear (Lee et a!., 2002). Frazer et a!., (2002) investigated the role of the antimicrobial and iron- regulatory peptide hepcidin in relation to iron absorption and hepatic and duodenal gene expression in rats after a switch to an iron deficient diet. They reported that iron absorption increased within 6 days of starting the iron-deficient diet. This was accompanied by increased duodenal expression of the ferric reductase Dcytb, DMT1 and Iregl, demonstrated by RNAse protection, immunofluorescence and western blotting. These changes correlated with changes in hepatic hepcidin expression and transferrin saturation. There was no alteration in iron stores or haematologic parameters over this period. Frazer at al (2002) proposed a model of the role of hepcidin in intestinal iron absorption based on these observations, in which ferric reductase forms part of the regulatory cycle (Figure 6.1). Chapter 6 Conclusions and Perspectives Page 175 V " ■* ' / - urcnrirB4'>H F t T W ‘TI? * THU? U vw H*pc Crypt Figure 6.1. A model showing the proposed role of hepcidin in intestinal iron absorption (from Frazer et al„ 2002). This model incorporates ferric reductase (Dcytb) as a component of this regulatory "loop". The amount of iron absorbed in the intestine determines the percentage saturation of circulating transferrin with iron. The liver is proposed to detect variation in transferrin saturation, possibly mediated by transferrin receptor 2 (TfR2) and / or the HFE - transferrin receptor 1 (TfRI ) complex. This results in modulation of hepatocyte expression of hepcidin; when transferrin saturation decreases, hepcidin production is decreased; when transferrin saturation increases, hepcidin production increases. Circulating hepcidin levels determine the level of duodenal iron absorption by regulating the expression of Iregl, DMT1 and Dcytb. It is not known if this is achieved by programming the crypt cells or by direct action on the mature enterocytes. This model permits the amount of iron absobed to be modulated (via transferrin saturation and hepcidin levels) before body iron stores are altered. Chapter 6 Conclusions and Perspectives Page 176 Recently, Muckenthaier et al., (2003) investigated Cybrd (mouse ferric reductase) mRNA expression along with other important genes in iron metabolism using a custom microarray ‘Iron Chip’. The mouse types analysed for duodenal expression were HFE knock-out, HFE transgenic mice homozygous for the murine equivalent of the human C282Y mutation, secondary iron overload and iron deficient. The results were interpreted to show that firstly, the duodenal gene expression patterns in the mouse models of haemochromatosis were more similar to secondary iron overload than to iron deficiency. This is controversial, as it is opposite to many lines of evidence suggesting that in haemochromatosis, the duodenal enterocyte is paradoxically iron-deficient in relation to body iron stores. Models of haemochromatosis pathogenesis to date have generally incorporated this concept of a paradoxically iron-deficient duodenum, so that challenging this concept may have far-reaching implications. Secondly, Muckenthaier et al, (2003) observed that Cybrd (ferric reductase) mRNA was an exception to this pattern, being significantly upregulated in the mouse models of haemochromatosis as well as in the iron deficient (rather than iron overloaded) duodenum. Parallel studies of liver expression patterns identified that only hepcidin differed between secondary iron overload in wild type mice (increased hepcidin) versus secondary iron overload in HFE knock-out or "C282Y" transgenic mice (decreased hepcidin levels). The interpretation of this was that hepcidin expression in response to iron overload is HFE- dependent. It was proposed that, in mouse models of Chapter 6 Conclusions and Perspectives Page 177 haemochromatosis, decreased hepatic hepcidin levels may lead to increased duodenal ferric reductase levels, contributing to increased iron absorption. As well as challenging the long-held concept of "paradoxical iron deficicency" of the duodenal enterocyte in haemochromatosis, these results also suggest that ferric reductase activity may be directly involved in the increased iron absorption of haemochromatosis. However, these experiments due to methodology are open to interpretation. It is important that the microarray method is a quantitative indication of gene expression. Although some evidence is presented that microarray expression levels vary in approximate accordance with expression data from Northern blot or quantitative PCR methods, this comparison is only provided for three genes. Expression was studied in pools rather than replicate experiments, which may confound the analysis. There are also significant omissions, such as the transferrin receptor, which does not appear in this analysis. Most importantly, the argument that mouse models of haemochromatosis have duodenal expression patterns similar to secondary iron overload, rather than iron deficiency, relies heavily on the expression of genes which are peripheral to the pathway of iron absorption, and so the observations of Muckenthaier et al., (2003) are as yet of unclear significance. The results of Muckenthaier and colleagues are however in contrast to those results obtained by Stuart et a/., (2003) using an RNAse protection assay approach to look at Dcytb expression. RNAse protection assays Chapter 6 Conclusions and Perspectives Page 178 have been widely accepted to give a quantitative estimate of RNA levels. Human Dcytb expression was found to be similar in iron deficient and iron replete subjects and was not increased in untreated haemochromatosis patients. In comparison with the microarray studies, duodenal expression of DMT1 (IRE-containing form) and Iregl was similar in haemochromatosis patients and iron replete subjects, but in haemochromatosis patients with increased serum ferritin concentrations, both DMT1 (IRE) and Iregl were inappropriately increased relative to serum ferritin concentration. These findings are consistent with the two transporters, DMT1(IRE) and Iregl, playing primary roles in the adaptive response to iron deficiency (Stuart et al., 2003). The RNAse protection methodology was also used to investigate the role of Dcytb and other genes of iron metabolism in the "mucosal block" phenomenom of iron absorption (Frazer et a/., 2003). This is the ability of a large oral dose of iron to reduce the absorption of a dose administered several hours later. After rats were given an oral dose of iron, a rapid decrease in iron absorption was detected, associated with increased enterocyte iron levels and a rapid decrease in the mRNA levels of Dcytb and DMT1(IRE). No such change was seen in expression of DMT1 (non- IRE), Iregl or hephaestin. These data implicate the brush border iron ferric reductase (Dcytb) and transporter (DMT1(IRE)) in the rapid "mucosal block" response, probably mediated by the response to local enterocyte iron levels. It was noted that this local and rapid response is Chapter 6 Conclusions and Perspectives Page 179 distinct from systemic signals, from either the level of stored iron or of erythropoiesis, to the intestine. 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(Raha-Chowdhury et al 1995, and Worwood and Raha-Chowdhury unpublished data) D6S265 (Blue) D6S1621 (Green) Allele No. b.p Allele No. b.p 1 119 5 292 2 121 * 6 290 3 123 7 288 4 125 8 286 5 127 9 284 6 129 10 282 7 131 8 133 D6S105 (Yellow) D6S1260 (Green) Allele No. b.p Allele No. b.p 11 114 1 146 10 116 2 148 9 118 3 150 8 120 4 152 7 122 5 154 6 124 6 156 5 126 7 158 4 128 8 160 3 130 9 162 *Not seen. Appendix 2 Page 222 Appendix 2 Appendix 2 Page 223 Appendix 2 Publications directly related to this thesis: Partridge J , Wallace D.F., Raja K.B., Dooley J.S., Walker A.P. (1998) Monocyte - macrophage ferric reductase activity is inhibited by iron and stimulated by cellular differentiation. Biochemical Journal 336: 541-543 Partridge J., Wallace D.F., Robertson A., Fox M.F., Simons J.P., Dooley J.S., Walker A.P. (1998) Cloning and molecular characterization of a cross-homologous zinc finger locus ZNF204. Genomics 50: 116-118 Partridge J., Wallace D.F., Mistry P.K., Dooley J.S., Walker A.P. (1996) Assesment of macrophage ferric reductase activity as a marker for genetic haemochromatosis. Eur J Hum Genet 4: 70 (NOT INCLUDED) Partridge J , Wallace D.F., Mistry P.K., Dooley J.S., Walker A.P. (1996) Macrophage ferric reductase is not a marker for genetic haemochromatosis. Clinical Science 91: (1): 8 (NOTINCLUDED) Biochem . J, (1 9 9 8 ) 336, 541-543 (Printed In Great Britain) 541 Monocyte-macrophage ferric reductase activity is inhibited by iron and stimuiated by ceiiuiar differentiation Jason PARTRIDGE*, Daniel F. WALLACE*, Kishor B. RAJA|, James S. DOOLEY* and Ann P. WALKER*^ 'Department of Medicine, Royal Free and University College Medical School, Royal Free Campus, Rowland Hill Street, London NW3 2PF, U.K., and tDepartment of Clinical Biochemistry, King's College School of Medicine & Dentistry, Bessemer Road, London SE5 9PJ, U.K. The enzyme ferric reductase catalyses the reduction of Fe(III) as cell supernatants confirmed that the ferric reductase activity was a prerequisite to its transportation across the cell membrane. not due to a secreted factor. Assay o f ferric reductase in Duodenal mucosal biopsies from iron overloaded patients with normalized-iron and iron-enriched (100 /iM ferric citrate) con genetic haemochromatosis (GH) have increased ferric reductase ditions showed no significant difference between Cys^^^Tyr activity and iron absorption compared with controls, yet the GH (Cys282 Tyr) homozygous GH macrophages and Cys^®^- mucosa is iron deficient. A similar GH-related iron deficiency is Tyr negative control activities (P > 0.05). However, a 900% also seen in macrophages. The aim o f this study was to investigate increase in ferric reductase activity was observed during mono whether macrophage ferric reductase activity is altered in GH, cyte to macrophage differentiation (P < 0.05), possibly reflecting and to determine ferric reductase activity in monocytes and the co-ordinate up-regulation o f iron metabolism in these cells. differentiated macrophages. The erythroleukaemic K562 cell line The demonstration of approx. 25 % activity after macrophage fes studied as a clonal reference cell line. The basal K562 ferric differentiation at high free-iron concentrations compared with reductase activity is characteristic o f a membrane bound enzyme, ‘normalized’ iron is consistent with repression of human ferric Seing both temperature and protease sensitive. Ferric reductase reductase activity by iron. The identification of the human activity was also demonstrated in human leucocyte, monocyte ferric reductase gene and its protein w ill ultimately provide insight and macrophage preparations. Assays of K562 and macrophage into its regulation and role in mammalian iron metabolism. INTRODUCTION accumulation of iron by the macrophage. This difference exists despite similar increases in serum-ferritin levels [15]. Cultured Iron is of vital importance in all living organisms, being an G H monocytes also have elevated levels o f ferritin release, and essential cofactor o f enzymes involved in respiration, cell rep abnormal kinetics of iron release, compared with control mono lication and electron shuttling. The homoeostasis of iron is cytes [16,17]. maintained primarily by its absorption in the duodenum and the The uptake of ‘ free’ iron provided in the form of ferric citrate proxim al region o f the jejunum [1]. N orm ally the daily loss o f by mononuclear cells has been established [18,19]. Both free iron ii'on (1-2 mg) is matched by absorption from the diet. This fine uptake and ferric reductase activity have been shown to be balance between iron excretion and absorption is lost in genetic increased in duodenal biopsies from haemochromatosis patients haemochromatosis (G H ). This autosomal recessive disease leads as compared w ith controls [20,21]. to increased absorption of iron from the diet despite increasing Ferric reductase activity has been described in both prokaryotic body iron stores. Iron is deposited in the parenchymal tissue o f and eukaryotic cells, including Escherichia coli, Saccharomyces the liver, pancreas, heart and other tissues. I f untreated, the iron cerevisiae, mouse, rat, rabbit and human duodenum, rat liver overload eventually leads to tissue damage. The prevalence of endosomes, rabbit reticulocytes and various cultured cell lines G H in Europeans is approximately 1 in 300, which is higher than [21-24]. Its conservation across species indicates a functional the combined prevalence of cystic fibrosis, phenylketonuria and role. The S. cerevisiae gene fo r ferric reductase (F R E l) has been Duchenne muscular dystrophy [2-4]. cloned and sequenced [22,23]. The ferric iron uptake mechanism Recently a candidate gene for haemochromatosis (HFE) was in the yeast, S. cerevisiae, utilizes the ferric reductase enzyme to identified [5]. In the United Kingdom, 91 % o f G H patients were reduce Fe(III) before absorption. The ferrous Fe(II) iron is found to be homozygous for a single mutation o f HFE, Cys®*®Tyr transported through the membrane, and is thought to be re (Cys®®® Tyr) [6]. Although the metabolic role of the HFE oxidized back to Fe(III) as it passes through the membrane. The protein is not fully understood, recent advances support a role in human homologue of FREJ has yet to be cloned. However, the regulation of iron absorption [7-10]. partial purification of human ferric reductase from both human Abnormalities of iron metabolism in the intestine, liver and Hutu 80 duodenal adenocarcinoma cells and human duodenal fnacrophages have all been implicated in the pathogenesis of GH microvillus membranes has shown that the activity is NADH- [11-13]. In the haemochromatotic macrophage, despite the dependent and the protein is membrane bound [24]. elevation of serum iron and transferrin saturation, concentrations We demonstrate ferric reductase activity in the K562 human o f iron are m inim al u n til late in the disease [14]. This is in erythroleukaemic cell line and primary cultures of human leu contrast w ith transfusional siderosis, where there is progressive cocytes, monocytes and macrophages. We report ferric Abbreviation used: GH, genetic haemochromatosis, ^ To whom correspondence should be addressed (e-mall [email protected]). 5 4 2 J. Partridge and others reductase activity in cells from G H patients compared with determined spectrophotometrically at 562 nm by measuring the controls, under conditions of high free iron and during mono reduction of the yellow Fe(III) to the purple coloured stable cyte-macrophage di flferenti ation. Fe(II)-ferrozine complex [21]. MATERIALS AND METHODS RESULTS Patients Preliminary experiments investigated the characteristics of the ferric reductase activity of the clonal control K562 erythro- Ten male patients were investigated: five GH and five control leukaemic cell line. In each experiment the rate o f ferric reductase patients with no family history of iron disorders. The diagnosis activity was determined by linear regression analysis o f at least of GH was made from clinical and biochemical data and three time courses; each individual time point was performed in confirmed by a hepatic iron index > 1.9 and/or removal of > 5 g duplicate. A ll time courses were linear for at least 60 min. A fter of iron during initial phlebotomies [3,6,25]. The groups were age m ild trypsinization, ferric reductase activity was decreased to matched: GH, 49+17.4 years; control, 55±5.3 years, 38.7% of the control (Table 1). Heating the cells to 65 °C for (mean + S.D.). 5 min prio r to assay decreased activity to 26.7 % o f the control. When the assay was performed at 10 °C, 17% o f the control Mutation anaiysis activity was observed. These observations were consistent with The Cys^'^^Tyr and His*® Asp mutations o f the haemochromatosis the assay of a membrane bound protein. When K562 and gene, HFE, were determined by PGR and restriction enzyme macrophage cell-free supernatants were taken for determining (jigestion [6]. A ll haemochromatosis patients studied were homo reductase activity at the end of the incubation period, only zygous fo r the Cys^'^^Tyr m utation and negative for His®®Asp. background levels of reductase activity were detected (Table 1). Two controls carried a single copy o f His®® Asp; all controls were This indicated that the activity was not due to a soluble, released negative for Cys®*®Tyr. Cell culture Table 1 Human ferric reductase activity has the characteristics of a membrane bound enzyme White blood cells were extracted from peripheral blood using Lymphoprep® (Nycomed). Monocytes were enriched by their The cells were subjected to various treatments and then ferric reductase activity was measured adherence to plastic. Adherent monocytes were cultured for 14 and expressed as a percentage of K562 control activity at 37 °C (mean + S.D.; /r = 3). t The ferric reductase activity for the control K 562 cells was 6.93 nmol of Fe • m in "' ■ (10® c ells)"', days for in vitro differentiation to macrophages [26]. Cell numbers where 1 /^fyi ferric iron gave an absorbance of approx. 0.03 at 562 nm. The reaction was \yere determined using a haemocytometer and by fluorescence performed in the absence of ferrozine, cells were removed by centrifugation, and the activity in activated cell sorter analysis (Coulter EpicXL). Cell viability was the supernatant was determined after the addition of ferrozine. Activities are compared with the confirmed by Trypan Blue exclusion (> 95 %). The purity of the control; " P < 0.01 (Student’s /-test). primary cell cultures was assessed by morphological criteria (Leishmanns stain) and CD14 fluorescence-activated cell sorter Cell type Treatment Ferric reductase activity (% )f analysis; typical purities were 80% for monocytes and 95% for macrophages. The clonal erythroleukaemic cell line K562 was K562 0.1 m g /m l trypsin, 3 7 °C , 60 min 38.7 + 1.5** included in all experiments as an internal control. Cells were K562 65 °C , 5 min 26.7 + 1.5** cultured at 37 °C either in conditions of ‘normalized’ iron K562 Assay performed at 10 °C 17.0 + 1.7** (24/tM iron, transferrin saturation 30%), or in normalized K562 Supernatant activity]: 0.33+0.58** tvtacrophage Supernatant activity]: 0.02 + 0 .0 2 ** medium supplemented with 100 /iM ferric citrate, to reflect the elevated in vivo concentrations of serum iron and free iron of a heavily iron-loaded haemochromatotic [27,28]. The 100/iM ferric citrate solution was freshly prepared as a mixture containing Table 2 Ferric reductase activities 100 /iM ferric chloride and 150 /aM sodium citrate, as described previously [29]. Cell cultures supplemented with ferric citrate Activities are shown for lymphocytes, monocytes and macrophages from classical Cys®®Tyr vye:re protected from light and fresh medium was substituted after homozygous GH patients and Cys®“®Tyr negative control patients. Statistical probabilities were assessed using the Student's Atest. There was a significant increase in activity with 7 days. In addition to the protection from light and chelation by differentiation of the monocytes to macrophages, for both GH (*) and control ( f ) preparations Oixrate, stability o f F e (III) was also conferred by the R P M I 1640 ( f < 0 . 0 5 in both cases). N, normalized-iron culture medium (see the lyiaterials and methods culture medium itself, which contains many other chelating section); 100/tfvl, iron loaded (additional 100/zM ferric citrate in normalized-iron medium). agents at high concentrations. These include amino acids, sugars, vitamins, bicarbonate and phosphate, which also prevent the Ferric reductase activity hydrolytic polymerization o f iron [30]. The amino acids and [nmol of Fe-m in"'-(10®cells)" glucose alone provide an additional 150-fold molar excess of chelating agent over iron [30]. Cell type Fe fVlean+ S.D. n P Lymphocytes GH N 4.37 + 3.13 3 I Ferric reductase assay > 0 . 0 5 Control N 3 .98 + 2.51 3 J tVlonocytes GH N 4.38 + 1.71* 3 1 Cells were harvested, rinsed, then incubated at a concentration of > 0.05 2 X 10® cells/ml in 200/d of oxygenated physiological buffer Control N 3.75 +0.711 3 / tVlacrophages GH N 43.3 + 5.13* 4 [ (125 mM NaCl/3.5 mM KCl/1 mM CaClg/lO mM MgSO^/ > 0.05 Control N 31.3 + 8.67f 4 f 10 m M D-glucose in 16 mM Hepes/NaOH, pH 7.4) in 96-well Cell line K562 N 6.93 + 0.88 3 1 > 0.05 tissue culture plates at 37 °C. The reaction was started by the K 562 100/ztVI 7.87 + 0.77 3 I addition of 100 /^M Fe(III), as a ferric chelate of nitrilotriacetate Ivtacrophages GH 100 /ztvt 10.0 + 4.87 5 \ > 0.05 in a 1:2 ratio (Fe:nitrilotriacetate), and ferrozine (1 mM). The Control 100 /iM 9.2 + 9.60 5 / reaction was performed at 37 °C. The rate of reaction was Macrophage ferric reductase 543 factor. These observations indicate that ferric reductase is a We thank Aida Condez, Len Poulter, Bob Simpson, Adrian Bomford, Kwee Yong, membrane bound enzyme. Nickie James, Karen Noble, Pram Mistry and Roger Ling for expert technical advice Investigation o f primary cultures o f lymphocytes, monocytes and and helpful discussions. The support of the fyiedical Research Council for the Ph.D. studentship (J.P.) Is acknowledged with gratitude. This work was supported by a macrophages demonstrated ferric reductase activity in these blood- Nuffield Foundation Award to Newly Appointed Science Lecturers (A.P.W.). derived cells (Table 2). N o significant difference (P < 0.05) was observed between G H (Cys**^Tyr homozygous) and control (Cys^*^Tyr negative) in either lymphocyte, monocyte or macrophage REFERENCES preparations. However, ferric reductase activity of macrophages 1 McCance, R. A. and Widdowson, E. M . (1937) Lancet II, 6 8 0 -6 8 4 .was approx. 900 % of the monocyte activity, for both GH and 2 Bothwell, T. H., Charlton, R. W . and tvtotulsky, A. G. (1 9 95 ) in The Metabolic and control preparations, when cultured in normalized iron. Molecular Bases of Inherited Disease (Scriver, C. R., Beaudet, A. L , Sty, W. S. and • When K562 cells were cultured in normal or high iron (100 Valle, D., eds.), pp. 2237-2269, McGraw-Hill, New York 3 Dooley, J. S., W alker, A. P., MacFarlane, B. and Worwood, M . (1997) Lancet 349, ferric citrate) concentrations, no difference in ferric reductase 1 6 8 8 -1 6 9 3 activity was observed (/* > 0.05; Table 2) 4 Witte. D. L.. Crosby, W. H., Edwards, C. Q., Fairbanks, V. F. and Mitros, F. A. (1996) When macrophages were cultured in media containing 100 /*M Clin. Chim. Acta 245, 1 3 9 -2 0 0 ferric citrate for 2 weeks, no significant difference {P > 0.05) was 5 Feder, J. N., Gnirke, A., Thomas, W ., Tsuchihashi, Z., Ruddy, D. A., Basava, A., observed between G H and the control. However, the ferric Dormishian, F., Domingo, R., Eiiis, M . C., Futlan, A. et al. (1996) Nature Genet. 13, reductase activity o f both G H and control macrophages cultured 3 9 9 -4 0 8 6 Worwood, M .. Shearman, J. D., Wallace, D. F., Dooley, J. S., Merryweather-Clarke, under these conditions was approx. 25% of the activity when A. T., Pointon, J. J., Rosenberg, W . M . C., Bowen. D. J., Burnett, A. K., Jackson. H. A. grown at normalized iron concentrations (P < 0.05; Table 2). et al. (1 9 97 ) Gut 41, 8 4 1 -8 4 4 7 Parkkila, S., Waheed, A., Britton, R. S., Bacon, B. R., Zhou, X. Y., Tomatsu, S., DISCUSSION Fleming, R. E. and Sly, W. S. (1 9 97 ) Proc. Natl. Acad. Sci. U.S.A. 94, 13198-13202 8 Lebron, J. A., Bennett, M . J., Vaughn, D. E., Chirino, A. J., Snow, P. M ., Mintier, The ferric reductase activity o f human K562 cells was dem G. A., Feder, J. N. and Bjorkman, P. J. (1998) Cell93 , 1 1 1 -1 2 3 onstrated to have the characteristics of a membrane bound 9 Feder, J. N., Penny, D. M ., trrinki. A., Lee, V. K., Lebron, J. A., Watson, N., enzyme and was not attributable to the secretion of reducing Tsuchihashi, Z., Sigai, E., Bjorkman, P. J. and Schatzman, R. C. (1998) Proc. Nati. Acad. Sci. U.S.A. 95, 1 4 7 2 -1 4 7 7 factors. There was no significant difference in the reductase 10 Zhou, X. Y., Tomatsu, S., Fleming, R. E., Parkkila, S., Waheed, A., Jiang, J., Fei, Y., activity between GH and controls for leucocyte, monocyte or Brunt, E. M ., Ruddy, D. A., Prass, C. E. et ai. (1 9 98 ) Proc. Natl. Acad. Sci. U.S.A. macrophage preparations. This was observed under both normal 95, 2 4 9 2 -2 4 9 7 ized iron conditions and in the presence o f 100 /iM ferric citrate. 11 Powell, L. W. and Halliday, J. W. (1981) Clinics Gastroenterol. 10, 7 0 7 -7 3 5 Ferric reductase activity of differentiated macrophages was 12 Cox, T. M . (1 9 90 ) Blood Rev. 4, 7 5 - 8 7 approx. 900 % that of monocyte activity (P < 0.05). This increase 13 Olynyk, J. K. (1 9 94 ) Aust. N. Z. J. M ed.24 , 7 1 1 -7 1 6 14 Yam, L. T., Finkel, H. E., Weintraub, L. R. and Crosby, W. H. (1968) New Engl. J. most likely reflects the co-ordinated up-regulation of the proteins M ed. 279 , 5 1 2 -5 1 4 of iron metabolism during the transition into macrophages [31]. 15 Saab, G., Green, R., Jurjus, A. and Sarrou, E. (1 9 86 ) Scand. J. Haematol. 36 , 6 5 - 7 0 Macrophage ferric reductase activity was decreased when cul 16 Flanagan, P. R., Lam, D., Banerjee, D. and Valberg, L. S. (1988) J. Lab. Clin. Med. tured in 100 /^M ferric citrate compared with that in normalized 113, 1 4 5 -1 5 0 iron. From these results it would appear that macrophage ferric 17 Fillet, G., Beguin, Y. and Baldelli, L. (1989) Blood 74 , 8 4 4 -8 5 1 reductase activity may be determined by external free-iron 18 Parmley, R. T., M ay, M. E., Spicer, S. S., Buse, M . G. and Alvarez, C. J. (1 9 81 ) Lab. Invest. 44, 4 7 5 -4 8 5 concentrations during macrophage differentiation. Macrophage 19 Bonkowsky, H. L , Carpenter, S. J. and Healey, J. F. (1 9 79 ) Arch. Pathol. Lab. M ed. ferric reductase activity was down-regulated by increased free- 103, 2 1 - 2 9 iron concentration. 20 Cox, T. M . and Peters, T. J. (1978) Lancet I, 1 2 3 -1 2 4 This control of ferric reductase activity could contribute to the 21 Raja, K. B., Pountney, D., Bomford, A. B., Przemioslo, R., Sherman, D., Simpson, low levels of iron observed in the haemochromatotic macrophage. R. J., William s, R. and Peters, T. J. (1 9 96 ) Gut 38, 7 6 5 -7 6 9 As haemochromatotic macrophages differentiate in high free- 22 Danois, A., Klausner, R. D., Hinnebusch, A. G. and Barriocanal, J. G. (1990) Mol. Cell. Biol. 10, 2 2 9 4 -2 3 0 1 iron conditions in vivo, their ferric reductase activities may be 23 Danois, A., Roman, D. G., Anderson, G. J., Hinnebusch, A. G. and Klausner, R. D. decreased according to the amount o f free iron present. Future (1 9 92 ) Proc. Natl. Acad. Sci. U.S.A. 89, 3 8 6 9 -3 8 7 3 studies could address this issue by isolating differentiated macro 24 Riedel, H. D., Remus, A. J., Fitscher, B. A. and Stremmel, W. (1995) Biochem. J. phages from G H and control human subjects, and measuring the 309, 7 4 5 -7 4 8 activity o f the in vivo differentiated cells. The clonal reference cell 25 Summers, K. M ., Halliday, J. W . and Powell, L. W . (1 9 90 ) Hepalology 12, 2 0 -2 5 line K562 did not show m odulation o f ferric reductase activity by 26 Tormey, V. J., Paul, J., Leonard, C., Burke, C. M ., Dilmec, A. and Poulter, L. W. (1997) Immunology 90, 4 6 3 ^ 6 9 iron. The K562 cell line originates from an erythroid cell clone 27 Baley, R. G., Lai Chung Fong, P., Shamir, S. and Sherlock, S. (1980) Dig. Dis. Sci. derived from a patient with myeloid leukaemia in acute blast 25, 3 4 0 -3 4 6 crisis [32]. The lack of modulation of K562 ferric reductase 28 Gutteridge, J. M . C., Rowley, D. A., Griffiths, E. and Halliwell, B. (1985) Clin. Sci.68 . activity by iron may reflect either the erythroid lineage or 4 6 3 -4 6 7 changes in the clonal expansion o f this cell line. The identification 29 Neumannova, V., Richardson, D. R., Kriegerbeckova, K. and Kovar, J. (1995) In Vitro of the human ferric reductase gene will ultimately establish Cell. Dev. Biol. 31, 6 2 5 -6 3 2 30 Richardson, D. and Baker, E. (1 9 91 ) Biochim. Biophys. Acta 1093, 2 0 -2 8 whether it is transcriptionally repressed by iron, as in S. cerevisiae. 31 Cottrell, B. and Jones, D. (1990) FEM S Microbiol. Im munol. 2, 3 3 3 -3 3 7 Future studies w ill elucidate whether ferric reductase plays a role 32 Andersson, L. C., Nilsson, K. and Gahmberg, C. G. (1 9 79 ) Int. J. Cancer 23, in the apparent iron deficiency of macrophages in GH. 1 4 3 -1 4 7 Received 9 July 1998/14 September 1998; accepted 8 October 1998 BRIEF REPORTS 117 S c ZNF204 (A UJ (/) TEL 1/ CEN Genomic ■<- -► pCYPAC2N302f1 PACs pCYPAC2N11g1 10kb 3.0 EST449 STSG-9945 I 1 1.6 cDNA 1 CDNA2 1.0 CDNA3 CDNA CDNA4 200bp PCR probe 4 5 . 0 34 Consensus H pA U 5 ' — L. 3' PCR products F IG . 1. Mapping, isolalion, and characterization of ZNF204 (GenBank Accession No. AF033199). (a) Fine-resolution genomic map around D6S1260. PAG clones used to screen the small intestinal cDNA library are shown; dots indicate the presence of markers (4, 5, 8, 9). (b) Overlapping cDNA clones showing the position of EST449 and STSG-9945; a double slash indicates the junction of ZNF204 cDNA clones with chimeric sequence, (c) Schematic representation of ZNF2Ü4 showing the 1.1-kb open reading frame consensus sequence and the positions of its start ATG (arrow at 5' end) and polyadenylation consensus sequence (pA). Blocks indicate the positions of individual zinc linger motifs with asterisks representing termination codons and a crooked arrow indicating the location of the frameshift mutation. The position of the PCR hybridization probe is indicated above the consensus. Below the consensus, the three bars represent the three overlapping PCR products amplified from genomic DNA using cDNA primers; cDNA and genomic DNA products were of identical size, (d) ZNF204 cross-hybridizes with multiple zinc finger loci. Southern blot of /Y/ndlll-digested DN A (10 p,g) from pig. cow, hamster, and human probed with an internal PCR fragment from ZNF204 (c). Southern blots were rinsed once with 4 x SSC at room temperature, twice for 20 min in 0.5x SSC, 0.1% SDS at 42°C, and once for 20 min in 0.5x SSC, 0.1% SDS at 65°C. A ladder of bands increasing uniformly by approximately 80 bp was seen in all lanes reflecting cross-hybridization of ZNF204 to multiple zinc finger loci across species. cell hybrid panel was screened by PCR using three sets of from coding sequence. Taking the mapping and homology anal primers (Fig. Ic); however, additional chromosomal loci were yses together, it seems likely that the putative parent locus is not detected. Non-zinc-finger portions of the locus (EST449, in 6p2L3 and possibly related to ZNF184. Indeed, ZNF204 STSG-9945; Fig. lb) gave only a smear when hybridized to and ZNF184 both hybridize to a 3.2-kb transcript on Northern genomic DNA, lim iting their use in the search for a parent analysis (data not shown and Ref. 3), consistent w ith a shared locus. Fluorescence in s itu hybridization (FISH) analysis ancestry. The processed appearance of ZNF204 suggests a with PAC pCYPACZNllgl revealed a strong signal only on retrotransposed intermediate. Localized enrichment of retro- chromosome 6p21.3. W ith cDNA3 (2.7 kb), a small specific transposed elements has not been previously demonstrated. signal was seen only in 6p21.3. Of 11 cells imaged, 14/44 Interestingly, the first 90 bp of cDNA sequence has homology (32%) of chromatids contained the signal. Chromosome with the TH Elc repeat element family. Further studies would 6p21.3 contains a cluster of at least six other C2H2 zinc finger be required to investigate the potential involvement of this genes (ZNF165, ZNF193, ZNF192, ZNF184, SRE-ZBP, and sequence in a mechanism of local retrotransposition. i WI-7080), suggesting the possibility that the parent locus To analyze the evolutionary conservation of ZNF204, a zoo may also be in 6p21.3. blot of ////Tdlll-digested DNA was screened using an internal To investigate the sim ilarity of ZNF204 to published se PCR fragment probe (Fig. Ic). This showed a ladder of hy quences, homology analysis was performed using the BLAST bridizing bands increasing uniformly in size by approxi program (1). Reanalysis of the highest 10 BLAST homologies mately 80 bp in all species tested (pig, cow, hamster, and using the BESTFIT (GCG) program showed that ZNF184 human; Fig. Id). This step-wise increase in the size of frag had the closest homology to ZNF204, with 68% nucleic acid ments corresponds to the distance between the phenylalanine identity over 1963 nucleotides. ZNF184 is also from the residues of adjoining zinc finger domains of 84 bp. The se 6p2L3 region and within 500 kb of ZNF204 (3, 9). When only quence Lys-Ala-Phe occurs commonly in zinc fingers and can the putative 5' UTR of ZNF204 was analyzed, homology to be encoded by sequences that include a H in d lll site. For'th’e the predicted coding “spacer” region of ZNF184 was detected, 10 zinc finger genes w ith highest homology to ZNF204, 15 of suggesting that the 5' region of ZNF204 may have been derived th e 20 H in d lll sites present were positioned at the conserved 118 BRIEF REPORTS phenylalanine residue. Two zinc finger genes contained 5 REFERENCES Hirtdlll sites, all at the phenylalanine residue of the consen sus, giving rise to a ladderHindlll of fragments as detected Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, by hybridization in Fig. Id. Conservation of zinc finger probesZ., M iller, W., and Lipm an, D. J. (1997). Gapped blast and psi- has been previously reported, with one or several cross-hy blast: A new generation of protein database search programs. bridizing bands detected on zoo blots (7). The demonstration Nucleic Acids Res. 25: 3389-3402. of a conserved ladder of zinc finger fragments by ZNF204 2 . Blumberg, H., Eisen, A., Sledziewski, A., Bader, D., and Young, results from the combination ofHindlll as a restriction en E. T. (1987). Two zinc fingers of a yeast regulatory protein zyme, formamide hybridization solution, moderate strin shown by genetic evidence to be essential for its function. A/a- gency washing, and possibly the partial degeneracy of thefure 328: 443-445. ZNF204 probe. 3. Goldwurm, S., Menzies, M. L., Banyer, J. L., Powell, L. W., afid We conclude that ZNF204 most likely arose by a local retro-Jazwinska, E. C. (1997). Identification of a novel Krueppel-rç- trahsposition event within the 6p21.3 ZNF184 cluster. We lated zinc finger gene (ZNF184) mapping to 6p21.3. Genomics observe a remarkable regular ZNF204 hybridization pattern40: 486-489. conserved across all species tested, which we have explained Pappas, G. J., Polymeropoulos, M. H., Boyle, J. M., and Trent, in terms of the periodicy ofHindlll sites within the consensus J. M. (1995). Regional assignment by hybrid mapping of 36 Ex sequence of multiple zinc finger proteins. At least 35 individ pressed sequence tags (ESTs) on humein chromosome 6. Ge/70- ual bands could be resolved in human and bovine DNA, sug mfcs 25: 124-129. gesting that the zinc finger genes, and therefore proteins, of5. Raha-Ghowdhury, R., Bowen, D. J., Stone, G., Pointon, J. J., these species can contain at least 35 motifs. Indeed, the Xfin Terwilliger, J. D., Shearman, J. D., Robson, K. J., Bomford, A., protein ofXenopus contains 37 C2H2 zinc finger motifs, con- and Worwood, M. (1995). New polymorphic microsatellite mark ers place the haemochromatosis gene telomeric to D6S105. :^istent with these observations (6). Most zinc finger proteins Hum. Molec. Genet. 4: 1869-1874. are transcriptional regulators involved in cell growth and diiftçrentiation. The cross-hybridization of ZNF204 to multi6 . Ruiz-i-Altaba, A., Perry-0 Keefe, H., and Melton, D. A. (lg^7) Xfin: An embryonic gene encoding a multifingered protein in ple zinc finger loci across species may provide a tool for theXenopus. EM BO J. 6: 3065-3070. ^ analysis of C2H2 zinc fingers throughout evolution. 7. Shannon, M., Ashworth, L. K., Mucenski, M. L., Lamerdin, J. E., Branscomb, E., and Stubbs, L. (1996). Comparative analy > ACKNOWLEDGMENTS sis of a conserved zinc finger gene cluster on human chromq, some 19q and mouse chromosome 7. Genomics 112-120.;, We thcink David Latchman, Arlan Smit, Dariusz Gôrecki, Marita 8 . Volz, A., Albig, W., Doenecke, D., and Ziegler, A. (1997). Physi Pdhïschimidt, Priyal de Zoysa, and Kevin Moore for helpful discus cal mapping of the region around the large his tone gene cluster sion. The UK HGMP Resource Centre provided PAG clones and the on human chromosome 6p22.2. DNA Sequence 8: 173-180., n?9pochromosomal hybrid panel; the MRC HGMP funded the CGD 9. Wallace, D. F., Partridge, J., Robertson, A., Simpson, V. M. A;v FIS,H equipment. This work was funded by grants from The Peter Worwood, M., Bomford, A. B., Volz, A., Ziegler, A., Dooley, J. S., Saipuel Charitable Trust (J.S.D., A.P.W.), and the Nuffield Founda and Walker, A. P. A 6p22 reference map of leukocyte DJNIA- tion (A.P.W.). The support of the MRC for the Ph.D. studentship of Exclusion of rearrangement in four cases of atypical haemochrp- J.P. is acknowledged with gratitude. matosis. Eur. J. Hum. Genet, in press. , , ,