Global Action is privileged to present a second article in the series BEFORE aHUS ENDEAVOURS FADE. Celebrating the work of people whose research made a significant difference for aHUS patients.
In his article Dr Paul Warwicker looks back 25 years and tells the story behind the research which discovered, and reported, for the first time, a genetic reason for uncontrolled complement being the cause of aHUS. He also reveals a surprising update.
A truly pivotal moment for aHUS patients everywhere. Please read on.
Paul Warwicker
“In 1994, I was fortunate to be offered a clinical research post under the twin supervision of two inspirational scientists and clinicians, Drs (now Professors) Tim and Judith Goodship, in the north-east of England, in Newcastle upon Tyne. Tim, a nephrologist was married to Judith, a geneticist.
My project was to investigate the genetics of a common inherited kidney condition affecting children and young adults called reflux nephropathy (also known as chronic pyelonephritis). Our aim was to find which stretch of the human genome (the entire set of DNA instructions found in each cell) was common to all the affected individuals, and absent in the unaffected ones. By doing this, we could locate the specific region of our chromosome responsible for the disorder. This is called linkage analysis, and in those days, it was a laborious business, running polymorphism gels utilising radioactive markers. We could then investigate which genes (sequences of code DNA) were found in this region, and perhaps get an insight into the cause of the condition.
The technique took the best part of a week to run a single panel and could fail at any stage losing a whole week’s work. These days, there are machines that can sequence every one of the bodies 25,000 genes in a matter of hours; back then it was taking us a week to investigate even a small fraction of a single gene.
But recruiting affected families was proving slower than anticipated, and I was running the panels half empty. It seemed a waste. During one of our weekly research meetings, I mentioned this to Tim.
He told me about a family on our dialysis programme, that suffered from a rare inherited condition called haemolytic uraemic syndrome. A nephrologist called Malcolm Farr had described the family back in 1974, publishing a scientific paper. It was a devastating illness. Back in 1974 there had been 5 cases in 3 generations, and by 1994, there had been 8 cases in 4 generations and 6 had died. The illness was identical in nature and severity, killing most who developed it in days and weeks. Those few who survived were not transplantable; the HUS inevitably recurring.
‘Why don’t you also look at that family’s DNA, Paul?’, suggested Tim.
In 1994, nobody knew what caused inherited HUS. The prevalent theories at the time involved a substance called prostacyclin. Also, there was a similar condition called thrombotic thrombocytopenic purpura (TTP), which, in the 1990’s was (erroneously) thought to be a sub-type of HUS. It was all very confusing.
So, what did we know about inherited atypical HUS back in 1994? We had some clues. We knew the condition was rare (a few cases per million population), and that it had a high mortality (often in excess of 80%). We knew it could be treated (to a degree) using a process called plasma exchange – removing blood plasma from an affected individual and replacing it with plasma from a donor. And we knew that it could recur in kidney transplants. The last two observations suggested that whatever caused inherited HUS would likely be found in the blood stream.
I contacted the surviving members of the Newcastle family,who were immediately agreeable and keen to help. They had lived with the curse of aHUS and were eager for research to be undertaken. The family willingly offered blood samples for DNA analysis. But we needed DNA samples from the deceased affected family members too.
With information provided from the surviving family members, and with their consent, I was able to trace tissue samples from affected individuals held in pathology departments. These included a microscope slide of a kidney biopsy I found in a dusty cardboard box in the cellar of a pathology department, and a post-mortem tissue sample sent from a different region across the country. Some of these samples had been obtained before I had been born.
Now, we attempted to extract DNA. This proved challenging to master – DNA extraction techniques from archival tissue were still being developed in the 1990s. Some of the samples were decades old. But with the help of Judith and the scientists in the Newcastle genetics lab, we were able to get reliable high-quality DNA.
It soon became clear that we would also need more large HUS families if we were to achieve our first target of identifying the stretch of chromosome that carried the gene(s) that caused the condition. That wouldn’t be easy. We knew it was a rare condition. In fact, back then, there were only about 20 such families in the world, and only one other family in the UK.
This family lived in the south-west, and via their kidney specialist (Dr Tony Nichols) I contacted a lady called Margaret, who was one of the few surviving members. She too was incredibly supportive. I drove down to visit her. Whilst I was there, Margaret mentioned that she was aware of a family with the same unusual condition, who lived in the next village. Given that inherited HUS was very rare, there had to be a link, somewhere back in time to her family. Margaret supplied me the details, and the next day I drove to visit the second family.
Family 2 readily agreed to help. And there was another surprise coming. ‘Oh, and by the way, we know of a third family with this condition who live with inherited HUS, in the village up the road.’ This family too, were soon on board.
If we could link these three families together, via a common ancestor, we could trace the path of the condition through the generations. It would significantly enhance our search for the faulty gene. I started to collect blood samples and hunt for pathology samples from the deceased family members. Meanwhile, the families, led by Margaret and the aHUS Allliance’s own Len Woodward, started to investigate the family trees, exploring family histories, and inspecting church and registry records. With painstaking detective work, they were able to connect two of the families to a common ancestor living in the nineteenth century. No connection could be found to the third family, even going back two to three hundred years.
In the meantime, I had received correspondence from a Belgian professor of nephrology, called Yves Pirson. Yves was looking after a family of 3 siblings with inherited HUS. There was an unusual feature of the illness in this family. The affected individual had been successfully transplanted without recurrence. Other than this, the features of their HUS illness were identical. With Yves help, and the family’s consent, we decided to include them in our study.
Now we had 3 cohorts, in three separate areas, all affected by inherited atypical HUS: the Newcastle family, the (three) South-west families and the Belgian family.
I now spent several months in the laboratory, extracting DNA from blood samples, microscope slides and pathology samples, and running DNA gels. Using a linkage analysis technique called microsatellite polymorphism genotyping, we were finally able to identify a stretch of unique DNA common to the affected individuals. At this point, I abandoned my reflux nephropathy work and allocated all my time to the HUS research.
We found that all the affected family members in all 3 cohorts had their own, family specific, identical stretch of DNA, found in one section on the long arm of chromosome 1. Therefore, somewhere in this area, was likely to be found the rogue HUS gene or genes. And there was one cluster of genes that looked promising.
We knew of a few scientific papers that had identified abnormal levels of complement proteins in HUS patients. And in the centre of this stretch of DNA was found a region known as the RCA (regulators of complement activation) gene cluster. The complement system is an evolutionally old immune pathway by which organisms have evolved the ability to recognise self from non-self. In essence they comprise a series of reactions resulting in the formation of a protein complex which kill invading pathogens by punching a hole in their cell membrane. Overactivity of complement is as dangerous as underactivity. In our bodies, the complement system is normally in balance. To prevent complement attacking our own bodies, we produce a family of regulatory proteins that puts a brake on complement – the regulators of complement activation.
If we could now prove that one of these genes carried a significant mutation (a ‘mistake’ in the DNA sequence), it would be very strong evidence that this gene likely caused inherited HUS (and that complement was implicated).
Judith and her colleagues taught me the techniques of identifying gene mutations: complex and time-consuming investigations called single strand conformational polymorphism (SSCP) and heteroduplex analysis. We studied all the family DNA samples as well as a panel of samples from patients with non-familial HUS. Within 2 weeks we had found a mutation in a gene for the complement regulatory protein Factor H, in an individual with relapsing atypical HUS. This is going to be easy, I naively thought, and waited for other mutations to present themselves. I was joined in the research by my colleague, Dr Rosie Donne, who started to sequence the Factor H gene. In fact, it then took another year of lab work to discover a second mutation, again in the gene for Factor H. This was found in all the affected members of the south-west families, even in the third family in whom we had been unable to find a common ancestor.
Now we had the proof. We had linked the inherited disorder in all 3 cohorts, to a stretch of DNA on chromosome 1, and had also identified two mutations in one of the genes controlling the complement system, Factor H. Further studies in these affected individuals also revealed evidence of abnormal levels of complement proteins in the blood. It was time to sit down and document our findings for the wider medical and scientific community. We wrote a scientific paper, outlining our findings, and presenting our conclusions about the role of complement in atypical HUS, and sent it off to be considered for publication.
This also proved harder than we anticipated. One journal after another declined our paper. At the time the prevailing orthodoxy, and indeed the opinion of several journal reviewers, was that abnormalities in prostacyclin metabolism were the cause of HUS and complement abnormalities merely an epiphenomenon. We couldn’t understand why nobody would believe our findings. Perhaps the understanding of the power and significance of molecular genetics (like genetic fingerprinting in criminal investigations) was not fully appreciated back in the 1990s.
Eventually, after nearly two years of serial rejections, one prestigious journal, Kidney International, was the first to agree to publish our findings (in 1998).
In the subsequent 20 years many scientists and clinicians, have since researched this serious disorder, and we have seen the full breadth of complement abnormalities in aHUS gradually uncovered. Mutations have been discovered in nearly all factors involved in the regulation of complement and in complement proteins themselves, eventually leading, in 2009, to the first effective targeted treatment, Eculizumab,based on this understanding of the role of complement in atypical HUS.
The Belgian family never really fitted in to the classical pattern of atypical HUS because three members had been successfully transplanted. In 2003 mutations were found in the membrane cofactor P gene (MCP) gene in this family. Membrane cofactor P, as the name suggests, is a membrane-bound complement control gene. Atypical HUS doesn’t recur in transplants in this family, because transplanted kidneys carry normal MCP genes from the donor. We were lucky. Thankfully the MCP gene sits close to the Factor H gene, so our linkage analysis still worked. Mutations in MCP have been discovered in around 10% of atypical HUS patients. And in 2006, the Newcastle family were also found to have a factor H gene mutation, causing an abnormal hybrid Factor H gene (FH/FHL1).
And there the story of the early days of atypical HUS research ends, or at least I had thought so. Earlier this year, I was contacted by Len Woodward. Over the years Len had continued to study his family tree ( annex A below) , and together with Margaret, they did it – they finally found a common ancestor to all the three South-west families, a man born in 1520, called John Huxtable. Indeed, historical records suggest that there appears to be a legacy of premature deaths in these families stretching back centuries. Obviously, we can’t be certain they were all related to aHUS but one must suspect so. These deaths occurred in an era when neither HUS nor indeed kidney failure were recognised.
So, on the 25th anniversary of our research, thanks to Margaret and Len, we have a 500-year anniversary. They have found the missing link, the common ancestor, born almost exactly half a millennium ago, of the three South-west families.
I feel privileged to have worked under the supervision of such inspirational doctors and scientists, as Tim and JudithGoodship. The vast majority of the work into atypical HUS has occurred since my time, in the last quarter century, much of it led by Tim and Judith, and undertaken by my colleagues in Newcastle.
But, the original breakthrough would never have occurred were it not for these three family groups. Their support and commitment to the research, made it all possible, and has, after centuries of suffering, started to turn the tide on this devastating disease.”
Dr Paul (Paweł) Warwicker
Consultant Nephrologist.
September 2023.
Annex A – The Devon Families’ Trees
| John Huxtable (1520-1557) | |||
| 1555- | William Huxtable | John Huxtable | 1555-1631 |
| 1585-1658 | William | Margaret | 1586-1632 |
| 1611-1679 | William | Margaret Goulde | 1609-1692 |
| 1633-1711 | Charles Frederick | Elizabeth Mary | 1631-1657 |
| Michael Slader | 1657- | ||
| 1674-1740 | Anthony | Thomas | 1680- |
| 1717- | John | William | 1721-1800 |
| 1754- | John | Eleanor | 1754-1834 |
| 1789-1860 | Sarah | William Purchase | 1791-1859 |
| 1828-1909 | William Ford | Jane | 1832-1881 |
| 1854- 1941 | Emily | Ann Berry Alford | 1855-1919 |
| 1875-1957 | George Webber | David Roberts | 1885-1936 |
| 1909-2003 | Herbert | Evelyn Marie | 1915-1993 |
| Living | Margaret | Leonard Woodward | Living |
| Living | Emma | Living | |
| Living | |||
Article No. 600
