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Mutation mapped

International work coordinated by a Brazilian researcher points to a gene that causes serious kidney disease in children

In an article published on-line on March 15 in the American Journal of Human Genetics, one of the most respected magazines in the area of genetics, researcher Luiz Fernando Onuchic, from the School of Medicine of the University of São Paulo (USP), in collaboration with 17 colleagues from the United States and Europe, identifies and describes, with a wealth of detail, the gene that unleashes a severe and rare hereditary disease when it is the target for mutations in its two copies. Affecting particularly the newly born and children, disease is called autosomal recessive polycystic kidney disease.

The gene in question is PKHD1 (which stands for Polycystic Kidney and Hepatic Disease 1), an enormous and complex gene, located in chromosome 6, which produces a probable set of hitherto unknown proteins, named by the authors of the study as polyductine. “This gene is involved in all the typical forms of the disease”, says Onuchic, whose name appears ahead of the other researchers, since the Brazilian was the one mainly responsible for the discovery of the gene. “We found different mutations in this gene that may lead to the disease”. Besides the scientist from USP, whose studies are funded by FAPESP, the group that took part in the work – actually, an international consortium – includes researchers from four universities in the United States (Johns Hopkins, Yale, Alabama at Birmingham and Case Western Reserve) and one from Germany (Aachen Technical University).

“The identification of the gene responsible for this hereditary problem is a landmark and opens the gates for the discovery of a cure for the disease”, comments Gregory Germino, from Johns Hopkins University, the leader of the consortium. “We can now start to develop a DNA test that will probably be able to be used to identify potential carriers (of the mutant gene) or affected individuals”. This month, the study by the group – which has now entered in the United States with the patent request for the new genes and the proteins it produces – is coming out in the printed version of the American Journal of Human Genetics .

In a predominant tendency amongst the major scientific periodicals, which compete for the best works by researchers, the article by Onuchic and his colleagues was published in advance electronically by the American magazine, due to its high impact, for bringing a very important discovery. And also because a rival group of researchers from the Mayo Clinic, United States, had also published on-line, on February 4, an article that also gives an account of the discovery of PKHD1 in the British magazine Nature Genetics .

One detail has to be made clear in this story: the group which the Brazilian was part of submitted its article to the appreciation of the American Journal on February 1st, before Nature Genetics published electronically the study by the scientists from the Mayo Clinic. In practice, the two teams, working independently and with different strategies, arrived simultaneously at the same result. “The scientific community understands the peculiarities of competitive science and is splitting the credit for this discovery between the two groups”, Onuchic explains .

The existence of autosomal recessive polycystic kidney disease, which affects the kidneys and the bile duct, is unknown to most people, because of its low incidence amongst the population. Known in English by the acronym ARPKD, the ailment attacks one out of each 20,000 live births. In a country like Brazil, where 3 million babies leave the delivery room alive every year, this is equivalent to saying that 150 new cases of the disease are expected every 12 months. In up to half of the occurrences, the children that are born affected by ARPKD die straight after childbirth. And up to 20% of the bearers of this ailment do not reach the age of 5, and there is no specific treatment to prevent or to control the development of the disease.

Dilated kidneys
The kidneys of the children who show the severe form of the disease when they are born increase, with a weight of up to ten times normal, and cystic dilations in the collecting ducts, which are segments of the functional structures of this organ. “In a child of the age of 2 who has the disease, taken care of in our hospital, the kidneys grew to a length of 15 centimeters, when the normal would be around 6 centimeters”, comments Onuchic. The patients who survive the initial period of life have a variable clinical evolution. The main culprits for clinical deterioration and death in these patients, are arterial hypertension, liver problems, and the progressive loss of kidney functions.

Roughly half of those affected lose completely their kidney functions before the age of 10. In these cases, the children come to depend on dialysis or a kidney transplant to stay alive. “As there is no specific treatment for the disease, we just take care of the complications and offer the patient therapeutic support for control, as far as possible, of the manifestations of the disease”, the researcher comments. In the Children’s Institute at the Hospital and Clinics of São Paulo, 25 cases of the disease are currently being followed up.

Until PKHD1was discovered, medicine knew that the disease was caused by defects in a single one of the 30,000 genes present in the human body, but it had not been able to specify which one of them was the cause of the problem. Another characteristic of the ailment that was in the public domain: the disease, as its very name made clear, was recessive. For the ailment to develop, the child had to inherit the two defective copies of the then mysterious gene that caused ARPKD. Those who had just one copy of the gene with a problem would not develop the disease, although they could transmit it to their children, if their partner also carried a mutation in this same gene.

As one knows, every human being has two copies of each one of his genes located in the non sexual chromosomes, one inherited from the father and the other from the mother. All this had already been known to science for a long time. However, what was missing was to locate this altered gene in the disease, which has happened only now. The first great step forward in the quest for the genetic root of ARPKD happened in 1994, when German researchers showed that the gene involved in the disease was to be found in a region of chromosome 6.

It was the first step forward, but a limited one. After all, this region was a very large genomic segment, which included an enormous number of genes, some of them known, others not. In the following year, with the impetus from this first find, the group headed by Germino, from John Hopkins University, where Onuchic was working at the time, put together the international consortium that was to decipher the genetic cause of the disease, seven years later.

Closing in
This feat was achieved with the help of a strategy known as positional cloning, an approach frequently used to discover genes related with complex diseases, whose the primary biochemical effect , the one that brings on the clinical problem is unknown. This was the case of ARPKD. The researchers from the Mayo Clinic, under the coordination of Peter Harris, used a different approach to identify the gene of the disease – they found PKHD1 using a model of a recessive polycystic kidney disease developed spontaneously in rats.

Some of the characteristics of this model were similar to those of the disease in humans. What does positional cloning consist of? First, the scientists have to discover in which region of a chromosome the gene that causes the disease in question is to be found. This is done with the use of a method called linkage analysis and a progressive reduction in the interval of interest. The researchers either identify genes in the laboratory or search in international databases those that through their structure or functions show the potential for housing the mutations that may cause the disease.

Finally, it has to be shown that mutations lead to the clinical problem (finding mutations of this gene in the DNA of patients with ARPKD). With the strategy of positional cloning, then, the siege of the gene linked to the disease gets tighter and tighter, until few suspects are left over. It is a process that calls for patience and concentration and which can take years. “It is more or less like trying to discover in a given population who is the bandit that committed a crime”, is Onuchic’s comparison.

In the case of PKHD1, the consortium’s earlier studies indicated that the mutated gene in the autosomal recessive polycystic kidney disease lay in the region of chromosome 6, which extended for 834,000 base pairs, where, after a painstaking analysis, the scientists arrived at several suspect expressed sequences. Among them, one that the researchers in the consortium successfully showed to be the much dreamt of gene associated to the disease, PKHD1. The characterization of this enormous and complex gene showed that it extended over a genomic segment of about 470,00 base pairs and that it expressed itself chiefly in the kidneys, the organ most affected by the disease. “At the beginning of this process, we did not know if the expressed sequencesthat are part of this gene belonged to one gene or to two”, Onuchic says.

Later studies revealed that a single gene was involved. The proof that this gene was the one related to ARPKD was supplied by evidence produced by Onuchic and his colleagues. The researchers looked for – and found – potentially pathogenic mutations in the PKHD1 belonging to 21 chromosomes from patients with the autosomal recessive polycystic kidney disease. Located in various parts of the gene, these mutations were not present in 120 chromosomes of healthy persons, the so-called control group.

In addition, during this hunt for the molecular origin of ARPKD, the researchers ended up discovering another new gene, which had never been described in the scientific literature and which bore no relation to the kidney disease. Called ML-1, this new gene apparently takes part in the inflammatory allergic response from asthma, a very frequent health problem. The account of its identification was published in an issue at the end of last year of the Journal of Immunology. ML-1 is now a target for studies carried out by the immunology sector at Johns Hopkins University.

Besides being the gene that causes this rare ailment that preys mostly on babies and children, PKHD1 may also help science to understand a bit better certain mechanisms related to the human genome. There are indications that form the foundations for this, shall we say, didactic and revealing character of the new gene. The genomic size of the PKHD1 (or the space it takes up in the chromosome) makes it one of the largest human genes ever characterized, and its longest transcript is formed with 67 parts (exons).

Another of the gene’s peculiarities is its great capacity for generating different transcripts, through a process that molecular biologists call alternative splicing. Studies following the work of Phillip Sharp (1993 Nobel Prize), who discovered the basic mechanisms of splicing, changed the way that genes were looked at. Until then, science postulated that each gene contained instructions for the cells to produce a single kind of protein. One gene, according to this precept, could process and use in just one way its sequence of bases, including adenine (A), cytosine (C), guanine (G) and thymine (T). Consequently, the result of this processing always had to be the same, a stable chemical recipe (technically called gene transcript or just transcript) which would always provide the same information for the cells, which, obviously, would end up always making the same protein.

Peculiar gene
It is, however, known today that many human genes are capable of using their exons in different combinations. In other words, some genes may generate different products of splicing, which may lead to the formation of more than one kind of protein, if they are translated. This is the case – and with particular intensity – of the gene related to the ailment. “We have now identified over 20 transcripts of the PKHD1 gene, but the total may be much greater”, comments Onuchic. “This multiplicity of variants from splicing is an uncommon characteristic in genes from mammals”.

In the scientific literature, there is a record of similar findings for the neurexin family of genes. Three in number, these genes may originate, by means of alternative splicing, more than 1,000 different forms of proteins. The analysis of the structure of the polyductine suggests that the protein codified by the PKHD1 gene may work as a receptor (of cells), but leaves open the possibility of acting as a linking molecule (which teams up with another protein), or as an enzyme associated to the membrane.

In the case of PKHD1, the scientists still cannot specify how many variants of polyductine are produced by the many transcripts codified by the gene. They do know, though, that several transcripts, if they are translated, will lead to the synthesis of two forms of protein: polyductine-M and polyductine-S. Each form, in turn, constitutes a group with subvariants of the protein. While the polyductine-M group includes the forms of the protein probably associated with the plasmatic membrane, the polyductine-S group includes the proteins that can be secreted.

Amongst all these subvariants of the protein, the biggest – and probably the most important – is a polyductine-M made up of 4,074 aminoacids (the chemical units of the proteins). Why is it looked at with more attention than the others are? Because this form of polyductine is produced by the only transcript that could be altered by all the mutations described so far. In other words, the results of the research suggest that this protein proves to be truncated or defective in all the patients in whom mutations have already been detected in both the copies of the gene.

“However, we still do not know if the disease results just from the reduction, below critical levels, of the quantity of the longer normal protein, or if the loss of a functional balance between the different forms of the protein, as a result of mutations, is also part of the genesis of the problem”, Onuchic comments. According to the researcher, the structure of the larger proteins produced by the neurexins is similar to the structure of polyductine-M.

One of the immediate challenges for the researchers is to establish possible relations between the various kinds of mutation found in the gene and the nature and severity of the clinical manifestations of ARPKD. Another challenge, which should be cleared up progressively and over time, is to understand the role of this new protein in healthy individuals, and how its alteration leads to ARPKD. “We hope to be able, one day, to manipulate the ways of signaling related to this protein, so as to find a way round this genetic disorder”, wishes Gregory Germino, from Johns Hopkins University, expressing the common desire of the team.

The project
Molecular Genetics of the Human Autosomal Recessive Polycystic Kidney Disease (nº 00/00280-3); Modality Regular line of benefits for research; Coordinator Luiz Fernando Onuchic – School of Medicine, USP; Investment R$ 272,389.66