Complicating Factors In Mapping The Tyrolean Nicc Gene

Although the Tyrolean population exhibiting NICC is an excellent resource for mapping the underlying gene defect using an IBD approach, there are a number of complicating factors in this study. The power of a haplotype-sharing approach is partly determined by the age of the mutation (Fig. 4). In general, such an approach works best when the common ancestor is no further than 10-12 generations away. Although we anticipate that the founder who brought in the Tyrolean NICC mutation lived no more than 10-12 generations ago, we have no proof that this is the case. If the mutation has been present in the Tyrolean population for a much longer time, the density of the current marker set might not be sufficient. However, this problem could, in part, be overcome by adding more markers to increase the resolution of the map. Nevertheless, we should realize that the reliability of the study is still lessened by two factors: (1) the small number of chromosomes being studied and (2) the complexity of screening obligate gene carriers. Because the NICC patients are unavailable, we cannot determine which of the two homologous chromosomes in each parent carries the NICC mutation (i.e., we do not know which combinations of haplotypes in the parents to compare in order to identify the mutation).

In the meantime, we have been able to increase the number of people in the study by collecting more DNA samples from the Tyrol. DNA is now available from 11 Tyrolean NICC families, comprising 18 obligate gene carriers, 1 surviving NICC patient, and 23 healthy siblings. In addition,

Fig. 8. Two examples of results from the genomewide association analysis in the eight Tyrolean NICC families. The X-axes represents the length of the chromosome (in cM); the position of the individual markers is indicated by a triangle. The 7-axes shows the lod score values. The black horizontal bars give the lod score value for the corresponding marker (indicated by the triangle in the center of the bar); the gray horizontal bars are the regions that were excluded for the presence of the NICC gene. (A) The data from chromosome 13. The part of the chromosome containing the ATP7B gene was excluded in this study. (B) The data from chromosome 6. The majority of chromosome 6 can be excluded. However, marker D6S1007 (at approx 160 cM) shows a lod score larger than +1 (indicated by an arrow). This region needs to be further investigated with a much denser set of markers.

Fig. 8. Two examples of results from the genomewide association analysis in the eight Tyrolean NICC families. The X-axes represents the length of the chromosome (in cM); the position of the individual markers is indicated by a triangle. The 7-axes shows the lod score values. The black horizontal bars give the lod score value for the corresponding marker (indicated by the triangle in the center of the bar); the gray horizontal bars are the regions that were excluded for the presence of the NICC gene. (A) The data from chromosome 13. The part of the chromosome containing the ATP7B gene was excluded in this study. (B) The data from chromosome 6. The majority of chromosome 6 can be excluded. However, marker D6S1007 (at approx 160 cM) shows a lod score larger than +1 (indicated by an arrow). This region needs to be further investigated with a much denser set of markers.

DNA samples have also been collected from 7 German NICC families, comprising 3 surviving NICC patients, 13 gene carriers (i.e., parents), and 13 healthy siblings. This material will be used for follow-up studies on the genomewide screen being performed for the Tyrol.

In addition, we have also collected DNA from 13 ICC families in India. Given the similarities in phenotypes between ICC and NICC and the intake of high environmental copper as a precipitating factor, it is conceivable that both diseases are the result of mutations in the same gene. These ICC families have two advantages over the Tyrolean NICC families: (1) The patients are still alive because they were diagnosed in time and treated successfully and (2) the patients parents' are more closely related than the parents in the Tyrolean families. We now have DNA from a total of 12 ICC patients and 51 relatives for a genomewide screen. The parents in some of these families are first-degree relatives, which may allow the identification of homozygosity around the ICC locus.

9. IS THERE AN ANIMAL MODEL FOR NICC?

The identification of an animal model with phenotypic similarities to Tyrolean NICC could prove a useful tool to studying the disease mechanism because it is usually easier to map genes in animal models because of (1) inbreeding of the strains, (2) the large size of pedigrees, and (3) the shorter generation time. There are a few current animal models in which ingestion of copper has produced cirrhosis of the liver: the copper toxicosis in Bedlington terriers (57,58) and in sheep (59,60). Copper toxicosis in North Ronaldsay sheep (so-called Ronaldsay copper toxicosis or RCT) shows many similarities with NICC. The North Ronaldsay sheep are an inbred population that do well on the island of North Ronaldsay, in the Orkneys, Scotland, because the sea grass contains very low copper concentrations (Cu < 5 ppm). When these sheep are brought to the mainland, where they switch to a normal diet, copper accumulates in the liver and they develop hepatic copper toxicosis. The histo-logical features include Mallory bodies and pericellular fibrosis, very similar to the pathology of NICC. However, there are currently no good marker maps available for the sheep genome to carry out genetic studies.

Copper toxicosis in Bedlington terriers was suggested as a good model for Tyrolean NICC because of the similarities in inheritance pattern and phenotype: hepatic copper toxicosis and normal cerulo-plasmin levels (61,62), although the terriers show no eye and brain lesions as in Wilson's disease (61,62). We were recently able to show that the copper toxicosis in Bedlington terriers is not the result of a mutation in the canine ATP7B gene (63). The copper toxicosis gene has recently been mapped to canine chromosome 10, region q26. This part of the dog genome is homologous to human 2p13-p16 (63,64). Based on genetic and physical mapping, the location of the copper toxicosis gene in Bedlington terriers has now been refined to a region of less than 5 million base pairs. Although the resources available for the dog genome are also rather limited, the cloning of the copper toxicosis gene will benefit from the progress being made in the human genome. So far, the genes and their order on canine 10q26 is identical to those on human 2p13-p16 (64). Once the complete sequence of human 2p13-p16 is released, it will therefore be possible to screen all of the canine homologs of the genes present in 2p13-p16 for mutations in Bedlington terriers that have copper toxicosis.

However, the genomewide screen in the Tyrolean NICC families did not reveal any evidence for association with markers from 2p13-p16, indicating that copper toxicosis in Bedlington terriers and Tyrolean NICC are two distinct genetic entities as a result of mutations in two different genes associated with copper metabolism.

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