National Disease Research Interchange Annual Progress Report: 2010 Formula Grant

Size: px

Start display at page:

Download "National Disease Research Interchange Annual Progress Report: 2010 Formula Grant"

Chrystal Miller
5 years ago
Views:

1 National Disease Research Interchange Annual Progress Report: 2010 Formula Grant Reporting Period July 1, 2011 June 30, 2012 Formula Grant Overview The National Disease Research Interchange received $62,393 in formula funds for the grant award period January 1, 2011 through June 30, Accomplishments for the reporting period are described below. Research Project 1: Project Title and Purpose The Development of Diabetic Retinopathy: Going from Genetic Susceptibility to Functional Analysis - The Human Biological Data Interchange (HBDI) database is a vast repository of family and medical information focused on the study of type 1 diabetes (T1D) and its complications. Previously, our work has confirmed that genetic factors influence susceptibility to microvascular complications of diabetes. We will now test for functional gene expression differences in retinopathy-affected vs. non-retinopathy-affected diabetic retina and correlate the expression data with a) the results of case-control association studies in HBDI samples previously typed and b) the results of family-based association and linkage analysis on a candidate locus for susceptibility to retinopathy integrating newly typed HBDI family members. We will also continue our program of administering follow-up questionnaires to T1D families, allowing us to track clinical changes in T1D and complications. Anticipated Duration of Project 1/1/2011 6/30/2013 Project Overview The overall goal of this project is to identify genetic variants that lead to susceptibility or protection from the microvascular complications of diabetes (MCD). We are specifically searching for genetic variants that predispose to development of, or protection from, type 1 diabetes (T1D) complications but that are not necessarily related to T1D susceptibility. Identifying such genetic variants will allow us to predict which patients are at greatest risk for the blindness, kidney failure and nervous system disease caused by MCD, as well as determine the mechanisms by which high blood glucose leads to MCD. Our specific aims are to: 1) Plan and execute gene expression profiling comparing retinopathy-affected and non-retinopathyaffected diabetic retinas and normal retinas 2) Perform family-based linkage and association analysis to identify loci and alleles involved in MCD expression 3) Continue follow-up on identified T1D families. National Disease Research Interchange 2010 Formula Grant Page 1

2 Specific Aims: Aim 1) While SNP analysis led to the discovery of genes of potential interest, this is still only a statistical finding. Functional analysis is needed to prove the biology underlying the statistical finding and indicate the biologic pathway by which known genetic variants lead to protection from/susceptibility to retinopathy. We plan microarray gene expression profiling to identify individual genes whose expression is correlated with the retinopathy phenotype (confirming that the candidate gene found by SNP analysis plays a role in the pathway) and to identify multiple genes that are interactively involved in mediating retinopathy pathogenesis. Aim 2) Perform family-based association and linkage analysis, stratifying the data on observations such as HLA type, presence/absence of other MCDs, and specific genotypes at other loci associated with T1D, as well as identify new loci linked to MCDs. This will allow us to test for possible interaction of loci in the development of MCD. Family-based association analysis will also eliminate the possibility of biased results due to population stratification. Aim 3) Continue our annual follow-up program to track the development and progression, or lack thereof, of MCD in families over time. Principal Investigator John T. Lonsdale, PhD Research Director National Disease Research Interchange 1628 John F. Kennedy Boulevard 8 Penn Center, Suite 1500 Philadelphia, PA Other Participating Researchers Cathie Miller, PhD, Davlyn Tang, BS employed by National Disease Research Interchange Maria Cristina Monti, PhD employed by Columbia University, New York Expected Research Outcomes and Benefits The clinical and familial data contained in the HBDI database is an immense and invaluable resource for the development and testing of hypotheses regarding the genetic and environmental factors leading to susceptibility to diabetic complications. It is the complications of diabetes that eventually take the biggest toll on the lives of T1D patients. The most common complications include: 1) retinopathy of the eye which distorts vision and leads to blindness, 2) neuropathy a degenerative nerve condition that can cause excruciating pain in the extremities, decreased blood flow and even require amputation, and 3) nephropathy a serious kidney disorder that prevents proper filtering of the blood which may require dialysis or transplantation. Better ways of preventing, treating and curing diabetes will inevitably stem from a better understanding of those factors that contribute to complications. We have already demonstrated National Disease Research Interchange 2010 Formula Grant Page 2

3 that SNPs within specific genes are associated with susceptibility to development/protection of diabetic complications. The next step is to explore the functional differences in expression patterns to determine if there is a correlation with the specific genes identified through SNP analysis. The information contained in the HBDI database, coupled with newly available genetic data, provides us a unique opportunity to screen these culprit genes for specific linkage markers to complication occurrence. These studies hope to provide a mechanism for better screening, prevention and treatment of complications with the ultimate aim of reducing the heavy burden on patients and society. Summary of Research Completed Aim 1) Procurement of eyes and dissection of retinas is complete and microarray gene expression profiling is being carried out. Specimens have been recovered from 10 donors without any history of diabetes or physical evidence of retinopathy; 11 donors with diabetes but without any history or physical evidence of retinopathy and 12 donors with diabetes and with physical evidence of retinopathy Aim 2) Family recruitment: Of the HBDI ascertained families (n=427), fifteen families were excluded due to missing genotype information; thus, there were a total of 415 families available for linkage analysis. (Those designated affected had one or more complications. The unaffected had T1D and no complications.) Among all families, 50 (12%) had exactly 2 complications-affected members and 0 unaffected (T1D only) siblings, 67 (16%) had exactly one affected and one unaffected sibling. 210 families (51%) had 2 siblings unaffected and 0 affected. (The unaffected only families were used for a sib pair analysis; see below.) The remaining 88 families (21%) had either 1 sibling or more than 2 siblings with various affected-unaffected permutations. Phenotype definitions: An individual was designated as affected if he/she had T1D with onset <30 years of age and the presence of retinopathy, nephropathy and/or neuropathy. An individual was designated unaffected if he/she had T1D with onset <30 years of age and was free of complications for at least 20 years. Phenotypes definitions included any complication, meaning T1D individuals with the presence of at least one complication. We also analyzed families in which the proband had only a given complication, i.e., retinopathy alone, nephropathy alone, and neuropathy alone. For each phenotype definition, we stratified families based on the DR3 or DR4 status of the family s proband. Genotyping: A total of 5966 markers were genotyped across the genome. Genotyping was performed by the Center for Inherited Disease Research (CIDR), a division of the National Human Genome Research Institute. Linkage analysis: This was performed using logarithm of odds (LOD) score analysis. Multi-point heterogeneity LOD scores (HLOD scores) were calculated using the Genehunter program). We assumed a penetrance of 95% and a dominant mode of inheritance for the analyses reported here. We report here on our current findings in the HLA region. The HLA region, particularly the DR and DQ loci, have the strongest genetic effect on diabetes susceptibility, thereby observing whether the region also affects complications expression. National Disease Research Interchange 2010 Formula Grant Page 3

4 Linkage results on chromosome 6: When we look at the phenotype of presence of any complication, that is, either retinopathy, nephropathy and/or neuropathy could be present, we see the following linkage pattern (recall T1D patients without complications are classified as unaffected ). What is striking is that in addition to the single T1D peak over HLA, we see indications of two separate genetic influences for complications, with the rightmost peak and leftmost peak lying outside the HLA region. Note that even though the analysis penetrance was 95%, the linkage information for complications is still statistically significant for two of the peaks (Figure 1). In order to determine if there was interaction between HLA genotype status and complications risk, we stratified on the proband s HLA status, that is, we included in the analysis only families in which the proband had a particular HLA allele or genotype. We first stratified families by the presence of DR3 in the proband. We found that the height of the HLOD peak outside of the HLA region decreased, suggesting a protective effect for DR3 on complications risk (Figure 2). The signal in the HLA region decreased from 5.8 to 3.1, with a dramatic reduction in the HLA-centromeric and telomeric linkage peaks outside the HLA region as well. We saw an even greater decrease in the linkage peaks after excluding families of probands with DR3/DR4 (Figure 3). With a pure pool of DR3 positive families, (that is, with only DR3/X proband families included (X=any allele but 4)), the linkage signal for both the HLA region as well as the peaks outside the HLA region barely exceed 1. We then we ran the linkage analysis on families in which the proband was DR4 positive (which include DR3/4s). We observed a different trend (Figure 4). The linkage peak in the HLA region remained high (HLOD 5.2). There were also three additional peaks outside of the HLA region. These peaks were absent from the linkage scan seen for the DR3 positive families. Further, if we exclude the DR3/4-carrying individuals from this population of DR4 positive families (i.e., the DR3/4s) the HLODs for all four peaks remain high (Figure 5). With comparable numbers of families as those in the pure DR3 positive families (without DR3/4 heterozygotes), we retain significant HLODs and observe distinct peaks that may be involved in expression of complications. If we confine ourselves to the phenotype of retinopathy, we observe a picture similar to that for the any complication phenotype (Figure 6). Although the strength of the linkage peaks in and outside the HLA region have diminished, they remain significant. To determine if DR3 and DR4 had the same effect on retinopathy alone, we stratified families with retinopathy based on the DR3 and DR4 status of the proband, just as we did for families with any complication. For DR3 positive families with retinopathy alone, we see a pattern of linkage peaks similar to that of any complication (Figure 7). The major peak is over the HLA region, while the peak centromeric to HLA remains distinct (HLOD>2). The region distal to the HLA peak increases steadily, but it no longer appears to form a distinct peak. When we removed the DR3/4 heterozygotes so that there are no DR4 alleles in probands, we observe an almost identical pattern to the linkage scan for any complication (Figure 8). The 3 distinct peaks barely exceed National Disease Research Interchange 2010 Formula Grant Page 4

5 1. When we look only at the DR4 positive families, we see a similar pattern for the DR4 positive families as we did for the any complication classification (Figure 9). The linkage peak at the HLA locus remains significant, but 10-fold lower than what we saw for any complication. The second peak, centromeric to the HLA region, becomes the most dramatic signal for retinopathy (HLOD>4). The other two peaks remain highly suggestive of linkage. When we remove the DR3/4 heterozygotes, the pattern remains (Figure 10). The most HLA-distal of the two distal peaks remains the most distinct peak, approaching an HLOD of 3. All other peaks diminish slightly, though they remain suggestive of linkage (HLOD>2), including the HLA peak. This indicates that among DR4-positive families, there may be a region of interest that lies outside of the HLA region involved in the expression of microvascular complications, especially for retinopathy alone. Further, DR3 families may be protected from the development of complications because of the disappearance of the linkage signal both in and near the HLA region for families with a DR3 positive proband. We also performed linkage on nephropathy alone and stratified on the DR3 and DR4 status of the proband. For this phenotype, we see an entirely different linkage pattern compared to retinopathy. For nephropathy alone (unstratified), we do not see a linkage peak in the HLA region. Rather, distal to the HLA locus, there is a linkage peak with an HLOD of 1.9, suggestive of linkage (Figure 11). When we stratify on DR3 positive families, this peak remains, although the strength of the peak is diminished (Figure 12). This diminution is likely due to decreased sample size, since the numbers of families is reduced from 64 to 24, approximately two-thirds. When the DR3/DR4 heterozygotes are removed, the linkage peak remains unchanged (Figure 13). Upon stratifying by the DR4 positive families, we find no evidence for linkage at that location (Figure 14), nor does it appear when we remove the DR3/DR4 positive families (Figure 15). For neuropathy alone, we observed no evidence for linkage in either the unstratified or in the DR3 and DR4 stratified linkage analyses (data not shown). However, the sample size of these families was too small to expect a good signal. In summary: We see distinct linkage peaks for retinopathy alone and for nephropathy alone. It appears that the HLA region may be involved in the expression of retinopathy. Stratification of proband s DR status provides evidence that DR3 is protective for retinopathy. We see no evidence for the involvement of HLA in the expression of nephropathy, but rather a distinct region outside of HLA (seen in a sample of DR3 positive families) may be contributing to the expression of this complication in particular. In conclusion, these preliminary analyses suggest several new hypotheses to investigate, including the suggestion of other chromosome 6 loci that contribute to complications but not to type 1 diabetes. Aim 3) 1000 questionnaires have been sent to registry participants to continue the annual National Disease Research Interchange 2010 Formula Grant Page 5

6 program of participant follow-up using an updated family questionnaire to track development/progression or lack of development/progression of microvascular complications among patients with both T1D and T2D. Figure 1. Any complication (n=156 families) Figure 2. Any complication, DR3-positive families (n=66 families) Figure 3. Any complication DR3-positive families (no DR3/DR4 heterozygotes), (n=40 families) Figure 4. Any complication, DR4-positive families (n=62 families) National Disease Research Interchange 2010 Formula Grant Page 6

7 Figure 5. Any complication, DR4-positive families (no DR3/DR4 heterozygotes), (n=38 families) Figure 6. Retinopathy (n=145 families) Figure 7. Retinopathy, DR3-positive families (n=61 families) Figure 8. Retinopathy, DR3-positive families (no DR3/4 heterozygotes), (n=37 families) Figure 9. Retinopathy, DR4-positive families (n=59 families) Figure 10. Retinopathy, DR4-positive families (no DR3/4 heterozygotes), (n=35 families) National Disease Research Interchange 2010 Formula Grant Page 7

8 Figure 11. Nephropathy (n= 64 families) Figure 12. Nephropathy, DR3-positive families (n= 24 families) Figure 13. Nephropathy, DR3-positive families (no DR3/4 heterozygotes), (n= 15 families) Figure 14. Nephropathy, DR4-positive families (n= 21 families) National Disease Research Interchange 2010 Formula Grant Page 8

9 Figure 15. Nephropathy, DR4-positive families (no DR3/4 heterozygotes), (n= 12 families) National Disease Research Interchange 2010 Formula Grant Page 9

National Disease Research Interchange Annual Progress Report: 2010 Formula Grant

National Disease Research Interchange Annual Progress Report: 2010 Formula Grant Reporting Period July 1, 2012 June 30, 2013 Formula Grant Overview The National Disease Research Interchange received $62,393