Our primary finding for linkage to diabetic nephropathy is on chromosome 19q (triangle MLS = 3.1), with a secondary peak on chromosome 2q (triangle MLS = 2.1). The former, but not the latter, exceeds the Lander and Kruglyak criterion of triangle MLS ≥2.6 (17
) for suggestive linkage. For reference, triangle MLS values of 3.3, 2.3, and 1.7 correspond to unadjusted P
values of 0.0001, 0.001, and 0.005, respectively.
Stratification of DSPs based on proteinuria or ESRD suggested four tertiary peaks: linkage with ESRD on chromosome 1q (MLS = 1.8), linkage with proteinuria on chromosome 20p (MLS = 2.8), and linkage with two separate regions on chromosome 3q, one for proteinuria (MLS = 1.5) and another, 58 cM away, for ESRD (MLS = 1.1). We also found two chromosomal regions linked with type 1 diabetes. The most striking, not surprisingly, was on chromosome 6p (MLS = 9.2, 52 cM), confirming the well-established linkage with HLA. We also replicated IDDM15
on chromosome 6q (MLS = 3.1, 142 cM) (http://t1dbase.org/page/Loci/display/?species=Human
Two previous publications have used the DSP study design developed by our group. The first was a pilot study done at Joslin Diabetes Center (8
). Sixty-six DSPs from 52 families were used to test chromosomal regions containing genes of the renin-angiotensin system. We found no evidence for linkage with AGT
(chromosome 1q) or with ACE
(chromosome 17q); however, we did obtain suggestive evidence (MLS = 3.1 at 157 cM) on the chromosome 3q region containing ATR1
. In the second study of 83 DSPs from 73 Finnish families with type 1 diabetes, suggestive evidence was found on chromosome 3q (MLS = 2.7 at 141 cM). The sharing patterns leading to the linkage effect were not presented (20
Contrary to these studies, the strongest linkage signal in our current study is on chromosome 19q. Our enthusiasm for this finding stems not only from the magnitude of the linkage statistic but also from the sharing pattern, which occurs well within the DSP triangle. Moreover, this signal was detected in both phenotypic subsets (ESRD and proteinuria). The 1 logarithm of odds support interval around the combined ESRD/proteinuria DSP result encompasses 6.5 cm, within which there are 136 genes (94 known and 42 hypothetical or predicted) (see online appendix).
As with chromosome 19q, our secondary peak on chromosome 2q (MLS = 2.1) has a sharing pattern consistent with linkage, and the peak exists in both subsets of DSPs. The 1 logarithm of odds support interval around the combined ESRD/proteinuria DSP result encompasses 25 cM, within which there are 206 genes (100 known and 106 hypothetical or predicted) (see online appendix).
Our remaining findings stemmed from subset analysis of 50 DSPs defined by ESRD and 50 DSPs defined by proteinuria. Specifically, we found modest evidence for linkage with ESRD on chromosome 1q (MLS = 1.8). Conversely, there is evidence for linkage with proteinuria on chromosome 20p (MLS = 2.8). The results on chromosome 3q point toward two separate regions: one linked to proteinuria (MLS = 1.5) and another, 58 cM away, linked to ESRD (MLS = 1.1). One possible explanation is phenotypic heterogeneity, with some locus (or loci) related to abnormalities in urinary albumin excretion (UAE) and others related to progression to ESRD (or differences in survival rates once ESRD occurs). Recently, we demonstrated such phenotypic heterogeneity in extended families with type 2 diabetes. In particular, we performed a whole genome scan using variance components analysis to study two renal phenotypes, UAE and renal function estimated with serum cystatin C. We found strong or suggestive evidence for linkage to UAE on chromosome 5q, 7q, and 22p (21
) and independently strong or suggestive evidence for linkage to renal function on chromosome 2q, 7p, 10q, and 18p (22
Our primary finding on chromosome 19q is novel and does not overlap with any regions reported by other authors. In contrast, our secondary finding on chromosome 2q overlaps exactly with a recently reported linkage result from the Family Investigation of Nephropathy and Diabetes (FIND) study for UAE variation in several ethic groups (23
). The region from FIND spanned markers D2S410–D2S1328 located at 127–138 cM. Among our tertiary results, chromosome 1q is novel, while there is some agreement with the results on chromosome 20p and 3q. Specifically, our finding on chromosome 20p seems to validate a finding reported in a genome scan of 59 Pima Indian families with 98 sibpairs concordant for both type 2 diabetes and diabetic nephropathy (21
). An MLS = 1.8 was found near D20S115 (24.7 cM) and a two-point MLS = 1.9 was found near GATA65E01 (57.2 cM). Speaking broadly, the location of our tertiary peak on 3q is in agreement with seven previous studies (). However, the location of peaks varied between 95 and 210 cM. In our current study, we found two minor peaks 58 cM apart.
Summary of linkage results reported on chromosome 3q
Phenotypic or genetic heterogeneity may underlie these discrepant results. For example, in our pilot study, we had 66 DSPs and we found a peak with MLS = 3.1 on chromosome 3q, close to our current ESRD peak (MLS = 1.1). The current study comprised 48 original and 52 new DSPs. In the 48 DSPs, the MLS was 2.6 at 154 cM. In the new 52 DSPs, we identified only modest evidence for linkage (MLS = 1.3) at position 103 cM. The noticeable difference between the groups was significantly shorter diabetes duration in diabetic nephropathy cases in the original DSPs. Due to the small number of DSPs, it is impossible to evaluate whether this phenotypic difference could explain the different linkage results. In our previous publication, however, we demonstrated an effect of diabetes duration at onset of complications on the results of genetic studies (27
There are certain limitations to this study. First, DSP studies are prone to inflated MLS values if allele-sharing patterns are improperly estimated (e.g., due to missing parents). In our study, in 26 families, neither parent was genotyped. We attempted to minimize any bias by choosing a highly reliable genotyping platform and being diligent in removing markers failing to meet strict quality-control standards. Our confidence in the genotyping platform is based on previous validation as well as nearly 30,000 replicate quality-control genotypes from five samples in our study. As a further check, we reanalyzed the top peaks using subsets of every third marker and found consistency in each of the three subsets (data not shown). Such consistency would not be expected were the results due to genotyping errors. Inclusion of diabetic nephropathy ASPs would have been another way to mitigate the risk of misgenotyping, and methods to analyze DSPs and ASPs together exist (28
). However, as documented (7
), collection of diabetic nephropathy ASPs is extremely difficult and, being concordant for both diabetic nephropathy and type 1 diabetes, these siblings would present an additional challenge in terms of interpretation. Second, while 100 DSPs are sufficient to detect a major locus, the power to detect moderate/minor genetic players is more modest. Moreover, the subanalyses we performed by distinguishing ESRD from proteinuria was a post hoc analysis that should be viewed as exploratory. Third, mortality in ESRD is quite high (30
), leading, potentially, to survival bias. This could manifest through reduced power among the 50 ESRD DSPs, which might be misinterpreted as phenotypic heterogeneity, or through drop-out of poor survivors with ESRD such that the genetic variants associated with good survival would appear to be related to diabetic nephropathy susceptibility. Fourth, while two loci (DSP triangle MLS = 3.1 on chromosome 19 and ASP triangle MLS = 3.1 on chromosome 6q) were “suggestive” (17
), none except HLA achieved “significance” (triangle MLS ≥4.0). Therefore, our results must be considered largely as hypothesis generating. Given the difficulty in assembling collections of sibs concordant for type 1 diabetes and discordant for diabetic nephropathy, a more practical approach may be to now focus on detecting association using resources such as GoKinD (31
In conclusion, our study has provided two sets of genome scan results: one for type 1 diabetes using 130 ASPs and another for diabetic nephropathy using 100 DSPs. The type 1 diabetes scan overwhelmingly confirmed the HLA region on chromosome 6p and it provided additional support for IDDM15. The diabetic nephropathy scan introduced a new candidate region on chromosome 19q and confirmed linkage to UAE on chromosome 2q reported by the FIND study. Using exploratory subset analysis based on the degree of diabetic nephropathy, we found a novel locus on chromosome 1q and confirmed a locus on chromosome 20p described in Pima Indians. Finally, we found evidence for two loci on chromosome 3q, adding to the list of positive studies on this chromosome. Taken together, the results from our diabetic nephropathy scan suggest several loci as candidates for susceptibility, none of which appear to the sole determinant of diabetic nephropathy.