S.P. From linkage analysis to linkage disequilibrium mapping: the case of HRPT2 ( a gene mutated in Hyperparathyroidism-jaw tumor syndrome) by Silvano Presciuttini Dept.of Biomedicine, University of Pisa, Italy and IDRB, NHGRI, Baltimore, USA  Linkage analysis is a powerful tool to locate genes of strong effect in a chromosomal region. However, it is not very efficient for fine mapping and has limited power to detect genes of ‘modest’ effect.  In contrast, association analysis is highly efficient for fine mapping, as appreciable linkage disequilibrium exists in human between loci with recombination fractions of less than 1-2%

S.P. The classical linkage analysis method The classical Lod score method of linkage analysis was devised to locate genes responsible for Mendelian traits The classical Lod score method of linkage analysis was devised to locate genes responsible for Mendelian traits  Typically, a Mendelian trait shows a biunivocal correspondence between a genotype and a phenotype In more general terms, classical linkage analysis necessitates that the transmission model for a trait be specified In more general terms, classical linkage analysis necessitates that the transmission model for a trait be specified  If the model is misspecified, then artefactually negative lod scores may be produced In classical linkage analysis the transmission model is fixed (possibly with parameter values obtained from segregation analysis) and the likelihoods of the disease and marker data are compared under the null hypothesis of no linkage and the alternative hypothesis of linkage

S.P. Looking for Mendelian phenotypes in Hyperparathyroidism Hyperparathyroidism affects 1 in 1,000 individuals in the general population, and 85-90% of cases are caused by excess hormone production due to adenoma development in one of the parathyroid glands. Hyperparathyroidism affects 1 in 1,000 individuals in the general population, and 85-90% of cases are caused by excess hormone production due to adenoma development in one of the parathyroid glands. Approximately 5%-10% of patients have a family history of parathyroid tumors. A proportion of such familial cases occur in combination with other tumors, such as in Multiple Endocrine Neoplasia 1 (due to MEN1, located at 11q) and in Multiple Endocrine Neoplasia 2A (due to RET, located at 10q ) Approximately 5%-10% of patients have a family history of parathyroid tumors. A proportion of such familial cases occur in combination with other tumors, such as in Multiple Endocrine Neoplasia 1 (due to MEN1, located at 11q) and in Multiple Endocrine Neoplasia 2A (due to RET, located at 10q ) However, hyperparathyroidism appears to be the sole manifestation in a subset of families (Familial Isolated Hyperparathyroidism, FIH), and in others it is associated with ossifying fibromas of the mandible, bilateral renal cysts, hamartomas, and Wilms tumor (Hyperparathyroidism – Jaw Tumor syndrome, HPT-JT). However, hyperparathyroidism appears to be the sole manifestation in a subset of families (Familial Isolated Hyperparathyroidism, FIH), and in others it is associated with ossifying fibromas of the mandible, bilateral renal cysts, hamartomas, and Wilms tumor (Hyperparathyroidism – Jaw Tumor syndrome, HPT-JT).

S.P. The HPT-JT syndrome HPT-JT is inherited as an autosomal dominant syndrome with a high penetrance for the presence of parathyroid tumors HPT-JT is inherited as an autosomal dominant syndrome with a high penetrance for the presence of parathyroid tumors Unlike MEN1 MEN2, which cause parathyroid tumors that are almost universally benign, those in HPT-JT are malignant in 15% of cases. This is also remarkable in comparison to sporadic parathyroid tumors, which are malignant in less than 1% of cases. Unlike MEN1 MEN2, which cause parathyroid tumors that are almost universally benign, those in HPT-JT are malignant in 15% of cases. This is also remarkable in comparison to sporadic parathyroid tumors, which are malignant in less than 1% of cases. Approximately 30% of HPT-JT patients develop rare ossifying fibromas primarily of the mandible and maxilla, which are different from the osteoclast-dominated, parathyroid hormone responsive “brown” tumors of severe hyperparathyroidism that can occur at any skeletal site. Approximately 30% of HPT-JT patients develop rare ossifying fibromas primarily of the mandible and maxilla, which are different from the osteoclast-dominated, parathyroid hormone responsive “brown” tumors of severe hyperparathyroidism that can occur at any skeletal site. Kidney manifestations may also be present in HPT-JT and include most commonly (10%) bilateral renal cysts, but also renal hamartoma, polycystic kidney disease, and Wilms tumor. Kidney manifestations may also be present in HPT-JT and include most commonly (10%) bilateral renal cysts, but also renal hamartoma, polycystic kidney disease, and Wilms tumor.

S.P. HPT-JT: early linkage studies Szabo et al., Am J Hum Genet 56: (1995) Hereditary hyperparathyroidism-jaw tumor syndrome: the endocrine tumor gene HRPT2 maps to chromosome 1q21-q31 Szabo et al., Am J Hum Genet 56: (1995) Hereditary hyperparathyroidism-jaw tumor syndrome: the endocrine tumor gene HRPT2 maps to chromosome 1q21-q31 Teh et al., J Clin Endocrinol Metab 81: (1996) Autosomal dominant primary hyperparathyroidism and jaw tumor syndrome associated with renal hamartomas and cystic kidney disease: linkage to 1q21-q32 and loss of the wild type allele in renal hamartomas. Teh et al., J Clin Endocrinol Metab 81: (1996) Autosomal dominant primary hyperparathyroidism and jaw tumor syndrome associated with renal hamartomas and cystic kidney disease: linkage to 1q21-q32 and loss of the wild type allele in renal hamartomas. Hobbs et al., Am J Hum Genet 70: (2002) Revised 14.7-cM locus for the hyperparathyroidism-jaw tumor syndrome gene, HRPT2. Hobbs et al., Am J Hum Genet 70: (2002) Revised 14.7-cM locus for the hyperparathyroidism-jaw tumor syndrome gene, HRPT2.

S.P. An international consortium to map HRPT2 An international consortium was established In 1999, with the goal of sharing DNA samples of HPT-JT families from several countries (Sweden, U.K. Netherlands, USA) and to perform a fine mapping study to narrow down the region of HRPT2 An international consortium was established In 1999, with the goal of sharing DNA samples of HPT-JT families from several countries (Sweden, U.K. Netherlands, USA) and to perform a fine mapping study to narrow down the region of HRPT2 John Carpten, at the NHGRI of Bethesda, leaded the group that performed the new genotyping John Carpten, at the NHGRI of Bethesda, leaded the group that performed the new genotyping John Carpten

S.P. Results of the fine mapping study We began our study with 22 kindreds, which were genotyped with 26 microsatellite markers in the 1q24-q32 interval. We began our study with 22 kindreds, which were genotyped with 26 microsatellite markers in the 1q24-q32 interval. Of these families, 18 were informative for genetic analysis. Of these families, 18 were informative for genetic analysis. Affected haplotypes were constructed and a recombination map was obtained. Affected haplotypes were constructed and a recombination map was obtained. A novel proximal recombination between D1S238 and D1S461 in Kindred-02 reduced the candidate interval by 3 cM. On the contrary, the distal boundary did not change. A novel proximal recombination between D1S238 and D1S461 in Kindred-02 reduced the candidate interval by 3 cM. On the contrary, the distal boundary did not change. Therefore, the genetic interval was reduced from 15 cM to 12 cM, including a chromosome segment of 14 Mb. Therefore, the genetic interval was reduced from 15 cM to 12 cM, including a chromosome segment of 14 Mb.

S.P. Genetic analysis of HPT-JT kindreds and partial transcript map of the critical region

S.P. Linkage Disequilibrium (Allelic Association) methods Linkage analysis is a powerful tool for detecting ‘major’ genes which does not require a candidate and is, therefore, a means of genome screening. However, its main limitation is its low-resolution mapping of the linked chromosomal interval (usually some cM), which could contain tens, or hundreds, of genes. Linkage analysis is a powerful tool for detecting ‘major’ genes which does not require a candidate and is, therefore, a means of genome screening. However, its main limitation is its low-resolution mapping of the linked chromosomal interval (usually some cM), which could contain tens, or hundreds, of genes. One way to perform fine mapping and confirm linkage of a susceptibility locus is to test for allele association due to linkage (i.e. "linkage disequilibrium") between particular genetic markers and the disease. One way to perform fine mapping and confirm linkage of a susceptibility locus is to test for allele association due to linkage (i.e. "linkage disequilibrium") between particular genetic markers and the disease. Linkage disequilibrium of a particular marker allele will occur when the disease locus and the marker locus are so closely positioned that recombination events between them are very rare and a certain marker allele is associated with the disease gene. A popular method to analyze linkage disequilibrium is the Transmission Disequilibrium Test (TDT). Linkage disequilibrium of a particular marker allele will occur when the disease locus and the marker locus are so closely positioned that recombination events between them are very rare and a certain marker allele is associated with the disease gene. A popular method to analyze linkage disequilibrium is the Transmission Disequilibrium Test (TDT).

S.P. A modified transmission distortion test applied to the HPT-JT haplotype data Our approach involved the selection of trios (mother, father, child), each trio from a different family, both the child and one of the parents being affected. The affected chromosomes (cases) were defined as those inherited by the child from the affected parent, and the unaffected chromosomes (controls) were those inherited by that child from the unaffected parent. Our approach involved the selection of trios (mother, father, child), each trio from a different family, both the child and one of the parents being affected. The affected chromosomes (cases) were defined as those inherited by the child from the affected parent, and the unaffected chromosomes (controls) were those inherited by that child from the unaffected parent. This procedure yields two set of chromosomes of equal size, which are contrasted to each other in terms of their genetic composition. In the presence of linkage disequilibrium, the affected chromosome set will show a reduced level of haplotype diversity, or an excess of haplotype sharing. This procedure yields two set of chromosomes of equal size, which are contrasted to each other in terms of their genetic composition. In the presence of linkage disequilibrium, the affected chromosome set will show a reduced level of haplotype diversity, or an excess of haplotype sharing. We considered sliding-window haplotypes of 2, 3, 4, … contiguous markers, and determined the haplotype frequency spectra (the distribution of the number of haplotypes observed in 1, 2,..., n individuals) in the two chromosome sets for each n–marker haplotype. Heterogeneity between the two sets of chromosomes was tested by chi-square. We considered sliding-window haplotypes of 2, 3, 4, … contiguous markers, and determined the haplotype frequency spectra (the distribution of the number of haplotypes observed in 1, 2,..., n individuals) in the two chromosome sets for each n–marker haplotype. Heterogeneity between the two sets of chromosomes was tested by chi-square.

S.P. Design of a modified L.D. mapping method The main advantage of our method derives from selecting trios in which both the child and one of the parents are affected. This allows us to determine which of the two chromosomes in the child is the "case", so that we do not have to use all the four founding chromosomes to look for transmission disequilibrium.

S.P. A peak of haplotype sharing among the 18 “affected” chromosomes The peak points to the interval between D1S384 and D1S412, the location where HRPT2 was actually identified

S.P. Conclusions On the basis of our haplotype-sharing analysis, we expected to identify some identical mutations in some of the families On the basis of our haplotype-sharing analysis, we expected to identify some identical mutations in some of the families This was not the case, as the final report included data on 11 different HRPT2 mutations identified in 12 probands, out of 26 screened families. This was not the case, as the final report included data on 11 different HRPT2 mutations identified in 12 probands, out of 26 screened families. The reasons of this result have still to be explained. The reasons of this result have still to be explained. However, our study shows the usefulness of following up linkage analysis with fine-mapping intrafamilial linkage disequilibrium analysis However, our study shows the usefulness of following up linkage analysis with fine-mapping intrafamilial linkage disequilibrium analysis