A Genome-Wide Linkage Scan of a Large Amish Pedigree with Von Willebrand Disease (VWD) Identified Several Chromosomal Regions That May Contain Potential Modifiers of Von Willebrand Factor (VWF) Levels and Disease Variability.
Authors: Di Paola Jorge , Rickard Mary , Murray Jeffrey , Burns Trudy , Wang Kai , Shapiro Amy 
Published: October 2019

VWD is a common and highly variable bleeding disorder with incomplete penetrance and variable expressivity. The ABO blood group is the only well-characterized modifier of VWF with a contribution of up to 30% of the genetic component. The remainder of the genetic influence has yet to be elucidated. Through linkage mapping, using large pedigrees, we aim to discover genes that exert a modifier effect on VWF levels and bleeding severity and that would be candidates for other disorders of hemostasis. We identified and characterized an extensive Amish pedigree from Indiana which is composed of 758 individuals of whom 395 have provided blood samples. Phenotypic characterization of the pedigree included VWF antigen levels (VWF: Ag), VWF Ristocetin cofactor activity (VWF: RCo), molecular ABO typing, CBCs and a clinical bleeding phenotype determined by a validated bleeding score. We used a value of 3 or greater to differentiate bleeders (≥ 3) from non-bleeders (< 3). Sequence from exon 28 of the VWF gene demonstrated a missense mutation at position 4120, represented by a single base substitution (C>T) that predicts an arginine to cysteine change at position 1374 (R1374C) in the A1 domain of the mature VWF molecule. All 71 individuals heterozygous for the 4120 C>T mutations have VWF: RCo levels ≤ 25, indicating 100% penetrance with the low VWF: RCo level phenotype. However the difference in factor levels between affected individuals is as high as 3-fold. Thus the genetic homogeneity of this family provides a large sample in which to study the genetic and/or environmental contributors to the remaining variability observed.

A primary genetic screen at a 10 cM average interval was performed on the entire Amish pedigree in collaboration with the Marshfield genotyping center. Standard Screening Set 16 with 400 short tandem repeat polymorphic (STRP) markers was utilized generating a total of approximately 160,000 genotypes on the 395 samples. When the phenotype was defined as VWF: RCo levels ≤ 25 IU/dL and utilizing an autosomal dominant model (disease frequency = 0.1, penetrance = 0.6, no phenocopies), the highest LOD score (16.34) corresponded to marker GATA49D12 located on chromosome 12 p13.3 where VWF has been physically mapped. Despite the impressive effect of the 4120C>T mutation on this particular phenotype, three other regions of linkage were also identified. The first region, with a LOD score of 1.32 at 9q34 harbors several genes of interest including ABO. Two other regions linked to this phenotype exhibited a LOD score of 1.03 and 1.30 and are located on 9q22.3 and 21q22 respectively. When the bleeding scores (BS) were used as phenotypes several regions of linkage were identified. Two regions (19p13.2 and 12p33) showed suggestive LOD scores (1.09 and 1.1) with a phenotype of BS ≥ 3. Four regions with LOD scores greater than 1 were identified with the more stringent phenotype of BS ≥ 4: 6p23, 8p23, 10q26 and 18q12. Only two regions suggested linkage with both bleeding score phenotypes: 7q32, with LOD scores of 2.52 and 1.52 and 16q12 with LOD scores of 1.22 and 1.48 for the BS ≥ 3 and BS ≥ 4 respectively. This analysis in a very large pedigree with a homogeneous primary cause for VWD has identified several regions that will be investigated at higher resolution for the presence of modifier genes and alleles.