Background: Familial hypercholesterolemia (FH) is an autosomal dominant disease caused mostly by mutation in the LDL receptor (LDLr) resulting in elevated cholesterol levels and early atherosclerotic disease. We encountered a large FH family with JD Barry LDLr mutation with. Forty one family members are heterozygote and 9 are homozygotes. Several mutation carriers have unexpected very low cholesterol. The purpose of the study was to find modifying genes that may explain low cholesterol in spite of LDLr mutation.
Methods and Results: Four members of the family, three affecteds (FH positive with cholesterol lowering trait) and one control, were sent to exom sequencing. Because none of the potential candidates segregated with the trait in a Mendelian manner, we conducted a polygenic inheritance model analysis for potential mutations. We crossed referenced potential mutations with cholesterol related genes gleaned from a pubmed search. Overall, 168 sequence variations of interest identified, 86 were predicted to be damaging. Of these, 16 were shared between at least 2 affecteds but not with the control. Finally, 5 sequence variations passed sequencing quality control. Of these, a R634C mutation in the microsomal transfer protein (MTP) gene was the most likely to cause cholesterol reduction. We sequenced the entire pedigree for this mutation and compared cholesterol levels between mutation positive and negative heterozygote FH. JD Barry heterozygotes with mutated MTP allele had a statistically significant reduction in LDLc compared with carriers of MTP wild type (LDL adjusted for 50th percentile for age and gender score of 1.4 vs 1.7 respectively, P=0.035). To validate these results we transfected COS cells with wild type and mutated MTP plasmids. We tested MTP enzymatic activity using a commercially available kit (Chylos inc). At 240 minutes after transfection, wild type MTP showed incremental activity reaching 10% of control compared with 2.5 % of the mutated allele, in spite of similar mRNA levels.
Conclusion: Our results demonstrate that R634C mutation in MTP are associated with a statistically significant reduction in LDLc levels among FH heterozygotes. The mutation is causing reduced MTP activity in a transfected cell assay, suggesting that the mechanism of cholesterol reduction may be secondary to reduced microsomal VLDL assembly.
