Pedigrees of family 1 (a) and family 2 (b). The plasma cholesterol concentration (mmol/l) is shown below each symbol. Filled symbols, homozygous for the Q136X (a, family 1) or the delGG_86,87 (b, family 2) mutations in ARH1; half-filled symbols, heterozygous carriers of the mutation, confirmed by sequencing amplified fragments of genomic DNA. Individuals whose cells were analyzed for LDL receptor activity or ARH1 mRNA levels are identified by a number in italics.

Identification of a partial deletion of ARH1 in family 3. (a) Diagram showing the approximate size of the ARH1 DNA probes used for fluorescent in situ hybridization (FISH). The vertical black bars represent the nine exons of ARH1. (b) Metaphase chromosomes from individual FH3.1 were hybridized with either DIG-labeled ARH1 DNA probe 1 or probe 2 as indicated (detected with FITC-labeled anti-DIG Ab; small filled arrows) and biotin-labeled chromosome 1–specific α-satellite probe DNA (detected with Cy3-labeled anti-biotin Ab; large open arrows).

Degradation of ¹²⁵I-labeled LDL by skin fibroblasts, EBV-lymphocytes, and monocyte-derived macrophages from individuals in family 3. Cells were preincubated for 16 hours in medium containing LPDS and then for 4 hours with ¹²⁵I-labeled LDL. Saturable degradation of LDL was determined as the difference in the amount of TCA-soluble, non-iodide radioactivity in the medium of cells incubated in the presence and absence of an excess of unlabeled LDL (1 mg/ml); values are the mean of duplicate dishes. Nonsaturable degradation of LDL by normal cells was always less than 5% of the total. Data shown are representative of at least two separate experiments. (a) EBV-lymphocytes, (b) skin fibroblasts, and (c) monocyte-derived macrophages, from probands 3.1 (filled triangles) and 3.2 (open triangles) in family 3 and from unrelated normolipemic controls (filled circles).

Confocal microscopy of cells labeled with anti–LDL receptor Ab. (a–f) EBV-lymphocytes were incubated with rabbit anti–LDL receptor (red) at 4°C, washed at 4°C, and either directly permeabilized (a–c) or incubated for 10 minutes at 37°C before permeabilization to allow internalization of LDL receptor/Ab complexes (d–f). Permeabilized cells were then incubated with mouse anti–α-adaptin (AP2), washed, and incubated with Alexa 568–conjugated goat anti-rabbit IgG (LDL receptors, red) and Alexa 488–conjugated goat anti-mouse IgG (AP2, green). Nuclei were stained with DAPI. The plates shown are an overlay of red and green images. The bar represents 5.0 μm. (a and d) Control cells; (b and e) cells from proband 1.1; (c and f) cells from proband 1.1 expressing viral c-myc-ARH. (g–l) EBV-lymphocytes (g–i) or cultured skin fibroblasts (j–l) from three different control subjects were incubated with anti–LDL receptor Ab at 4°C, permeabilized, and then incubated with anti–α-adaptin Ab (AP2) as described for a–f above. The bars represent 5.0 μm (in g for g–i, and in j for j–l).

Pedigree and haplotype analysis of family 3. The plasma cholesterol (chol.) concentration (mmol/l) is shown below each symbol. Filled symbols, homozygous for the insC mutation in ARH1; half-filled symbols, heterozygous carriers of the mutation, confirmed by sequencing amplified fragments of genomic DNA. Haplotypes for the ARH1 locus are shown for polymorphic markers flanking the gene, known polymorphisms (8), and the two novel base substitutions within the coding region of ARH1; the position of each marker on Ensembl is shown (Mb). A recombination in the paternal uncle of the proband is indicated (X). The maternal allele carrying the insC₆₂₀ mutation is boxed; the paternal allele carrying the putative deletion is boxed and shaded. The haplotype in the father, shown in italics, was deduced. Individuals whose cells were analyzed for LDL receptor activity or ARH1 mRNA levels are identified by a number in italics.

Degradation of ¹²⁵I-labeled LDL by cultured skin fibroblasts and EBV- lymphocytes from individuals in family 1. Cells were preincubated for 16 hours in medium containing LPDS and then for 4 hours with ¹²⁵I-labeled LDL. Saturable degradation of LDL was determined as the difference in the amount of trichloroacetic acid–soluble (TCA-soluble), non-iodide radioactivity in the medium of cells incubated in the presence and absence of an excess of unlabeled LDL (1 mg/ml); values are the mean of duplicate dishes. Nonsaturable degradation of LDL by normal cells was always less than 5% of the total. Data shown are representative of at least two separate experiments. (a) Cultured skin fibroblasts from the proband in family 1 (ARH^–/–), and from a normolipemic control (ARH^+/+). (b) EBV-lymphocytes from the proband in family 1 (ARH^–/–), her heterozygous sibling (ARH^+/–), and a sibling who does not carry the mutant allele (ARH^+/+).

Effect of expression of c-myc-ARH1 on LDL receptor activity in mutant EBV-lymphocytes. (a and b) Cells were preincubated for 16 hours with LPDS before preparation of cell extracts. Proteins were fractionated on nonreduced SDS-polyacrylamide gels (13%), transferred to nylon membranes, and immunoblotted with anti–c-myc (lanes 1–8) or anti–LDL receptor Ab (lanes 9 and 10). Bound Ab was detected with peroxidase-conjugated anti-mouse IgG and chemiluminescence. (a) Whole-cell extracts (approximately 50 μg of protein per lane) of Chinese hamster ovary (CHO) cells transiently transfected with pcDNA3-c-myc-ARH1PA317 (lane 1), PA317 cells transfected with ARH1 retroviral construct (lane 2), PA317 cells (lane 3), EBV-lymphocytes from affected individual 1.1 (ARH1.1 cells) 1 month after infection with c-myc-ARH1 retrovirus (lane 4), and uninfected EBV-lymphocytes from affected individual 1.1 (lane 5). (b) Whole-cell extracts (approximately 50 μg of protein per lane) of EBV-lymphocytes from affected individual 1.1 (lane 6), the same cells 3 months after stable infection with c-myc-ARH1 retrovirus (lanes 7 and 9), and the same infected cells after preincubation for 16 hours with 0.3 μM trichostatin A (lane 8 and 10). (c) Virus-infected (ARH^–/c-myc-ARH) and uninfected (ARH^–) EBV-lymphocytes from individual 1.1 were preincubated for 16 hours in medium containing LPDS and then for 4 hours with ¹²⁵I-labeled LDL. Saturable degradation of LDL was determined as the difference in the amount of TCA-soluble, non-iodide radioactivity in the medium of cells incubated in the presence and absence of an excess of unlabeled LDL (1 mg/ml); values are the mean of duplicate dishes. Data shown are representative of two separate experiments.