Alternative titles; symbols
HGNC Approved Gene Symbol: LDLRAP1
Cytogenetic location: 1p36.11 Genomic coordinates (GRCh38): 1:25,543,606-25,590,400 (from NCBI)
| Location | Phenotype |
Phenotype MIM number |
Inheritance |
Phenotype mapping key |
|---|---|---|---|---|
| 1p36.11 | Hypercholesterolemia, familial, 4 | 603813 | Autosomal recessive | 3 |
LDLRAP1 is an adaptor protein that interacts with the cytoplasmic tail of low density lipoprotein receptor (LDLR; 606945), phospholipids, and components of the clathrin (see CLTC; 118955) endocytic machinery (Garuti et al., 2005).
Garcia et al. (2001) cloned the ARH gene after performing linkage analysis in families suffering from autosomal recessive hypercholesterolemia (ARH; 603813) that mapped the ARH locus to a 1-cM interval on chromosome 1p35. The gene encodes a 308-amino acid protein containing a 170-amino acid phosphotyrosine-binding (PTB) domain, which shares considerable sequence similarity with the PTB domains of many adaptor proteins. PTB domains bind the consensus sequence NPXY, which is present in the cytoplasmic domains of several cell surface receptors, including LDLR. The integrity of the NPXY sequence in the cytoplasmic tail of LDLR is absolutely required for internalization, and LDLR has been shown in vitro to bind other proteins containing PTB domains. The human ARH protein shares 89% sequence identity with orthologous proteins in mouse and Xenopus. By Northern blot analysis, Garcia et al. (2001) found that ARH is normally expressed at high levels in the kidney, liver, and placenta, with lower levels detectable in brain, heart, muscle, colon, spleen, intestine, lung, and leukocytes.
Nagai et al. (2003) reported that the N-terminal half of human, rat, and mouse ARH contains the PTB domain, and the C-terminal half contains a clathrin box (LLDLE), a beta-2 adaptin (AP2B1; 601025)-binding site, and a C-terminal PDZ-binding motif.
Using yeast 2-hybrid, pull-down, and coimmunoprecipitation assays, Nagai et al. (2003) found that rat Arh bound the first FxNPxY motif of megalin (LRP2; 600073). Arh colocalized with megalin in clathrin-coated pits and in recycling endosomes in the Golgi region of rat L2 cells. Upon internalization of megalin, megalin and Arh colocalized in clathrin-coated pits, followed by their colocalization in early endosomes and tubular recycling endosomes in the pericentriolar region, and then by their reappearance at the cell surface. Expression of Arh in canine kidney cells expressing megalin minireceptors enhanced megalin-mediated uptake of lactoferrin (LTF; 150210), a megalin ligand. Nagai et al. (2003) concluded that ARH facilitates endocytosis of megalin and escorts megalin along its endocytic route.
By biochemical assays and electron microscopy, Michaely et al. (2004) found that lymphocytes from patients with ARH deficiency had over 20-fold more LDLR on the cell surface and about 27-fold excess of LDLR outside of coated pits. However, despite the increase in cell surface receptors, LDL binding was only 2-fold higher in ARH-deficient lymphocytes. Michaely et al. (2004) concluded that ARH is not only required for internalization of the LDL-LDLR complex, but also for efficient binding of LDL to the receptor.
By mutation analysis, Garuti et al. (2005) found that integrity of the FDNPVY sequence in LDLR was required for ARH-associated LDLR clustering into clathrin-coated pits in polarized hepatocytes. The phosphotyrosine-binding domain of ARH plus either the clathrin box or the AP2-binding region was required for LDLR clustering and internalization. These findings were confirmed in vivo by expressing the same ARH mutants in livers of Arh -/- mice. Garuti et al. (2005) concluded that ARH must bind the LDLR tail and either clathrin or AP2 to promote receptor clustering and internalization of LDL.
Sirinian et al. (2005) found that ARH codistributed with LDLR on the basolateral surface of polarized confluent HepG2 cells. Activation of LDLR-mediated endocytosis, but not binding of LDL to LDLR, promoted colocalization of ARH with the LDL-LDLR complex. Depletion of ARH by more than 70% by RNA interference caused an 80% reduction in LDL internalization. Coimmunoprecipitation analysis of LDL-stimulated polarized HepG2 cells showed that ARH interacted with other components of the endocytic machinery, including beta-adaptin, DAB2 (601236), and the small GTPase RAB4 (179511). Sirinian et al. (2005) concluded that ARH is not constitutively associated with LDLR at the plasma membrane, but is recruited to the membrane after LDL binding, thus facilitating endocytosis of the LDL-LDLR complex.
Garcia et al. (2001) determined that the ARH gene spans 25 kb and contains 9 exons.
By genomic sequence analysis, Garcia et al. (2001) mapped the ARH gene to chromosome 1p35.
In 6 families with autosomal recessive hypercholesterolemia-4 (FCHL4; 603813), including 2 Sardinian, 2 Lebanese, 1 Iranian, and 1 American family, Garcia et al. (2001) identified homozygosity for 6 different mutations in the LDLRAP1 gene (605747.0001-605747.0006). A nonsense mutation (Q136X; 605747.0003) was identified in 1 of the Lebanese families; only trace amounts of ARH mRNA was detected in cultured fibroblasts from patients with this mutation. The other Lebanese family was homozygous for a missense mutation (P202H; 605747.0004) that was associated with a normal level of ARH mRNA in cultured fibroblasts. Garcia et al. (2001) found that the defect in LDLR function in ARH patients appeared to be not only receptor-specific, but also tissue-specific. They were not able to identify a consistent defect in LDLR function (binding, uptake, or internalization) in cultured fibroblasts from ARH patients. It is possible that another PTB domain protein compensates for the absence of ARH in cultured fibroblasts, or that adaptor molecules are not required for receptor-mediated endocytosis of LDL in fibroblasts and possibly other extrahepatic cells.
Arca et al. (2002) performed extensive clinical and molecular genetic studies in 28 Sardinians with autosomal recessive hypercholesterolemia from 17 unrelated families. They sequenced the coding regions and consensus splice sites of the ARH gene in probands from these families, and from 40 individuals of non-Sardinian origin who had an autosomal recessive form of hypercholesterolemia of unknown cause. In all 17 unrelated Sardinian families, 2 ARH mutations were present, 432insA in exon 4 (605747.0002), referred to as ARH1 by them, and a nonsense mutation, 65G-A in exon 1 (trp22 to ter; 605747.0001), referred to as ARH2 by them. Three of the ARH alleles in the Sardinian patients contained both mutations, as a result of an ancient recombination between ARH1 and ARH2. No regional clustering of the 3 mutant alleles was apparent. Furthermore, 4 Italians from the mainland with autosomal recessive hypercholesterolemia were homozygous for ARH1. The small number, high frequency, and dispersed distribution of ARH mutations on Sardinia were consistent with these mutations being ancient and maintained in the Sardinian population because of geographic isolation.
Mishra et al. (2002) showed that the ARH protein is a component of the endocytic machinery, with mutations of the ARH gene contributing to the LDL-uptake-disease phenotype of ARH patients. PTB domains of the ARH protein bind to the internalization motif of the LDL receptor. The authors showed that in addition, ARH binds directly to soluble clathrin trimers and to clathrin adaptors. At steady state, ARH colocalizes with endocytic proteins in HeLa cells, and the LDL receptor fluxes through peripheral ARH-positive sites before delivery to early endosomes. Their findings suggested that in ARH patients, defective sorting adaptor function in hepatocytes leads to faulty LDL receptor traffic and hypercholesterolemia.
To define the molecular mechanism underlying autosomal recessive hypercholesterolemia, Wilund et al. (2002) examined ARH mRNA and protein in fibroblasts and lymphocytes from 6 hypercholesterolemic patients with different ARH mutations. Five probands were homozygous for mutations that introduced premature termination codons; the sixth patient was homozygous for a 2.6-kb insertion in intron 1 which was associated with no detectable ARH mRNA. None of the probands had detectable full-length ARH protein in fibroblasts or lymphoblasts. No relationship was apparent between the site of the mutation in ARH and the amount of mRNA. Radiolabeled LDL degradation was normal in ARH fibroblasts, but LDLR function was markedly reduced in ARH lymphoblasts, despite a 2-fold increase in LDL cell surface binding in these cells. Wilund et al. (2002) concluded that ARH is required for normal LDLR function in lymphocytes and hepatocytes, but not in fibroblasts, and that residual LDLR function in cells that do not require ARH may explain why ARH patients have lower plasma LDL levels than do patients with homozygous familial hypercholesterolemia (144010) who have no functional LDLRs.
Al-Kateb et al. (2002) studied a Syrian family in which 3 sibs had elevated LDL levels; 3 other sibs and both parents had normal LDL levels, suggesting an autosomal recessive mode of inheritance. A genomewide scan using 427 markers showed support for linkage on both chromosomes 1 and 13, with significant lod scores at 1p36.1-p35 and 13q22-q32 (see cholesterol-lowering factor, 604595). Al-Kateb et al. (2002) found evidence for an interaction between these loci. They identified an intron 1 acceptor splice site mutation in the ARH gene (605747.0007) in homozygous state in the affected sibs and in heterozygous state in the parents.
In 2 affected sibs from a nonconsanguineous Mexican family with autosomal recessive hypercholesterolemia, Canizales-Quinteros et al. (2005) identified homozygosity for a donor splice site mutation in intron 4 of the ARH gene (605747.0008).
Jones et al. (2003) generated Arh-deficient mice and found that the fractional clearance rate of radiolabeled Ldl in these mice was lower than that in Ldlr -/- mice. By immunolocalization studies, they demonstrated that Ldl receptors are sorted normally to the sinusoidal surface in Arh -/- mouse livers. Jones et al. (2003) concluded that the Ldl internalization defect in Arh-deficient mice is caused by the inability of the receptors to enter the endocytic cycle.
Jones et al. (2007) examined the synthesis and catabolism of Vldl in mouse models of autosomal dominant familial hypercholesterolemia (Ldlr -/-) and ARH (Arh -/-). Despite similar rates of Vldl secretion in response to a high-sucrose diet, the rate of Vldl clearance was significantly higher in Arh-null mice than in Ldlr-null mice, suggesting that LDLR-dependent uptake of VLDL is maintained in the absence of ARH. Hepatocytes from Arh-null mice, but not Ldlr-null mice, internalized beta-Vldl, demonstrating that ARH is not required for LDLR-dependent uptake of VLDL by the liver. Jones et al. (2007) concluded that the preservation of VLDL remnant clearance attenuates the phenotype of ARH and likely contributes to greater responsiveness to statins in ARH compared with FH.
In a Sardinian family with autosomal recessive hypercholesterolemia (FHCL4; 603813), Garcia et al. (2001) found all affected individuals to be homozygous for a c.65G-A transition in exon 1 of the ARH gene, resulting in a trp22-to-ter (W22X) substitution. Three additional Sardinian patients were homozygous for this nonsense mutation, and 3 other unrelated probands were compound heterozygotes for this mutation and a frameshift mutation (605747.0002).
In 2 affected sibs from a consanguineous Sardinian family (ARH1) with autosomal recessive hypercholesterolemia (FHCL4; 603813), originally reported by Zuliani et al. (1995), Garcia et al. (2001) identified homozygosity for a 1-bp insertion (c.432insA) in exon 4 of the LDLRAP1 gene, causing a frameshift predicted to result in a premature termination codon at residue 170, within the terminal portion of the PTB domain. The plasma LDL level was about 460 mg/dl in a proband from this family. Coronary artery disease was prevalent in this family, with 8 relatives dying at less than 33 years of age.
In 4 Italian probands who had hypercholesterolemia and at least 1 normocholesterolemic parent, Arca et al. (2002) identified homozygosity for the c.432insA mutation in the LDLRAP1 gene.
In 4 affected sibs from a consanguineous Lebanese family (ARH3) with autosomal recessive hypercholesterolemia (FHCL4; 603813), previously described by Khachadurian and Uthman (1973), Garcia et al. (2001) identified homozygosity for a c.406C-T transition in the LDLRAP1 gene, resulting in a gln136-to-ter (Q136X) substitution. Plasma total cholesterol in this family ranged from 440 to 580 mg/dl, and LDL receptor (see 606945) activity was 60 to 70% of normal in fibroblasts.
In a Lebanese family (ARH4) with autosomal recessive hypercholesterolemia (FHCL4; 603813), Garcia et al. (2001) found that all affected individuals were homozygous for a C-to-A transversion at nucleotide 605 of the ARH gene, resulting in a pro-to-his substitution at codon 202 (P202H). Family members had plasma total cholesterol of 520 to 610 mg/dl, with LDL cholesterol ranging from 392 to 520 dl.
In an Iranian family (ARH5) with autosomal recessive hypercholesterolemia (FHCL4; 603813), Garcia et al. (2001) identified a frameshift at nucleotide 72 of the ARH gene, resulting in a premature termination codon at residue 33. The 10-year-old proband had a plasma total cholesterol of 637 mg/dl.
In a family (ARH6) with autosomal recessive hypercholesterolemia (FHCL4; 603813) from the United States, Garcia et al. (2001) identified a single basepair deletion at nucleotide 71 of the ARH gene, resulting in a premature termination codon at residue 55. The patient was homozygous for this mutation and had a plasma total cholesterol of 800 mg/dl at the age of 15.
In a Syrian family with autosomal recessive hypercholesterolemia (FHCL4; 603813), Al-Kateb et al. (2002) identified a G-to-C transversion in the acceptor splice site of intron 1 of the ARH gene. Al-Kateb et al. (2003) found that the mutation caused the deletion of 2 bp from the start of exon 2, resulting in a frameshift and a truncated protein due to a premature stop codon (TGA) 2 codons later.
In 2 affected sibs from a nonconsanguineous Mexican family with autosomal recessive hypercholesterolemia (FHCL4; 603813), Canizales-Quinteros et al. (2005) identified homozygosity for a +2T-G transversion in intron 4 of the ARH gene, resulting in the activation of a cryptic splice site and the expression of a mutant protein lacking 26 amino acids involving the beta-6 and beta-7 strands of the phosphotyrosine-binding (PTB) domain. The authors stated that this was the first case of an ARH mutation causing an altered PTB domain. Both parents and an unaffected sister were heterozygous for the mutation, which was not found in 41 unrelated normolipidemic Mexican individuals.
In 2 Japanese sibs with autosomal recessive hypercholesterolemia (FHCL4; 603813), Harada-Shiba et al. (2003) identified a novel insertion in the ARH gene of a cytosine in the tract of 8 cytosines at positions 599 through 606 in exon 6, resulting in a sequence of 9 cytosines and generating an early stop codon at 657-659. The mother was heterozygous for this mutation. Neither transcription product nor protein of ARH was detected in the fibroblasts of the homozygous patients. Both sibs exhibited fatty liver, which may also be related to this mutation.
In a Spanish man with autosomal recessive hypercholesterolemia (FHCL4; 603813), Sanchez-Hernandez et al. (2018) identified compound heterozygosity for missense mutations in the LDLRAP1 gene: a c.653C-T transition, resulting in a thr218-to-ile (T218I) substitution, and a c.863C-T transition, resulting in a ser288-to-leu (S288L; 605747.0011) substitution. The authors noted that this patient presented a milder phenotype than patients with homozygous truncating mutations in LDLRAP1, with much lower baseline low density lipoprotein cholesterol levels and later diagnosis.
For discussion of the c.863C-T transition in the LDLRAP1 gene, resulting in a ser288-to-leu (S288L) substitution, that was found in compound heterozygous state in a Spanish man with autosomal recessive hypercholesterolemia (FHCL4; 603813) by Sanchez-Hernandez et al. (2018), see 605747.0010.
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