Common Apolipoprotein E Gene Mutations Contribute to Lipoprotein Glomerulopathy in ChinaHan J.a · Pan Y.a · Chen Y.a · Li X.b · Xing G.c · Shi J.d · Hou P.a · Zhang H.a · Wang H.a
aRenal Division, Peking University, First Hospital and bRenal Division, Traditional Chinese Medicine University, Dongzhimen Hospital, Beijing, cRenal Division, First Hospital, Qingdao Medical University, Qingdao, and dRenal Division, Tanggu Hospital, Tianjin, China Corresponding Author
Background: Lipoprotein glomerulopathy (LPG) is a unique disease characterized by thrombus-like lipoprotein deposition in glomeruli and an increased serum apolipoprotein E level (ApoE protein or APOE gene). Several APOE mutations contribute to the occurring of LPG. Methods: We confirmed LPG in 7 individuals by renal biopsy, and investigated families of 2 patients with urinalysis, serum creatinine and serum lipid examination. Exons of APOE of all individuals as well as their relatives were amplified and sequenced directly. Results: Two types of APOE mutations were identified in the 7 patients and their relatives. APOE Maebashi (Arg142-Leu144→0) heterozygotes were found in 5 individuals who were from 4 different families. APOE Kyoto (Arg25-Cys) was confirmed heterogeneous in another 2 individuals. Both mutations present incomplete penetrance. Conclusion: Our research indicates that APOE Maebashi (Arg142-Leu144→0) is a common mutation in Chinese LPG. However, not all carriers of the 2 mutations have LPG, although hyperlipidemia and high serum ApoE level are tested. There are likely other reasons, such as a local mechanism in the glomeruli, which participated in the renal injury.
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Lipoprotein glomerulopathy (LPG) is a rare disease characterized by intraglomerular lipoprotein thrombi and abnormal lipid metabolism [1,2]. Abnormalities in the apolipoprotein E (APOE) gene have been suspected in the pathogenesis of LPG. The major physiological role of the APOE gene (OMIM+107741) is to mediate the binding of lipoproteins to the low-density lipoprotein (LDL) receptors and chylomicron remnant receptors. The binding site of the ApoE protein to LDL receptors lies between the 140th and 160th amino acid residues [3,4], and several APOE mutations around this region have been reported to be associated with LPG [5,6,7,8,9] and dysbetalipoproteinemia, such as APOE Sendai (Arg145- Pro), APOE Kyoto (Arg25-Cys), APOE Tokyo (141Arg-143Leu→0), APOE (156Gln-173Gly→0) and APOE Maebashi (142Arg-144Leu→0). Series studies about APOE Sendai strongly demonstrated that glomerular lesions are not only due to hyperlipidemia, but also due to in situ interaction between mutant ApoE protein with glomeruli [10,11]. In addition, some researchers indicated the location of mutations in the ApoE protein was one of the important determinants for the development of LPG .
In this study, we investigated the APOE mutations in 7 individuals who have been confirmed with LPG via renal biopsy, and screened 3 families.
Subjects were patients who accepted renal biopsy in the Renal Division, Peking University First Hospital. They were 12- to 42-year-old Chinese (n = 7), consisted of 2 males and 5 females, with proteinuria and a diagnosis of LPG, documented by a serum creatinine 75.6 ± 6.1 µmol/l, 6/7 elevated ApoE level and 3/6 elevated apolipoprotein B (ApoB) level. The renal pathological examinations showed enlarged glomerular capillaries deposited with pale stain substances, which could be stained by red oil O. Electron microscopy revealed that the capillary lumina in the glomeruli were occupied by granules and vacuoles of various sizes (fig. 1). Clinically, all individuals presented proteinuria (4 of them had nephritic syndrome) and a normal range of serum creatinine. All of them showed increased serum lipids and high levels of serum ApoE, however only 3 individuals were companied by high ApoB levels (table 1). Subject A and B came from the same family, while another 6 subjects appeared to be sporadic. We screened 3 families, including urinary analysis serum creatinine, serum lipid and serum lipoproteins E and B (fig. 2, 3, 4).
|Table 1. The clinical features and mutations of 7 patients|
|Fig. 1.a Light microscopy findings of renal biopsy specimen of patient C (red oil stain, color available online only). The capillary lumina of most glomeruli showed a marked dilatation and were filled with thrombi stained by red oil. b Light microscopy findings of renal biopsy specimen of patient C (periodic acid silver methenamine). Most glomeruli presented dilatation of the capillary lumen and ApoE thrombi deposits. c Electron-microscopic findings of thrombi-like substances in renal biopsy specimen of patient C.|
|Fig. 2. Pedigree analysis of family A. Pedigree analyses of patients A and B were presented with clinical characteristics and APOE mutation status. Arrows indicate the patients in the family.|
|Fig. 3. Pedigree analysis of family C. Pedigree analysis of patient C was presented with clinical characteristics and APOE mutation status. Arrow indicates the patient in the family. Grey square and circle indicate carriers without proteinuria or abnormal serum creatinine.|
|Fig. 4. Pedigree analysis of family G. Pedigree analysis of patient G was presented with clinical characteristics and APOE mutation status. Arrow indicates the patient in the family. Grey circle indicates carrier without proteinuria or abnormal serum creatinine.|
We were able to ascertain, obtain consent, and prepare genomic DNA from blood leukocytes collected from 7 participants and 3 families in this study. Each subject gave informed, written consent to the local institutional review boards. Genomic DNA was prepared from leukocytes in EDTA-anticoagulated blood. The human reference genome sequence was obtained from the UCSC Genome Browser (http://genome.ucsc.edu). PCR was performed with primers F4 5′ ACAGAATTCGCCCCGGCCTGGTACAC 3′ and F6 5′ TAACCTTGGCAGGGCTGTCCAAGGA 3′, 5′ TTTGTGGAGCACCTTCTGTG 3′, 5′ GCAGAATGAAACCTGGACCT 3′ for exon 3, and 5′ ATCAAGCTTTCGCCCGCCCCATCCC AGCCCTTC 3′, 5′ CGTGAATTCGCATGGCTGCAGGCTTCGGCGTTC 3′ for exon 4. Ten pmol of each primer and 200 ng genomic DNA were mixed in a 25 µl reaction mixture consisting of 0.2 mM dNTPs, TaKaRa LA Taq 0.5 u and GC buffer for amplification of template with a high percentage of GC (TaKaRa Bio. Co., Ltd., Japan). The mixture was incubated at 96°C for 10 min, then PCR was amplified as follows: denaturing at 96°C for 30 s, annealing at 65°C for 30 s and polymerization at 72°C for 30 s for 30 cycles using a thermocycler. For exon 3, touchdown PCR was used as 65°C→50°C for 30 cycles with 50°C for 15 cycles. Genotypes were scored by direct sequencing in all participants as well as their family members. When mutations were found in subjects, restriction fragment isotyping with HhaI (Hixson and Vernier) combined with direct sequencing were performed to confirm the distribution of mutations in the indicated families. Data from 200 healthy Chinese Han persons were considered as controls.
Two types of APOE mutations were identified in the 7 participants. A 3 amino acid deletion (142Arg-144Leu→0) was detected in 5 individuals and a missense mutation of APOE Arg25-Cys was found in 2 individuals (table 1). Based on genotypes confirmed by direct sequencing and restriction fragment length polymorphism analysis, we concluded that the 3 amino acid deletion (142Arg-144Leu→0) occurred in ε3 (table 1, fig. 2, 3), and Arg25-Cys occurred in ε3 in individual G (fig. 4). However, we could not confirm which allele the Arg25-Cys was located in, due to a lack of family data.
To estimate the occurrence of the Arg25-Cys and 142Arg-144Leu→0 mutations in the Chinese population, genotyping was performed in 200 Chinese Han persons who did not presented any evidence of LPG or abnormal serum lipids. None of them was identified in either of the 2 mutations.
We investigated 3 families (family A, C and G) in this research. In family A, both mother and son were diagnosed with LPG (fig. 2). In family C, the APOE mutation (142Arg-144Leu→0) was detected in 3 individuals (I:2, II:3 and II:5). Only individual C (family member II:5) was identified with LPG; another 2 persons, I:2 and II:3, did not show any sign of abnormal urine and serum creatinine (fig. 3). We observed similar phenomena in family G, where both I:2 and II:1 carried the APOE Arg25-Cys, but only II:1 was confirmed with LPG (fig. 4). In those APOE mutation carriers, although they did not manifest with renal disease clinically, the serum lipids and ApoE level were markedly elevated.
We followed the family of individual C for 2 years. Patient C first accepted therapy of prednisone 50 mg per day and simvastatin 200 mg for 3 months without any remission of nephrotic syndrome. Then prednisone was gradually decreased and leflunomide was added, for another 6 months, the nephrotic syndrome was improved completely, and all treatments were stopped. Proteinuria was negative for 3 years. Her mother and brother who carry the heterozygous APOE 142Arg-144 Leu→0 have not been found to have abnormal proteinuria or serum creatinine.
Many researches suggested that the APOE gene may play an important role in the pathogenesis of LPG, and several mutations of the APOE gene have been identified to be associated with LPG. Although most of the cases of LPG come from Asia, mutations of APOE associated with LPG from China appeared to be different from Japan. Chen et al. [13 ] did not identify any APOEgene mutations in 17 LPG patients, while only 10 variation sites in the non-coding regions were identified. Luo et al. [14 ] confirmed a new type of APOE mutation, APOE Guangzhou (Arg150 Pro). In this study, we identified 2 types of APOE mutations among 7 Chinese patients with LPG. APOE Maebashi (142Arg-144Leu→0) was detected in 5 patients from 4 different families, and APOE Kyoto (Arg25-Cys) was confirmed in 2 patients.
APOE Maebashi (142Arg-144Leu→0)  has been reported in a Japanese girl. APOE Kyoto (Arg25-Cys)  was originally described in a Japanese man with LPG and was identified totally in 4 Asians and 2 European-Americans (table 2) . APOE Maebashi appears to be more common in Chinese LPG patients based on our study.
|Table 2. Mutations of APOE gene contribute to LPG|
Studies showed that the mutations of APOE were transmitted as autosomal dominant with incomplete penetrance [5,7,8] with unclear mechanisms. We also observed a similar phenomenon in our LPG families. Both APOE Maebashi and APOE Kyoto heterozygous carriers showed abnormal serum lipids and elevated serum APOE level, without signs of renal disease. More interestingly, patient C got a complete remission of nephrotic syndrome after 6 months of treatment with a steroid and leflunomide, with no proteinuria for more than 3 years. In most of the reported cases, lipid-lowering therapy was the first choice and appeared to be effective in patients with less proteinuria after a long period of treatment (table 2). In our observations, patient C did not show any signs of remission of nephritic syndrome after simvastatin treatment for 3 months.
These phenomena suggest that some other possibility, such as a local mechanism in glomeruli, participated in renal lesion formation. Case series studies of APOE Sendai strongly demonstrated that glomerular lesions are not only caused by hyperlipidemia, but also by in situ interaction between mutant ApoE with the glomeruli [15,16]. Furthermore, prior research illustrated the location of mutations in the ApoE protein is one of the important determinants for the development of LPG .
Based on the family investigations, all the carriers who did not show any sign of proteinuria or renal failure had normal ApoB level compared with the patients in these families. It was reported that concentrations of ApoB correlated with urine protein in a study of 16 LPG cases  and ApoB was also detected in the lipoprotein deposition in the enlarged glomeruli. It was shown that the high levels of ApoB can lead to plaques that can result in atherosclerosis. Interestingly, ApoB secretion was dependent on APOE binding to receptors . These findings suggest that the ApoB level may be one of the factors leading to renal lesion formation in LPG. However, not all the patients with LPG had a high ApoB level both in our and other studies. The roles of ApoB and APOE on the renal lesion still need to be investigated.
In most of the reported cases, long-time treatment with statins or bezafibrate appeared to decrease proteinuria. It appeared that leflunomide played a role in the remission of patient C’s nephrotic syndrome. Local inflammation might be involved in the mechanism of LPG and immunosuppressive therapy might act on this step, resulting in the remission of proteinuria.
In summary, both APOE Maebashi (142Arg-144Leu→0) and APOE Kyoto (Arg25-Cys) heterozygous exist in Chinese LPG patients, and APOE Maebashi appears more common. Both types of mutations are not sufficient to result in clinical LPG. Immunosuppressants and lipid-lowering agents might ameliorate proteinuria.
Support was provided through the Grant for Capital Medical Development (ZD199910, 2003–2001) and the Grant for Excellent Scientist 985–2-007–113.
Renal Division, Peking University, First Hospital
No.8, Xishiku Street, Xicheng District, Beijing, 100034 (China)
Tel. +86 10 6655 1122 2388, Fax +86 10 6655 1055
J.H. and Y.P. contributed equally to this paper.
Received: April 27, 2009
Accepted: September 16, 2009
Published online: January 20, 2010
Number of Print Pages : 8
Number of Figures : 4, Number of Tables : 2, Number of References : 23
Nephron Clinical Practice
Vol. 114, No. 4, Year 2010 (Cover Date: April 2010)
Journal Editor: El Nahas M. (Sheffield)
ISSN: 1660-2110 (Print), eISSN: 1660-2110 (Online)
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