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Epigenomics AG Berlin, Kleine Präsidentenstr. 1, 10178 Berlin, Germany
¹ Institute of Human Genetics, Otto-von-Guericke University, Leipziger Str. 44, 39120 Magdeburg, Germany
² Institute for Human Genetics, Ernst-Moritz-Arndt University, Fleischmannstrasse 42/44, 17489 Greifswald, Germany
³ University Children’s Hospital, Department of Perinatology: Gerhard-Hauptmann-Str. 35, 39108 Magdeburg, Germany

(Requests for offprints should be addressed to T Brune who is now at Universitätsklinikum Magdeburg, Zentrum für Kinderheilkunde, Gerhard-Hauptmann-Str. 35, D-39108 Magdeburg, Germany; Email: thomas.brune{at}med.ovgu.de)

(R Cortese and F Eckhardt contributed equally to this work)

	Abstract

Top Abstract Introduction Patients, materials and methods Results Discussion References

 
Mutations in the LMNA gene cause various phenotypes including partial lipodystrophy, muscular dystrophies, and progeroid syndromes. The specific mutation position within the LMNA sequence can partially predict the phenotype, but the underlying mechanisms for the development of these different phenotypes are still unclear. To investigate whether different DNA methylation patterns contribute to the development of different phenotypes caused by LMNA mutations, we analyzed a panel of ten candidate genes related to fat metabolism, aging, and a tendency to different methylation patterns: CSPG2, ESR1, IGF1R, IGFR2, LMNA, MLH1, RANBP1, RARB, ZMPSTE24, and TGFBR1. We studied two independent families each comprising three individuals affected by familial partial lipodistrophy type 2 (FPLD2). Affected members in each family carried two different mutations of the LMNA gene (R482L and R471G respectively). In addition, we analyzed four progeria patients (2xLMNA/C G608G, 1xLMNA/C S143F, and 1xZMPSTE24 IVS9-Ex10) and seven healthy adults. The gene encoding retinoic acid receptor B (RARB) showed a higher methylation in all six patients with FPLD2 when compared with the progeria patients with other LMNA mutations as well as the healthy controls (P<0.05). All other investigated genes showed no difference in the methylation patterns between the groups. A drug-induced inhibition of the retinol pathway is discussed as the key pathway for developing HAART-associated lipodystrophy and our data support a possible role of the retinol pathway in the development of lipodystrophy phenotypes.

	Introduction

Top Abstract Introduction Patients, materials and methods Results Discussion References

 
Mutations within the LMNA gene cause many diseases with different phenotypes such as different muscular dystrophies, progeroid syndromes, mandibuloacral dysplasia, dilated cardiomyopathies, restrictive dermopathy, and lipodystrophy syndromes. The mutation position within the LMNA sequence can partially predict the phenotype, but the underlying mechanisms for the development of these different phenotypes are still unclear (Hutchison et al. 2001, Hegele 2005). One possible reason could be specific epigenetic alterations of other genes.

Epigenetic alterations such as histone modifications and DNA methylation have been proposed to be involved in the etiology of a number of diseases and to modify their pathological phenotype (Schumacher & Petronis 2006). Specifically, the best-studied epigenetic modification, DNA methylation, is believed to be involved in human pathologies such as cancer (Baylin 2005), diabetes (Maier & Olek 2002), atherosclerosis (Dong et al. 2002), inflammatory bowel disease (Kim et al. 1996), inflammatory arthritis (Petronis & Petroniene 2000), and autoimmune disease (Richardson 2003).

To investigate if different DNA methylation patterns contribute to the development of different phenotypes caused by LMNA mutations, we analyzed a panel of ten candidate genes related to fat metabolism and aging including genes that display an age-dependent differential methylation. We analyzed the selected genes in six familial partial lipodistrophy (FPLD) patients from two independent families and four patients suffering from progeria, all bearing different mutations in the LMNA gene. The results were compared with those offive healthy adults and one healthy member of both FPLD2 families.

	Patients, materials and methods

Top Abstract Introduction Patients, materials and methods Results Discussion References

 
The Independent Ethics Committee of the University of Magdeburg, Germany, approved the protocol and the study was performed according to the guidelines of the Declaration of Helsinki. Written informed consent was obtained from all patients.

Patients

Ten patients from two independent families bearing different mutations in the LMNA gene were enrolled in the study. Five healthy adults and one healthy member of each of the FPLD2 families served as controls. The patients and the controls were grouped and evaluated as displayed in Table 1.

Patients 1–3 belong to the same family (Family A) and exhibit the typical phenotype of familial partial lipodistrophy type 2 (FPLD) including acanthosis nigricans, partial lipodystrophy with sparing of the face, severe hypertriglyceridemia, insulin-resistant diabetes, and severe hypertriglyceridemia. Patient 1 is a 36-year-old female and the mother of Patient 2, a 12-year-old female. Patient 3 is an 8-year-old female and a cousin of patient 2. All three carry the LMNA mutation R482L (Ambrosch et al. 1996, Capanni et al. 2003). One healthy member of the family, an 11-year-old female cousin of patient 2 with no mutation, serves as a family internal control (Patient 7).

Patients 4–6 belong to the same family (Family B) and carry the LMNA R471G mutation. Patient 4, a 14-year-old girl, is affected by an overlapping syndrome including partial lipodystrophy, insulin-resistant diabetes, acanthosis nigricans, liver steatosis, muscle weakness, and contractures. Patient 5 is her 18-year-old sister and exhibits only a partial lipodystrophy. Patient 6 is their father carrying the same mutation but he does not show a lipodystrophy phenotype. Their healthy mother (Patient 12) shows no LMNA mutation and serves as a family internal control (Muschke et al. in press).

Progeria group

Patients 7 and 8 are a 1-year-old girl and a 5-year-old boy, both affected by typical symptoms of a Hutchinson–Gilford syndrome (progeria) reported by de Busk (1972). Both carry the LMNA G608G mutation typical of progeria (Eriksson et al. 2003, De Sandre-Giovannoli et al. 2003). Patient 9 is a 5-year-old girl with an overlapping syndrome including early onset myopathy with progeroid features. She carries a de novo heterozygous LMNA mutation (S143F) and during the first year of life displayed myopathy with marked axial weakness; progeroid features including growth failure, scleroderma-like skin, and osteolytic lesions developed later (Kirschner et al. 2005). Patient 10 is a three and a half-year-old boy suffering from scleroderma-like lesions of the skin which emerged at 3 months of age. Poor weight gain with loss of subcutaneous fat, prominent scalp veins, progressive hair loss, and lentigo senilis were noted shortly afterwards. X-rays revealed the absence of the lateral regions of clavicles as well as acroosteolysis of the fingers, myopathy, and cardiomyopathy . Sequencing of LMNA gene revealed a heterozygous stop mutation leading to a protein which is elongated by seven additional amino acids at the C terminal. The mother and a healthy brother of the patient carry the same mutation. Sequencing of the ZMPSTE24 gene showed a homozygous deletion of an acceptor splice site (IVS9-Ex10) leading to the complete loss of function of the mutated ZMPSTE24 (Denecke et al. 2006).

Control group

Five healthy females (Patients 13–17) served as controls.

Lymphoblastoid B-cell lines

Ten milliliters of venous blood was drawn from each patient and controls, and placed in a heparin blood collection tube. The blood samples were fractionated by the standard Ficoll-Hypaque method (Ficoll Biocoll, 1.077 g/ml Nycomed Pharma AS, Oslo, Norway, Biochrom AG, Berlin, Germany). After washing twice with phosphate buffered saline (PBS), the cells were infected with Epstein–Barr virus (EBV) containing supernatant. After 30 min, culture medium (RPMI 1640) supplemented with 12% fetal bovine serum (FBS) containing cyclosporin A was added. Six to eight weeks after setting-up, an aliquot of the cells was used for further investigations. To rule out an impact of the immortalization process on the methylation pattern of the investigated genes, we compared DNA from freshly prepared lymphocytes (A) and the corresponding immortalized lymphocytes (B) of three healthy probands.

Cryopreservation

After washing once with serum-free culture medium, the cells were resuspended in cryomedium, i.e. complete culture medium containing 10% DMSO, transferred to cryotubes, and deposited in a Nalgene Cryo 1 °C freezing container at –80 °C for 24 h. The next day, cryotubes were transferred to a liquid nitrogen container.

Thawing

The cryotubes were placed in a 37 °C water bath until the cell suspension was thawed. After washing once with culture medium, the cells were transferred to a culture vessel containing fresh culture medium.

DNA preparation

DNA was isolated from B-lymphoblastoid cells. Cells were pelleted by centrifugation, washed twice in 1xPBS and resuspended in 2 ml OLD-T buffer (40 mM Tris–HCl, 150 mM NaCl, 25 mM EDTA, pH 7.5). After addition of 50 µl proteinase K and 100 µl 20% SDS, incubation was carried out at room temperature overnight, followed by phenol/chloroform extraction. The DNA was precipitated using two volumes of absolute ethanol.

Candidate genes

The panel of the analyzed genes consisted of ten candidate genes related to differential methylation, aging and progeria selected from the literature. All genes and the corresponding references are shown in Table 2.

The described methods and algorithms have been published previously (Lewin et al. 2004, Rakyan et al. 2004, Eckhardt et al. 2006). The entire workflow is shown in Fig. 1. Bisulfite conversion of genomic DNA was performed as previously described, followed by a direct quantification of DNA methylation. Briefly, the bisulfite reaction was carried out using genomic DNA that was treated with a 2.5 M sodium bisulfite solution (Berlin et al. 2005), converting unmethylated cytosines into uracil, whereas methylated cytosines remained unaltered. Upon PCR amplification, the uracil was converted into a thymine and the cytosine:thymine ratio was used for the methylation quantification as described below. PCR amplification using primers (Table 3) flanking the region of interest was performed with Qiagen hot-start PCR reagents using the following PCR profile: hot-start at 95 °C for 15 min (95 °C for 1 min, 55 °C for 45 s, 72 °C for 1 min) x 40 cycles, and an elongation step of 72 °C for 10 min. PCR amplicons were controlled by agarose gel electrophoresis, and excess nucleotides and primers were subsequently removed using ExoSAP-IT (USB Corporation, Cleveland, OH, USA).

View larger version (23K): [in this window] [in a new window]   Figure 1 Workflow of bisulfite sequencing and evaluation of the candidate genes. After the bisulfite treatment, methylation values are calculated from the sequence trace file using the C:T signal intensities. Methylation values for each sample and CpG position are color coded ranging from 0% methylation (yellow) to 100% methylation (blue).

Quantitative cytosine methylation levels were calculated from the ABI sequencing trace files as previously described (Lewin et al. 2004, Rakyan et al. 2004, Eckhardt et al. 2006). In brief, after trace curve normalization, the methylation of each individual cytosine-phosphate-guanine site (CpG) was quantified by C:T signal peak intensities ratios at the cytosine position located within a CpG (the dinucleotide where DNA cytosine methylation occurs). Only sequences displaying a conversion rate higher than 98% were used for analysis. Methylation values for individual Cs within each amplicon and sample were calculated and visualized by color coding ranging from dark blue (100% methylation) to yellow (0% methylation). The rows of the matrix correspond to the methylation levels of individual CpG sites, and each column represents an individual sample.

To minimize the risk of possible methylation variation caused by the immortalization process, our cell culture methodology or the freezing and thawing process (Liu et al. 2005), we compared the methylation pattern DNA from freshly prepared lymphocytes and the corresponding immortalized lymphocytes before and after thawing of three healthy probands.

Statistical analysis

Differential methylation was tested for significance using Wilcoxon’s rank sum test. Significant results (P<0.05) were visualized as a green box on the right of the figures.

	Results

Top Abstract Introduction Patients, materials and methods Results Discussion References

 
Figure 2 displays the methylation profiles of all individuals investigated beside the two healthy family controls. In all 14 samples, the ten genes analyzed showed a low methylation level of 0–30%. Within the FPLD group, the RARB gene showed a significantly higher methylation (P<0.05) in all six patients when compared with both patients with other LMNA/C mutations and to healthy controls. In addition, we observed methylation differences between the two families in the FLPD group with, for example, LMNA being highly methylated in patients 1–3 (Family A) than in patients 4–6 (Family B). However, these findings were not statistically significant when tested against the control group or the progeria group.

View larger version (40K): [in this window] [in a new window]   Figure 2 (a) Methylation profiles of all investigated individuals. The ten genes of all samples analyzed showed a low methylation level of 0–30%. The methylation profile of the RARB gene is shown in detail in (b). The LMNA gene shows a higher methylation level in patients 1–3 (Family A) than in patients 4–6 (Family B). This result is not statistically significant when tested against the control group or the progeria group. (b) The methylation profile of the RARB gene in detail. Within the FPLD group, the RARB gene showed a significantly higher methylation (P<0.05) in all six patients when compared with both patients with other LMNA/C mutations and when compared with healthy controls.

Noteworthy, all methylation changes were detected in B-lymphoblastoid cells isolated from whole blood samples. This fact enables the study with minimal intervention, which is a very desirable feature in a diagnostic procedure. In order to control possible changes in the methylation patterns introduced during culture and freezing–thawing of the cells (Liu et al. 2005), we compared DNA from freshly prepared lymphocytes and the corresponding immortalized lymphocytes of three healthy probands (Fig. 3). The methylation profiles of our marker did not change during the immortalization process or different steps of culture suggesting that the immortalization process had no effect on the methylation pattern of these genes.

View larger version (29K): [in this window] [in a new window]   Figure 3 Methylation pattern of the LMNA and the RARB genes from freshly prepared lymphocytes and the corresponding immortalized lymphocytes before and after thawing of three healthy probands. No change of the methylation pattern is detectable in immortalized lymphocytes.

	Discussion

Top Abstract Introduction Patients, materials and methods Results Discussion References

In the present study, we investigated the relation of DNA methylation and the underlying mutation as a reason for the development of the differential phenotypes in patients bearing LMNA mutations. For this purpose, we analyzed the methylation patterns of ten candidate genes in patients with FPLD2, with other laminopathies and controls. Out of the ten gene methylation profiles, we found that the studied amplicon of the RARB gene is significantly hypermethylated in FLPD when compared with the patients with other laminopathies, indicating an epigenetic component in this disease. The studied amplicon is located in intron 1 of the annotated RARB gene (Ensembl v42).

The RARB gene encodes the RAR-ß and mediates retinoid effects in controlling cell growth, differentiation, apoptosis, and carcinogenesis. RARB has been shown to be differentially methylated in several cancers (Paz et al. 2003) such as malignant melanoma (Furuta et al. 2004), testicular tumors (Kawakami et al. 2003), and carcinoma of cervix (Narayan et al. 2003). Although the function of RARB in the etiology of FLPD2 is currently not known, it is noteworthy that protease inhibitor therapy of human immunodeficiency virus (HIV) is frequently associated with a FPLD-like condition characterized by lipoatrophy of the limbs, fat accumulation of abdomen, breasts and cervical region (‘buffalo hump’), hyperlipidemia, and insulin resistance. This symptoms complex is called highly active anti-retroviral therapy (HAART)-related lipodystrophy syndrome (Carr 2000). Protease inhibitors including indinavir have been shown to reduce adipocyte cell differentiation and increase apoptosis of adipocytes in vitro. The HAART-related lipodystrophy syndrome may be a result of the inhibition of two proteins involved in lipid metabolism, which have significant homology to the catalytic site of HIV protease, namely cytoplasmic retinoic acid-binding protein (cRABP) and low-density lipoprotein receptor-related protein (Lenhard et al. 2000). CRABP is an intracellular protein involved in the transmission of the vitamin A-derived signal, which regulates genes responsible for lipid metabolism and adipocyte differentiation (Carr et al. 1998).

Although the phenotypes of HAART-induced lipodystrophy and FPLD seem distinct in the sense that FLDP patients have fat deposition in face and neck whereas HAART lipodystrophic individuals have facial fat loss, both the phenotypes have multiple clinical and metabolic features in common (Behrens et al. 2000). Our results and the above-mentioned role concerning the inhibition of CRABP in the etiology of HAART-related lipodystrophy suggest a link between the retinoic acid pathway and lipodystrophy phenotypes and support a role for changes in the methylation of the retinoic acid receptor gene in the development of lypodystrophic phenotypes.

The different DNA methylation of the LMNA gene between the two FPLD families may play an additional role in the different development of the FPLD phenotype. Although the difference in LMNA is visible in the plot, the P values after the Wilcoxon’s test do not show statistical significance. As we compared only three patients in each group, marginal P values are expected. Thus, these results would suggest a trend towards differential methylation of LMNA gene between both families carrying different mutations. Additional studies focusing on the link between the observed differential methylation and the different mutation in LMNA will allow an understanding of whether they have a synergetic effect on the development of the phenotype.

	Acknowledgements

 
R C is an employee of Epigenomics AG. F E was an employee of Epigenomics AG during the study. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

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Received in final form 4 April 2007
Accepted 12 April 2007
Made available online as an Accepted Preprint 18 April 2007

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Cortese, R. Articles by Brune, T. Search for Related Content PubMed PubMed Citation Articles by Cortese, R. Articles by Brune, T.