The patient was a Caucasian woman from Spain, and who was the first child of non-consanguineous, healthy parents. The neonatal period and her psychomotor development were normal. She had her first menstruation at the age of 13 years, and regularly since then. She has one child following a normal pregnancy history. At age 8 years, she noted that her subcutaneous adipose facial tissue gradually began to decrease and she complained of generalized muscle pain, predominantly in her lower legs, after exercise. The patient was first seen at age 44 years, this being the time at which adipose tissue biopsy was conducted (see below). A physical examination revealed generalized and symmetrical loss of subcutaneous fatty tissue, predominantly in her face and the upper part of her body. The facial lipoatrophy gave an impression of ageing, and a male aspect was noted. However, testosterone levels were normal (0.34 ng/ml, normal range from 0.3 to 1.2 ng/ml). Blood examination showed normal levels of glucose (98 mg/dl, normal range 45 to 135 mg/dl), whereas levels of triglycerides were higher than normal (252 mg/dl, normal range 35 to 150 mg/dl) and levels of cholesterol slightly higher than normal (234 mg/dl, normal range 100 to 220 mg/dl). However, unaltered levels of HDL-cholesterol (47 mg/dl, normal range 35 to 80 mg/dl) and LDL-cholesterol (137 mg/dl, normal range 60 to 150 mg/dl) were found. The blood examination also revealed normal muscular enzyme levels. A neurological examination indicated that deep tendon reflexes were normal and no myotonic phenomena were observed. Nerve conduction studies showed normal values in all tested nerves. A concentric needle examination showed complex repetitive discharges in all tested muscles with no spontaneous activity. Renal and liver function were, as inferred from serum enzyme levels, also normal (ALT/GPT 34 U/liter, normal range 2 to 41 U/liter; AST 27 U/liter, normal range 1 to 40 U/liter; GGT 24 U/liter, normal range 5 to 49 U/liter). Ultrasonic examination of the abdomen indicated hepatic steatosis with normal liver size and morphology, and the kidneys and spleen were normal. Cytogenetic studies revealed a normal karyotype (46XX) without evidence of chromosome breakage. Serum C3 levels were 45 mg/dL, which was abnormally low with respect to normal values (range from 85 to 180 mg/dl). Results were positive for the presence of serum complement 3 nephritic factor. Thus, the overall clinical and biochemical features of the patient led to the diagnosis of APL. The pattern of progressive loss of subcutaneous adipose tissue in the face and the upper part of the body was in accordance with the major criterion established by Misra
et al. (2004) [
5] and supportive criteria were also met: onset during childhood, low serum levels of complement 3 and the presence of serum complement 3 nephritic factor.
A biopsy sample of subcutaneous adipose tissue was taken from the arm. Control values of gene expression in adipose tissue were obtained from the analysis of biopsies of subcutaneous adipose tissue taken from the arms of 10 healthy control individuals (mean age 38.5 ± 9.0 years, 4/6 female/male). The patient and the individual controls gave their consent to participate in the study and the protocol was approved by the Ethics Committee of the Hospital de la Santa Creu i Sant Pau, Barcelona, Spain. Separate analysis of gene expression markers in men and women did not show any significant difference and therefore reference values of gene expression in healthy men and women were shown together. After homogenization in RLT buffer (Qiagen, Hilden. Germany), an aliquot was used for isolation of DNA, which was performed using a standard phenol/chloroform extraction methodology. Another aliquot of the homogenate was used for RNA extraction using a column-affinity based methodology (RNEasy, Qiagen, Hilden, Germany). For mRNA analysis TaqMan Reverse Transcription and -RT-PCR reagents were used (Applied Biosystems, Foster City, CA, USA). One microgram of RNA was transcribed into cDNA using random-hexamer primers and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on an ABI PRISM 7700HT sequence detection system. The TaqMan RT-PCR was performed in a final volume of 25 μl using TaqMan Universal PCR Master Mix, NoAmpErase UNG reagent and the specific gene expression primer probes. The TaqManGene Expression assays used were: COX4I1 (subunit IV of cytochrome c oxidase,
COIV), Hs00266371_m1; ATPase5J (subunit F6 of F0-ATPsynthase) Hs0036588_m1; PPARGC1 (
PGC-1α), Hs0013304_m1; CEBPA (CCAAT/enhancer binding protein-α,
C/EBBP-α), Hs00269972_s1; PPARG (peroxisome-proliferator activated receptor-γ,
PPAR-γ), Hs00234592_m1; RB1 (retinoblastoma protein,
pRB), Hs00153108_m1; DLK1 (
Pref-1), Hs00171584_m1; UCP2 (uncoupling protein-2,
UCP-2), Hs00163349_m1; UCP3 (uncoupling protein-3,
UCP-3), Hs00243297_m1; NRF1 (nuclear respiratory factor-1,
NRF-1) Hs00192316_m1; LPL (lipoprotein lipase,
LPL), Hs00173425_m1; LEP (leptin,
LEP), Hs00174877_m1; Complement precursor-3, Hs00163811_m1; TNF (tumor necrosis factor-α,
TNF-α), Hs00174128_m1; SLC2A4 (glucose transporter,
GLUT4), Hs00168966_m1; APM1 (adiponectin), Hs00605917_m1; β2-microglobulin, Hs9999907_m1; MCP-1 (monocyte chemoattractant protein-1,
MCP-1), Hs00234140_m1; CD68 antigen, Hs00154355. Primers and probe for the detection of cytochrome c oxidase subunit II (
COII) mRNA and mtDNA abundance were designed as previously reported [
8]. Controls with no RNA, primers, or reverse transcriptase were included in each set of experiments. Each sample was run in duplicate and the amount of mRNA for the gene of interest in each sample was normalized to that of the reference control using the comparative (2
-ΔCT) method. Data for gene transcripts are expressed as the ratio of relative abundance of the mRNA of the gene of interest respect to 18S rRNA (Hs99999901_s1).
Examination of gene expression for master regulatory factors associated with the promotion of adipogenesis indicated that peroxisome proliferator-activated-γ (
PPARγ) mRNA was the only mRNA significantly down-regulated in the patient with APL with respect to controls (Table
1). The expression of mRNA for another positive regulator of adipogenesis, CCAAT/enhancer binding protein-α (
C/EBPα) mRNA was not significantly altered. The mRNA levels of retinoblastoma protein pRb, a protein that may have negative effects on adipogenesis [
9], and of
Pref-1, a known negative regulator of adipogenesis [
10] were also unchanged. Among adipokines, leptin mRNA levels were unaltered in the patient whereas adiponectin mRNA was down-regulated. The mRNA for two marker genes of adipose tissue metabolism, insulin-sensitive glucose transporter-4 (
GLUT4) and lipoprotein lipase (
LPL), were also down-regulated in the patient with respect to controls. In contrast, the mRNA levels for three marker genes of inflammation (tumor necrosis factor-α,
TNFα;
MCP-1; and β2-microglobulin) as well as the marker of macrophage infiltration CD68 were unaltered in the patient, with values almost identical to those of controls. Complement component-3 gene expression was also unchanged.
Table 1
mRNA expression of genes involved in adipogenesis, metabolism, inflammation and mitochondrial function in adipose tissue from our patient with APL
Adipogenesis (+)
| | | |
PPARγ mRNA | 2.78 (1.81–3.74) × 10-5
| 1.35 × 10-5
| ↓ |
C/EBPα mRNA | 1.55 (0.93–2.03) × 10-4
| 1.66 × 10-4
| = |
Adipogenesis (-)
| | | |
pRb mRNA | 7.52 (3.47–11.57) × 10-6
| 3.84 × 10-6
| = |
Pref-1 mRNA | 0.44 (0.03–0.84) × 10-9
| 0.04 × 10-9
| = |
Adipokines
| | | |
Leptin mRNA | 0.73 (0.41–1.02) × 10-4
| 0.85 × 10-4
| = |
Adiponectin mRNA | 1.05 (0.69–1.39) × 10-3
| 0.49 × 10-3
| ↓ |
Metabolism
| | | |
GLUT4 mRNA | 0.96 (0.43–1.47) × 10-5
| 0.13 × 10-6
| ↓ |
LPL mRNA | 2.88 (2.15–3.47) × 10-4
| 0.73 × 10-4
| ↓ |
Inflammation
| | | |
TNFα mRNA | 5.18 (2.74–7.63) × 10-7
| 4.56 × 10-7
| = |
MCP-1 mRNA | 0.24 (0.07–0.40) × 10-5
| 0.19 × 10-5
| = |
β2-microglobulin mRNA | 2.85 (2.06–3.49) × 10-4
| 3.12 × 10-4
| = |
CD68 mRNA | 0.75 (0.26–1.24) × 10-4
| 1.01 × 10-4
| = |
Complement system
| | | |
Component 3 mRNA
| 1.99 (0.91–3.07) × 10-4
| 2.08 × 10-4
| = |
OXPHOS
| | | |
COII mRNA | 1.17 (0.74–1.51) × 10-2
| 0.35 × 10-2
| ↓ |
COIV mRNA | 4.24 (3.42–4.81) × 10-5
| 1.55 × 10-5
| ↓ |
ATPsynthase F0-6 mRNA
| 6.77 (4.85–8.69) × 10-5
| 2.10 × 10-5
| ↓ |
UCPs
| | | |
UCP2 mRNA | 4.05 (2.74–5.77) × 10-5
| 5.72 × 10-5
| = |
UCP3 mRNA | 4.13 (1.67–6.50) × 10-7
| 2.56 × 10-7
| = |
Mitochondriogenesis regulators
| | | |
PGC-1α mRNA | 1.12 (0.71–1.51) × 10-6
| 0.82 × 10-6
| = |
NRF-1 mRNA | 4.06 (2.96–5.26) × 10-6
| 3.37 × 10-6
| = |
Levels of transcripts corresponding to components of the respiratory chain system (OXPHOS), either mtDNA-encoded (COII) or nuclear DNA-encoded (COIV and ATP synthase F0 subunit 6) were reduced in adipose tissue from the patient with respect to healthy controls. No significant changes were observed for mtDNA abundance in adipose tissue from the patient with respect to reference control values (1.08-fold change with respect to the mean control values). Neither UCP2 mRNA levels nor UCP3 mRNA levels were altered in the patient relative to controls. Likewise, transcript levels for PPARγ-coactivator-1α (PGC-1α) and nuclear respiratory factor-1 (NRF-1), regulatory factors of mitochondrial biogenesis, were also unaltered in the patient with respect to controls.