A 49-year-old man of Japanese and British ancestry was referred to a metabolic diseases clinic for evaluation 5 months after nontraumatic spleen rupture requiring splenectomy. Prior history included hypertension and mild frontal headaches, but no other neurological or cardiovascular symptoms. The patient did not smoke and used alcohol infrequently. His mother had coronary artery disease, and his father had mild hypertension. There was no family history of consanguinity, splenomegaly, diabetes, or developmental delay.
The ruptured spleen weighed 727 g, and splenomegaly was associated with marked sinus histiocytosis spreading apart the lymphoid component. The overwhelming majority of histiocytes were foamy (Fig. 1a⇓ ), and only a few had sea-blue appearance and reacted positively with periodic acid-Schiff (PAS), PAS and diastase, and May-Giemsa stains. A lipid storage disorder was suspected, but the histiocytes did not have the cytoplasmic linearity appearance of Gaucher cells and were otherwise nonspecific. Before splenic rupture, the patient’s lipoprotein profile was reported as being normal, with no past recorded triglyceride measurement exceeding 2 mmol/L.
Two-month postsplenectomy laboratory investigations revealed combined hyperlipidemia with plasma total cholesterol, HDL-cholesterol, and triglycerides of 7.9 (normal <5.2), 1.4 (normal >1.0), and 4.3 (normal <1.7) mmol/L, respectively. Liver function tests were normal aside from increased γ glutamyltransferase (88 μg/L; normal <49 μg/L). Hemoglobin and leukocyte counts were normal with mild thrombocytosis. Physical examination at 5 months revealed obesity (body mass index 28.9 kg/m2) and hypertension (resting blood pressure 140/100 mm Hg). Cardiovascular examination was normal. There were no xanthomata or xanthelasmata and no hepatomegaly. Left ventricular ejection fraction by echocardiogram was normal at 50%. Coronary artery computed tomographic scan revealed no obvious arterial occlusion, and brain MRI revealed nonspecific white matter changes consistent with ischemia. Carotid artery ultrasound showed no significant obstruction.
Six months postsplenectomy, the patient’s plasma triglycerides were 17.2 mmol/L. He was placed on a seafood-rich, low-fat, low-sugar diet. At 8 months his plasma triglycerides had fallen to 1.5 mmol/L, while total cholesterol and HDL-cholesterol were 8.2 and 1.2 mmol/L, respectively, and apolipoprotein (apo)B and apoA-I concentrations were 1.19 and 1.35 g/L, respectively. The patient’s dietary regimen was relaxed, and at 12 months triglycerides had again increased to 21.1 mmol/L. In view of findings suggesting cardiovascular disease and recurrent severe hypertriglyceridemia, aspirin and antihypertensive, and lipid-lowering therapies (atorvastatin 10 mg/day and salmon oil 3 g/day) were initiated. Genomic investigation was requested.
Primary hypertriglyceridemia is a family of relatively common disorders, some of which have a known molecular genetic basis. For instance, some cases of familial chylomicronemia result from mutations in the gene encoding lipoprotein lipase or its cofactor apoC-II. Some cases of familial combined hyperlipoproteinemia result from mutations in the upstream transcription factor 1 (USF1) gene encoding a transcription factor. Dysbetalipoproteinemia (type III hyperlipoproteinemia) requires 2 copies of the binding defective APOE E2 allele. Most cases of primary hypertriglyceridemia, however, do not have a known molecular basis. Secondary factors that worsen hypertriglyceridemia include poor diet, obesity, alcohol use, diabetes, renal disease, pregnancy, nonalcoholic fatty liver disorder, paraproteinemias, autoimmune disorders, and certain medications, such as steroids.
The potential etiologies of splenomegaly vary with the patient’s age. For example, splenic sequestration in sickle cell disease occurs early in life, whereas noncirrhotic portal fibrosis occurs at older ages. Mechanisms of splenomegaly include: (a) infections, which may be viral, such as Epstein-Barr virus and cytomegalovirus, bacterial, protozoal, and fungal, and in endemic areas, malaria and schistosomiasis; (b) autoimmune disorders such as juvenile rheumatoid arthritis; (c) hemolysis; (d) neoplasia, such as acute lymphoblastic leukemia, non-Hodgkin lymphoma, Hodgkin disease, and acute or chronic myeloblastic leukemia; (e) metastatic involvement, such as neuroblastoma; (f) histiocytosis; (g) obstructed splenic venous circulation due to portal vein thrombosis, hepatic cirrhosis, or congestive heart failure; (h) extrahepatic portal venous obstruction, such as cavernous transformation; and (i) storage diseases, such as Gaucher or Niemann-Pick disease.
The differential diagnosis of nontraumatic splenomegaly with hypertriglyceridemia includes: hereditary absence of lipoprotein lipase or apoC-II, rare apoE3 mutations, deficiency of lysosomal acid lipase, or hemophagocytic lymphohistiocytosis (1)(2)(3). Work-up of these particular rare conditions includes a complete lipoprotein profile; advanced lipoprotein assays, such as APOE genotyping; abdominal ultrasound imaging to document liver and/or spleen enlargement; and evaluation of serum hematologic and inflammatory biomarkers. Molecular analysis of apolipoprotein E (APOE), apolipoprotein C-II (APOC2), lipoprotein lipase (LPL), or lipase A (lysosomal acid, cholesterol esterase, LIPA) genes may be required, in addition to histopathological evaluation of the spleen.
Two previous reports have identified the existence of a very rare apoE3 isoform with a leucine deletion at codon 149, designated E3Δ149Leu (4)(5). Clinical features observed in patients with the E3Δ149Leu mutation include hypertriglyceridemia, thrombocytopenia, splenomegaly, and splenic foamy histiocyte infiltration with sea-blue histiocytosis.
The case patient underwent DNA APOE genotyping performed with the LightCycler (Roche Diagnostics), and the results showed that the patient was homozygous for trinucleotide TGC at codon 112 (Cys112), a finding that is consistent with apoE2 and -E3 isoforms, whereas a finding of atypical amplification at codon 158 was inconsistent with E2, E3, or E4 isoforms (Fig. 1b⇑ ). APOE direct sequencing revealed a trinucleotide TCC deletion on 1 allele spanning codons 149 and 150, predicting an inframe leucine deletion at residue 149. To confirm the precise sequence and chromosomal phase of the mutation, plasmid vector subcloning of APOE exon 4 using primers F4 and F6 (5), pcDNA3+ plasmid and E. coli DH5α™ (Invitrogen) was performed to sequence each of the patient’s APOE alleles individually. The deletion was found on the E3 allele (Fig. 1c⇑ ), confirming the patient’s APOE genotype as apo E2/E3Δ149Leu.
Apo E mediates uptake of cholesterol-rich lipoproteins by hepatocytes. It is a ligand for LDL receptors (LDLRs) and is also a structural component of chylomicron remnants and VLDLs, enabling their removal from plasma (6). Apo E is normally incorporated into VLDL during hepatic synthesis. After secretion into plasma, VLDL is converted to LDL and the minor intermediate species, including VLDL remnants and IDLs, are catabolized via uptake through the LDLR mediated by apoE residues 136 through 150 (6)(7). ApoE is also secreted into plasma by macrophages and extrahepatic tissues, where it binds HDL, facilitating its removal from plasma (6).
A common polymorphism in the APOE gene affects coding sequences for amino acid residues 112 and 158, resulting in 3 protein isoforms and 6 codominant genotypes. The E3 isoform is most common (approximately 75% of all apo E alleles), and contains a cysteine at position 112 and arginine at position 158 (8). The alleles encoding the E2 and E4 isoforms each differ from the E3 allele by a single nucleotide. The E4 isoform (approximately 15% of all alleles) has arginine at position 112 and is associated with approximately 5% higher concentrations of LDL cholesterol. The E2 isoform (approximately 10% of all alleles) has cysteine at position 158 and is associated with approximately 5% lower concentrations of LDL cholesterol (8). In all populations, E3/E3 homozygosity is the most common genotype (8). E2/E2 homozygosity occurs in approximately 1% of the population and contributes to expression of type III hyperlipoproteinemia, characterized by roughly equimolar elevations of plasma cholesterol and triglycerides and by early cardiovascular disease (9). Other rare mutations of APOE are also associated with dyslipidemia (7).
Nguyen et al. (4) first identified the E3Δ149Leu mutation in 2 unrelated probands. In both families, the mutation was associated with a dominant syndrome involving sea-blue histiocytosis and splenomegaly. Faivre et al. (5) later found the same E3Δ149Leu mutation in a proband presenting with clinical type III hyperlipoproteinemia, and subsequently found the mutation in several relatives. The current case shares the dyslipidemia phenotype and need for splenectomy reported for the other reported cases, but is also distinct from the other cases because of the absence of persistent transaminase elevation, thrombocytopenia, and ischemic heart disease. This case provides further evidence that links the E3Δ149Leu allele to dyslipidemia with splenomegaly and infiltrates of foamy histiocytosis. This syndrome may be included in the differential diagnosis of patients with postsplenectomy hypertriglyceridemia.
The E3/E3Δ149Leu mutation negatively affects apoE function, and carriers develop signs and symptoms of a lipid storage disease. The preferential association of the E3Δ149Leu mutation with splenic foam-cell formation remains to be explained. The spleen is a major triglyceride reservoir in E3Δ149Leu carriers, because splenectomy is followed by severe hypertriglyceridemia. Macrophages can secrete apoE into plasma and receive circulating apoE-containing lipoproteins (6), perhaps explaining why the macrophage-rich spleen behaves like a lipid sponge.
A comparison with earlier case reports suggests that the dyslipidemia in E3Δ149Leu carriers shows remarkable clinical heterogeneity (Table 1⇓ ). We speculate that the clinical and pathological sequelae attributable to the E3Δ149Leu mutation depend on a combination of genetic and environmental factors. From this case study and others, we may conclude that apo E3Δ149Leu causes a lipid-storage disease characterized by splenomegaly, splenic infiltration with foamy and sea-blue histiocytes, and hypertriglyceridemia that may be unmasked following splenectomy. The disorder seems responsive to diet, lifestyle, and pharmacologic intervention.
Points to remember
In addition to heterozygous E3Δ149Leu, the differential diagnosis of nontraumatic splenomegaly with hypertriglyceridemia includes: deficiency of lipoprotein lipase or its cofactor apoC-II, deficiency of lysosomal acid lipase, or hemophagocytic lymphohistiocytosis (1)(2)(3).
The concomitant presentation of hypertriglyceridemia and splenomegaly results from a rare subset of the conditions that lead to either clinical presentation individually. Work-up of patients with this combination includes: lipoprotein profile, apoE isoform analysis, or APOE genotyping; complete blood count with differential and smear; and liver function tests, including serum albumin, bilirubin, aspartate aminotransferase and alanine aminotransferase, γ glutamyltransferase, and alkaline phosphatase. Imaging studies include abdominal ultrasound, computed tomographic scanning or MRI of the left upper quadrant, or radioisotopic scanning (spleen scan). Histological evaluation after splenectomy typically reveals foamy histiocytosis and characteristic sea-blue histiocytosis. Molecular analysis of APOE (to detect del49Leu), APOC2, LPL, or LIPA genes to detect loss-of-function mutations may be required.
Grant/funding Support: Supported by the Jacob J. Wolfe Distinguished Medical Research Chair, the Edith Schulich Vinet Canada Research Chair (Tier I) in Human Genetics, operating grants from the Canadian Institutes for Health Research (MOP-13430, MT-8014), the Heart and Stroke Foundation of Ontario (NA-6059, T-5603, PRG-5967, and the Irwin Bernick Summer Student Fellowship through the University of Western Ontario) and by Genome Canada through the Ontario Genomics Institute.
Financial Disclosures: None declared.
- © 2008 The American Association for Clinical Chemistry