A three-year old boy presented to his paediatrician for assessment of a pruritic abdominal rash. His birth and infancy had been unremarkable, with normal growth and development. There was no consanguinity; both parents and two older sisters were all healthy. At age 3, hepatosplenomegaly was noted on abdominal examination and was confirmed by ultrasound. No diagnosis was made and he was monitored periodically. At age 8, he was admitted to the hospital with gastroenteritis. Light microscopy of a liver biopsy showed increased intracytoplasmic glycogen and small lipid droplets in hepatocytes. Electron microscopy showed membrane-bound lipid droplets with small electron dense granules. A working diagnosis of glycogen storage disease type III (DeBrancher disease) was made, but skin fibroblast Debrancher activity was normal.

At age 10, hepatomegaly persisted and a second liver biopsy was taken. Light microscopy showed altered lobular architecture of the hepatic parenchyma with distended hepatocytes containing cytoplasmic granules and vacuoles with mild periportal fibrosis. Fibroblast acid lipase activity was found to be 7% of normal, confirming the diagnosis of CESD. Plasma concentrations of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) were each above the 95^th percentile for age and sex at 7.49, 3.23 and 5.59 mmol/L, respectively, while plasma high-density lipoprotein cholesterol (HDL-C) was below the 5^th percentile at 0.46 mmol/L; he had combined hyperlipidemia (hypercholesterolemia, hypertriglyceridemia, hypoalphalipoproteinemia and hyperbetalipoproteinemia). After 12 months, a low fat diet was started (Figure 1).

After 6 months of diet alone, lovastatin 10 mg daily was added. Because of rising plasma concentrations of TC and LDL-C, the dose of lovastatin was increased to 20 mg after ~22 months and increased again to 40 mg after a further ~8 months. Ezetimibe 10 mg per day was added after a further ~40 months and the combination of lovastatin 40 mg and ezetimibe 10 mg daily has continued for 12 months. Serum asparagine transaminase (AST) and creatine kinase (CK) were measured concurrently with the lipoproteins.

The proband's lipoprotein profile since age 10 is summarized in Figure 1. Mean ± standard deviation (SD) lipoprotein concentrations on each phase of treatment were determined from a minimum of three values. From baseline concentrations, the diet was associated with a 5.3% decrease in TC (6.67 ± 0.79 to 6.32 ± 0.26 mmol/L), a 27.3% decrease in TG (2.75 ± 0.48 to 1.99 ± 0.50 mmol/L), a 2.2% increase in LDL-C (4.63 ± 0.77 to 4.73 ± 0.53 mmol/L), a 7.8% increase in HDL-C (0.64 ± 0.12 to 0.69 ± 0.04 mmol/L), and a decrease in TC:HDL-C ratio of 11.5% (10.4 ± 0.3 to 9.2 ± 0.5). When pooled data over ~70 months from all 19 determinations on lovastatin were compared with diet alone, statin treatment was associated with a further 13.5% decrease in TC (to 5.47 ± 0.42 mmol/L), a 12.6% increase in TG (to 2.24 ± 0.36 mmol/L), a 15.9% decrease in LDL-C (to 3.98 ± 0.41 mmol/L), an 8.7% decrease in HDL-C (to 0.63 ± 0.08 mmol/L), and a 5.5% decrease in TC:HDL-C ratio (to 8.7 ± 0.2). Finally, when pooled data over 12 months from all four determinations on ezetimibe plus lovastatin were compared with lovastatin monotherapy, the drug combination was associated with a further 9.4% decrease in TC (to 4.96 ± 0.60 mmol/L), a 3.1% decrease in TG (to 2.17 ± 0.20 mmol/L), an 15.8% decrease in LDL-C (to 3.35 ± 0.46 mmol/L), no change in HDL-C (0.63 ± 0.08 mmol/L), and a 9.1% decrease in TC:HDL-C ratio (to 7.9 ± 0.3). Unpaired t-tests showed that the TC and LDL-C concentrations were significantly different for the period with lovastatin monotherapy compared to the period with combination therapy (P < 0.05). Also, there were no deviations of plasma CK and AST above the upper limit of normal for any treatment period. Finally, liver and spleen size evaluated clinically were reduced compared to baseline over the treatment period with statin and then later statin plus ezetimibe; specifically, while the liver edge was palpable 5 cm below the right costal margin before drug treatment it was not palpable at the most recent clinical assessment.

Genomic DNA sequencing of the LIPA gene revealed that the proband had two mutations (Figure 2). The first was a T insertion in exon 6 at codon 178 that shifted the reading frame (Figure 2A) and caused a premature termination at codon 190 (FS A178-X190). The second was G>A change at the last nucleotide of exon 8 (Q277), which resulted in a silent mutation at the amino acid level (Figure 2B). The patient was heterozygous for both mutations. In order to determine the chromosomal phase of the two LIPA mutations, sequencing of exon 6 and exon 8 from the proband's mother's genomic DNA revealed that she was a simple heterozygote for the frameshift mutation, confirming the that two mutations in the proband were on different chromosomes. Reverse transcriptase PCR amplification of LIPA from the proband, followed by sequence analysis of the partial cDNA spanning exon 5 through exon 10 revealed an abnormal sequence in which the entire exon 8 had been deleted (Figure 2C).

Genomic DNA was isolated from whole blood obtained from patients (Puregene, GentraSystem, Minneapolis, MN). The entire coding region and intron-exon boundaries of LAL gene were amplified using custom primer pairs shown in table 1. PCR amplifications were carried out in a 50 μl mixture containing 32 pmole of each primer, 0.2 mM of each dATP, dCTP, dGTP and dTTP, 1.5 mM MgCl₂, 50 mM KCl, 20 mM Tris-HCl (pH 8.4), 2.5 units of Taq platinum DNA polymerase (Invitrogen, Mississauga, ON). 30 cycles were performed consisting of denaturation at 94°C, annealing at 56°C and extension at 72°C for 30 s each, followed by extension for 10 min at 72°C. PCR products were purified from agarose (QIAQuick Gel Extraction Kit, Qiagen, Mississauga, ON). Direct DNA sequence results were analyzed with an ABI automated sequencer 3730 and were read with ABI Sequence Navigator software (both from PE Biosystems, Mississauga, ON).

Total RNA was isolated from the proband using PAXgene Blood RNA Tube and Blood RNA Kit (Qiagen, Mississauga, ON). 2.5 ml blood was used for RNA isolation according to manufacturer's instruction. 100 ng of total RNA was used for first strand cDNA synthesis (SuperScript First Strand Synthesis System, Invitrogen, Mississauga, ON). 2 μl of first strand reaction was used for amplify partial cDNA sequence of the LIPA gene spanning exon 5 to 10 with primer pair 5' AAT ATG ACC TAC CAG CTT CCA, 3' GTA AGC AAA CAC ATT TTC ACA. PCR products were gel purified, sequenced and read as described above.