The female patient was born to second-degree consanguineous Indian parents. She experienced age-appropriate development with no significant illnesses until 10 months of age, when she was admitted to a hospital with vomiting, loose stools, poor feeding, fast breathing, and lethargy for one day. Her laboratory investigations showed normal hemogram levels, in addition to levels of blood glucose of 5.4 mmol/l, serum sodium of 138 mEq/l, serum potassium of 4.5 mEq/l, lactate of 0.6 mmol/l, marginally elevated ammonia of 146 µmol/l, and urinary ketone of 3+. An arterial blood gas analysis showed a severe metabolic acidosis with pH 6.88, pO₂ 127 mmHg, pCO₂ 10 mmHg, and HCO₃⁻ 4.5 mmol/l. Blood cultures and C-reactive protein were negative.

The patient was successfully treated with intravenous fluids (10% dextrose with electrolytes), bicarbonate infusion, and intravenous carnitine supplementation (100 mg/kg/day); blood glucose level was kept at a high normal range to induce insulin secretion which suppressed ketogenesis. The acidosis gradually resolved, and the patient's condition improved. Urinary gas chromatography-mass spectrometry revealed an increased excretion of lactic acid, 3-hydroxybutyrate, and 2M3HB. Plasma acylcarnitine profiling by tandem mass spectrometry showed an elevated C5-OH carnitine and C5-OH/C2 ratio. She is now 3-years old, and has attained age-appropriate development without any further ketoacidotic episodes thus far.

This study was approved by the Ethics Committee of the Graduate School of Medicine, Gifu University (Gifu, Japan) and was carried out in accordance with the principles contained within the Declaration of Helsinki. The participant provided informed consent to participate in the study.

The patient's and controls' fibroblasts were cultured in an Eagle's minimal essential medium containing 10% fetal calf serum. Protein concentration was determined by the method of Lowry using bovine serum albumin as a standard. Enzyme assay for T2 activity was performed as described (9). In brief, using supernatants from cell extracts, we spectrophotometrically monitored the decrease of acetoacetyl-CoA absorbance at 303 nm, which is the result of thiolysis of acetoacetyl-CoA to acetyl-CoA. We measured the difference of thiolase activity in the absence and the presence of potassium ions, which specifically stimulate T2; such a difference represents T2 activity (9). Immunoblot analysis was performed using rabbit polyclonal antibodies and Proto Blot^® Western Blot AP system (Promega Corp., Madison, WI, USA) as described by Fukao et al (10); a mixture of an anti-human T2 antibody and anti-human succinyl-CoA: 3-oxoacid CoA transferase antibody was used as the first antibody. We applied 30 µg proteins extracted from each of patient's and two controls' fibroblasts. Kaleidoscope Prestained Standards (Bio-Rad Laboratories, Inc., Hercules, CA, USA) were used as molecular size markers.

Genomic DNA was purified from the patient's fibroblasts using a Sepa Gene^® (EIDIA Co., Ltd., Tokyo, Japan). A mutation screening was performed at the genomic level by PCR and direct sequencing using a set of primer pairs that amplify exons with their intron boundaries as previously described (11). The genomic ACAT1 sequence was obtained from NCBI Reference Sequence no. NG_009888.1.

The patient's and controls' fibroblasts were cultured in two flasks, one of which was treated with 200 µg/ml of cycloheximide (CHX) (Sigma, St. Louis, MO, USA) for 5 h before RNA extraction, as previously described (12). Total RNA was isolated from fibroblasts using ISOGEN kit (Nippon Gene Co., Ltd., Tokyo, Japan). Thereafter, the isolated RNA (5 µg) was reverse-transcribed in 20 µl of 50 mM Tris-HCl pH 7.5, 75 mM KCl, 3 mM MgCl₂, 10 mM dithiothretiol, 0.5 mM dNTPs, and 200 U of M-MLV reverse transcriptase (Life Technologies, Rockville, MD, USA) with a primer mixture including 5 pmol of both a ACAT1-specific antisense primer, T2-135 (5′-^c.*76 TGACCCACAGTAGTCACAC-3′), and oligo dT primers.

The above preparation was incubated at 37°C for 1 h. A total of 1 µl of this cDNA solution served as a PCR template. The positions of the PCR primers were numbered in relation to the adenine of the initiation methionine codon, which was assigned position +1. The ACAT1 cDNA fragment was amplified (c.-40-646) using Primer T2-4 (sense) (5′-^c.-40AGT CTA CGC CTG TGG AGC-3′) and Primer T2-64 (antisense) (5′-^c.646TAG CAT AAG CGT CCT GTT CA-3′) (13). The ACAT1 cDNA sequence was obtained from Gen Bank accession number NM_000019.3. After 30 PCR cycles, amplified fragments were separated by electrophoresis on a 5% (w/v) polyacrylamide gel. The amplified fragments were also separated in 1% (w/v) agarose gel, extracted using a Geneclean^® II kit (BIO 101, Vista, CA, USA), and analyzed by direct sequencing.

An ACAT1 segment, from exon 2 to 4 (exon 2-intron 2-exon 3-intron 3-exon 4; approximately 2.4 kb long), was amplified from control genomic DNA using an Ex taq^® (Takara Bio, Inc., Otsu, Japan). The used primers included the EcoRI linker sequence: a sense primer (Exon2-EcoRI) (5′-cgttcgaattc^c.77TAA GAT ATGTGG AAC GGA GTT ATG-3′) and an antisense primer (Exon4-EcoRI) (5′-cgttcgaattc^c.321TGC CTG CCT TGT AGG AGC-3′). After sequence confirmation in a pGEM^®-T Easy Vector (Promega Corp.), the EcoRI fragment was subcloned into a eukaryote expression vector pCAGGS (14). This was defined as a wild-type minigene splicing construct. We used a KOD-Plus-Mutagenesis Kit^® (Toyobo Co., Ltd., Osaka, Japan) to make three mutant constructs: c.121-13T>A, T>C, and T>G. A total of 2 µg of each minigene splicing vector was transfected into 5×10⁵ cells of SV40-transformed T2-deficient fibroblasts using Lipofectamine^® 2000 (Invitrogen, San Diego, CA, USA). At 48 h after transfection, RNA was extracted from the cells. Our minigene constructs should produce human T2-rabbit β-globin fusion mRNAs. The first strand cDNA was transcribed with the rabbit β-globin-specific antisense primer (β-glo2) (5′-⁴⁶¹AGC CAC CAC CTT CTG ATA-3′) and then amplified with the Exon2-EcoRI primer on ACAT1 exon 2, and another rabbit-specific antisense primer (β-glo3) (5′-⁴⁴³GGC AGC CTG CAC CTG AGG AGT-3′) to amplify the chimeric cDNA of human ACAT1 and rabbit β-globin. A study of normal and aberrant splicing in the four constructs was performed by sequencing and 5% polyacrylamide gel electrophoresis of the PCR-amplified cDNA fragments.

The potassium ion-activated acetoacetyl-CoA thiolase activity was markedly reduced in the patient's fibroblasts; in the absence of potassium (−K⁺) it was 11.5, and in the presence of potassium (+K⁺) it was 12.6 nmol/min/mg of protein, leading to a +K⁺/-K⁺ ratio of 1.1 (the control fibroblasts -K⁺ 5.1, +K⁺ 8.0 nmol/min/mg of protein, +K⁺/-K⁺ ratio of 1.6). In immunoblot analysis, no T2 protein was detected in the patient's fibroblasts (Fig. 1). These data confirm the diagnosis of T2 deficiency in the patient.

As shown in Fig. 2, a homozygous substitution, c.121-13T>A, was revealed in the DNA fragment around exon 3 of ACAT1. No other mutations were identified.

The amplification of the ACAT1 cDNA (c.-40-646) from control's fibroblasts produced a single fragment with the expected size of 686 bp. However, two faint fragments were amplified from CHX-untreated patient's fibroblasts; one had the same mobility as that of control's fibroblasts and the other was smaller (568 bp). The smaller fragment was predominantly amplified from CHX-treated patient's fibroblasts (Fig. 3A). The sequencing of this smaller fragment revealed an exon 3 skipping (Fig. 3B). Since exon 3 skipping causes frame-shift, ACAT1 mRNAs with exon 3 skipping was subjected to nonsense-mediated mRNA decay (NMD).

We hypothesized that the patient's mutation, c.121-13T>A, in intron 2 caused exon 3 skipping, hence minigene splicing experiment was performed. In case of wild-type minigene splicing construct, normal splicing was predominantly observed although small amount of transcript with exon 3 skipping was also detected. In case of c.121-13T>A construct, almost all transcripts skipped exon 3, confirming this mutation identified in the patient was responsible for the exon skipping. Furthermore, c.121-13T>G and unexpectedly c.121-13T>C also resulted in predominate exon 3 skipping (Fig.4).