Molecular and functional analysis of two new MTTP gene mutations in an atypical case of abetalipoproteinemia.

Abetalipoproteinemia (ABL) is an inherited disease characterized by the defective assembly and secretion of apolipoprotein B-containing lipoproteins caused by mutations in the microsomal triglyceride transfer protein large subunit (MTP) gene (MTTP). We report here a female patient with an unusual clinical and biochemical ABL phenotype. She presented with severe liver injury, low levels of LDL-cholesterol, and subnormal levels of vitamin E, but only mild fat malabsorption and no retinitis pigmentosa or acanthocytosis. Our objective was to search for MTTP mutations and to determine the relationship between the genotype and this particular phenotype. The subject exhibited compound heterozygosity for two novel MTTP mutations: one missense mutation (p.Leu435His) and an intronic deletion (c.619-5_619-2del). COS-1 cells expressing the missense mutant protein exhibited negligible levels of MTP activity. In contrast, the minigene splicing reporter assay showed an incomplete splicing defect of the intronic deletion, with 26% of the normal splicing being maintained in the transfected HeLa cells. The small amount of MTP activity resulting from the residual normal splicing in the patient explains the atypical phenotype observed. Our investigation provides an example of a functional analysis of unclassified variations, which is an absolute necessity for the molecular diagnosis of atypical ABL cases.

. Examination by ultrasound showed that the liver was moderately enlarged and slightly hyperechogenic. A percutaneous liver biopsy showed extensive fi brosis (F3 score according to the METAVIR scoring system ( 21 )) that was associated with a massive micro-and macrovesicular steatosis. Large amounts of free lipid droplets were observed in the cytoplasm by electron microscopy ( Fig. 1C ). Abetalipoproteinemia was suspected, although retinitis pigmentosa and acanthocytosis were absent and the blood vitamin E level was only slightly decreased. The fecal fat content, measured over a three-day period, was also normal (mean, 2.8 g/24 h; normal, <3 g/24 h). However, when the child consumed a normal fat diet (33% fat), diarrhea occurred and steatorrhoea was 24 g/day. After an oral fat load ( Table1 ), the level of serum triglycerides increased slightly and a small amount of chylomicrons appeared in the plasma, suggesting that some intestinal lipid absorption occurred accompanied by low levels of intestinal lipoprotein secretion.
A diet limited in saturated fatty acids (22% of total energy) and enriched with medium-chain triglycerides (MCT) was initiated and resulted in the elimination of diarrhea and progressive improvement in liver function. Transaminases were 2 to 4 times normal at the age of 7 years, 2 to 3 times normal at 14 years, and normal by 18 years of age. Levels of essential fatty acids and fat soluble vitamins were also normalized, and the patient presented normal growth and puberty. At the age of 14 years, the patient no longer adhered to the MCT diet, and 36% of total energy intake was composed of fat. Hepatitis recurred (transaminases increased to 150 to 200 times normal) but resolved when the MCT diet was reinstituted.
The patient is now 22 years of age and is in good health. Fat soluble vitamins are in the normal range without any supplementation. The levels of transaminases are < 1.5 times normal, and hepatic steatosis persists (16% of hepatic parenchyma as determined by magnetic resonance imaging). There are no neurological, ophthalmological, or retinal abnormalities.
Blood samples and intestinal and liver biopsies were obtained from the patient using the procedures and the experimental methods approved by INSERM (RBM 0256) and by a bioethics committee (Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale de Paris Bichat-Claude Bernard, Paris, France, CCPPRB Bichat-C. Bernard-2003/05). Informed, written consent was obtained from the patient's parents.

Mutation analysis
Following the extraction of genomic DNA from the blood (Nucleon Bac3, GE Healthcare®, Chalfont St. Giles, UK), each of the coding exons and the fl anking intronic junctions of the MTTP , APOB, proprotein convertase subtilisin/kexin type 9 ( PCSK9) and angiopoietin-like 3 ( ANGPTL3 ) genes were amplifi ed by PCR. The amplicons were sequenced directly with the BigDye® Terminator v3.1 Cycle Sequencing Kit on an ABI PRISM 3130 or 3730 DNA sequencer (Applied Biosystems, Foster City, CA).

Protein expression
To evaluate the p.Leu435His protein expression, two different assays were performed. First, to measure wild-type and mutant the prediction of altered MTP function is readily made on the basis of premature stop codons, mutations in canonical splice sites, or frameshift mutations. In other cases, the functional consequences have been diffi cult to predict (13)(14)(15), even if the mutations cosegregate with a clinical phenotype. The measurement of MTP activity ( 2,3,6,11,13,16,17 ) and the consequence of intronic mutations ( 9,11 ) have been studied in only a few cases due to the necessity of additional intestinal or hepatic biopsies.
ABL patients are usually diagnosed during infancy, and they exhibit marked lipid malabsorption, very low levels of cholesterol and triglycerides, the absence of ApoB in the plasma, the absence of chylomicrons after fat loading, and essential fatty acid and fat-soluble vitamin defi ciencies (particularly vitamin E); moreover, parental lipoprotein profi les are normal. Hepatic steatosis has not been reported uniformly (although it is frequent), and hepatic fi brosis or cirrhosis has been reported in a few cases ( 18,19 ). We report here an atypical case of ABL presenting in childhood with severe liver injury, hypocholesterolemia associated with a low (but not absent) levels of plasma ApoB, and a subnormal level of plasma vitamin E. Furthermore, the patient exhibited normal development into adulthood. ABL was suspected because of the presence, at the ultrastructural level, of large amounts of free lipid droplets accumulated in the cytoplasm of enterocytes and hepatocytes in intestinal and liver biopsies.
The patient was found to be compound heterozygous for two novel mutations in the MTTP gene, one intronic (c.619-5_619-2 del, from her mother) and the other a missense mutation (p.Leu435His, from her father). The possible functional impact of these two mutations was not readily apparent. Because no additional biopsy material was available from the patient, two different assays were established. COS-1 cells were transfected with wild-type and several mutant MTTP cDNAs to evaluate the impact of the missense mutation on MTP activity, and HeLa cells were transfected with wild-type and mutant minigenes to evaluate possible defects in splicing.

Patient diagnosis and follow-up
A four-year-old girl was referred for an anicteric chronic hepatitis fortuitously discovered 10 months earlier. The level of blood transaminases were consistently elevated and ranged from 5 to 7 times the normal values associated with bilirubinemia in the absence of infl ammation or hepatic insuffi ciency. Upon clinical examination, the liver was moderately enlarged and fi rm, but there was no other sign of chronic liver disease. The patient's growth was normal. The patient's parents altered her diet to avoid fatty foods following episodes of diarrhea in the fi rst months of life. The levels of total cholesterol and ApoB in the plasma were found to be low, whereas the level of triglyceride was normal ( Table1 ). The parents were not related, and they had normal levels of blood lipids.
A perendoscopic jejunal biopsy, performed as previously described ( 20 ), showed a white mucosa. Electron microscopic examination of the biopsy showed enterocytes with many small or very large free lipid droplets scattered throughout the cytoplasm gen, Carlsbad, CA) downstream of the human elongation factor 1 ␣ -subunit promoter. The P4HB cDNA was inserted into the Hind III site of the same vector, downstream of the human cytomegalovirus immediate-early promoter, allowing the production of the two proteins from the same plasmid. COS-1 cells were harvested by trypsinization 48 h posttransfection and disrupted by sonication. Triglyceride transfer from donor to acceptor vesicles was measured by a fl uorescent-labeled method using a commercial kit (R100 MTP activity; Chylos Inc.) according to the manufacturer's instructions and previously described fl uorescent methods (26)(27)(28). The results are expressed as percentage transfer per milligrams of total protein per hour . The method was evaluated with intestinal and hepatic biopsies, and the MTP activities were in agreement with previous published results (data not shown).

Minigene splicing reporter assay
A 371 bp MTTP fragment (the last 147 bases of intron 5, 140 bp of exon 6, and the fi rst 84 bases of intron 6) was amplifi ed from the patient's DNA and inserted (In-Fusion Advantage PCR MTP activities, we transfected COS-1 cells with the MTTP and P4HB cDNA. Second, to compare the production of wild-type with p.Leu435His, we transfected COS-1 cells with wild-type and mutated MTTP cDNA C-ter tagged with GFP.  Oral fat load with 15 g of fat performed at 5 years of age: T0, before fat ingestion; T90, 90 min after ingestion. ALB, albumin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; HDLC, HDL cholesterol; LDLC, LDL cholesterol; ND, not determined; TC, total cholesterol; TG; triglyceride; Vit, vitamin.

MTP activity
a Normal values for four-year-old child. and several other mutants (p.Leu435Glu, p.Leu435Val, and p.Cys194Stop). These mutations were tested in COS-1 cells to evaluate the sensitivity and specifi city of our MTP activity assay: p.Leu435Glu was expected to be as severe as p.Leu435His because it changes the hydrophobic Leu to a hydrophilic acidic Glu, whereas p.Leu435Val was expected to be a mild change (Leu and Val are both hydrophobic and closely related amino acids). The introduction of a premature stop codon at the position p.Cys194 was expected to lead to the complete abolition of MTP activity as the ApoB and PDI binding sites are absent from the mutant. Further, the homozygous p.Cys194Stop mutation has been reported in an ABL patient ( 8 ).

Analysis of c.619-5_619-2del
Splicing predictions. All the algorithms predicted an effect on splicing with a decrease in the score for the acceptor site of intron 5 ranging from Ϫ 10% for SSF-like to a complete abolition for GeneSplicer ( Ϫ 44% for MaxEnt Scan, Ϫ 38.9% for NNSplice, Ϫ 79% for HSF). As all the algorithms predicted an effect on splicing, experimental analysis of splicing was performed. HeLa cells transfected with wild-type or mutant minigenes were analyzed (size and sequence) by RT-PCR to determine the presence of abnormal transcript processing of the mutant.
Cloning Kit) into the Nde I restriction site of the pTB minigene vector ( 29 ). The transfection of normal and mutant minigenes into HeLa cells and RT-PCR procedures were as previously described ( 30 ).
The fl uorescence of the ethidium bromide bands obtained following gel electrophoresis of RT-PCR products was integrated under unsaturated conditions (Quantity One ® 1-D Analysis Software Cat. No. 170-9600; Bio-Rad) to derive band intensities.

Metabolic labeling of intestinal biopsies
Intestinal biopsies from normal individuals and from the patient were placed into organ culture and metabolically labeled with [ 35 S] methionine as described previously ( 31 ). After homogenization and solubilization, the labeled intestinal biopsy extracts and the corresponding media were immunoprecipitated with polyclonal antibodies against ApoB (rabbit polyclonal antibodies to ApoB were the gifts of Dr. A. Mazur of Institut National de la Recherche Agronomique, Champanelle, France) and MTP ( 31 ). For semiquantitative analysis, densitometric analysis was performed using the PC version of NIH Image software (Scion Image) after photography with a computer-assisted camera (GS-800 Calibrated Densitometer; Bio-Rad). The values were normalized with respect to the amount of TCA precipitable incorporated material in the biopsies.

Identifi cation of mutations
Sequencing was performed on the genomic DNA of the patient and her parents. Two novel variants of the MTTP gene were identifi ed in the patient. The fi rst, inherited from her father, is a change from T to A at position c.1304 in exon 10 (supplementary Fig. IA) (nucleotide numbering starts at A of the ATG initiating codon, and exon 1 is the fi rst coding exon), which changes the amino acid Leu 435 to a His. The second, inherited from her mother, is a 4 bp deletion in intron 5: c.619-5_619-2 del (supplementary Fig. IB). This deletion of a repeated motif (TTTA) is upstream the acceptor site. These mutations were found neither in our panel of 100 normal alleles from unrelated subjects nor in the 1000 genomes database ( 32 ).
The patient is also heterozygous for a well-described polymorphism p.Gln95His inherited from her father. The frequency of this allele is between 5.4% and 6% in a healthy adult Caucasian population (England) and in a sample of 270 unrelated French Canadian men ( 6, 33 ) and 6.2% in a cohort of abetalipoproteinemia ( 6 ).
The patient had no mutation in the APOB , PCSK9 , or ANGPTL3 gene.

Analysis of c.1304 T>A, p.Leu435His
The missense mutation (p.Leu435His) is located in exon 10 and affects a highly conserved residue in the middle ␣ -helical domain of MTP. The substitution changes a hydrophobic to a hydrophilic, basic residue [Grantham distance: 99 (0-215)]. This missense mutation is predicted by Polyphen to be "probably damaging" with a score of 0.99 and by SIFT to "affect protein function." To determine whether the p.Leu435His missense mutation produces a protein that is functional, we expressed, in COS-1 cells, the wild-type MTP as well as the p.Leu435His

DISCUSSION
In this article, we describe two new mutations in the MTTP gene: p.Leu435His and a 4 bp deletion in intron 5: c.619-5_619-2 del. Because the impact of these mutations was unclear and the phenotype was atypical, cellular functional tests were used to further understand this new case of ABL.
The diagnosis of ABL is made readily if the following criteria are present during the fi rst months of life: severe diarrhea, failure to thrive, undetectable vitamin E, severe hypocholesterolemia and hypotriglyceridemia with undetectable amounts of plasma ApoB, and the absence, postprandially, of chylomicrons in the serum. Our patient presented with a mild phenotype and lacked the major criteria for the diagnosis of ABL. Surprisingly, the patient was referred only for anicteric chronic hepatitis with major liver steatosis at four years of age. The other causes of hypobetalipoproteinemia were ruled out because of the patient's lipids profi le and the normal lipid values of the parents (34)(35)(36)(37)(38)(39)(40). Furthermore, no mutation was found in the APOB, PCSK9 , or ANGPTL3 gene. The diagnosis of ABL was fi nally suspected because of hypocholesterolemia and the typical aspect of intestinal and liver biopsies suggesting a defect in the assembly of ApoB-containing lipoproteins.
Seven atypical cases of ABL have been reported ( 13-15, 19, 41, 42 ). For fi ve of these patients ( Table 2 ), a molecular diagnosis of the mutation in MTTP was established. However, a functional assay of the mutation was performed in only one case ( 13 ). Two of the cases (cases 1 and 2 in Table 2 ) are very surprising. Both patients have deleterious mutations; however, the fi rst patient exhibits a mild phenotype ( 42 ) and the other is almost asymptomatic ( 13 ). Two other patients (cases 4 and 5 in Table 2 ) are homozygous for the same p.Ser590Ile mutation ( 14,15 ). Unfortunately, the functional impact of this mutation has not been established. Only case 3 ( 19 ) in Table 2 resembles our case clinically and biologically. This patient exhibits compound heterozygosity for a splice site and a missense mutation, along with liver dysfunction and slightly decreased level of vitamin E.
Minigene splicing reporter assay. Transfection of the wildtype minigene into HeLa cells produced, as expected, a 386 bp RT-PCR fragment ( Fig. 3 , lanes 1, 2, and 3). In contrast, transfection of the mutant c.619-5_619-2del minigene produced two different RT-PCR fragments: a small amount of a fragment of normal size (386 bp) and a large amount of a 248 bp product ( Fig. 3 , lanes 4, 5, and 6). The sequence of the 386 bp PCR product includes exon 6 of MTTP , whereas exon 6 is skipped in the 248 bp product (supplementary Fig. III). This result shows that c.619-5_619-2del mutation produces two differently spliced transcripts: one containing exon 6 and the other without exon 6. Skipping exon 6 would result in a protein containing the fi rst 206 MTP amino acids, followed by 26 aberrant amino acids and a premature stop codon. In vivo , this mutant mRNA might be targeted for nonsense mediated decay and degraded. By densitometry of the agarose gel, the amount of normal splicing for the mutant minigene is estimated to be 26% of that of the wild-type ( Fig. 3 ). These data indicate that the c.619-5_619-2del is a splice site mutation that can lead to the skipping of exon 6. However, the effect of this mutation on splicing was not complete, and about 26% of the transcript was correctly spliced in our assay.

Biosynthesis of ApoB and MTP in intestinal organ culture
Immunoprecipitation with polyclonal antibodies to ApoB of the medium and the total homogenate of the organ culture of intestinal biopsies from the patient showed the synthesis of an ApoB48 identical in size to that of normal control subjects ( Fig. 4 ). Analysis by densitometry with correction for the amount of incorporated TCA-precipitable radioactivity showed that there was an intracellular retention of ApoB48 (1.6-fold more) and a decreased ApoB48 secretion (1.3-fold less).
Immunoprecipitation with polyclonal antibodies to MTP of the total homogenate of the organ culture of intestinal biopsies from the patient showed the synthesis of a large subunit of identical in size to that of normal control subjects. However, by densitometric analysis, there was 2.49-fold less MTP synthesized in the patient's biopsy compared with that of the normal control subject after correction. A protein band corresponding to the molecular mass of PDI coimmunoprecipitated with the MTP was observed in the patient and in the normal control subject (identical in amount compared with that of the normal subject after correction).  In our patient, the lipid transfer activity of the MTP produced from the normally spliced transcript may be sufficient to allow the assembly and secretion of a relatively limited amount of ApoB-containing lipoproteins by liver and intestine and could explain the presence of the lipid absorption (as shown by metabolic-labeling biosynthesis data) observed in the intestine of the patient. Both the intracellular accumulation and the decreased secretion of ApoB48 from the intestine are consistent with a partial defect in the assembly of ApoB, which leads to a partial defect in secretion of ApoB48 (unusual in ABL). In addition, the data also suggest that a low amount of intestinal MTP was present with a normal molecular weight and with an intact PDI binding domain. Finally, the presence of a limited amount of fat absorption and lipoprotein secretion could explain the normal level of vitamin E and the normal development with only a low-fat diet as treatment. However, the residual MTP activity is not suffi cient to prevent the accumulation of lipids in the enterocytes and the hepatocytes.
In conclusion, our study of this atypical case of abetalipoproteinemia shows that the combination of molecular diagnosis and functional analysis resulted in a defi nitive diagnosis of MTP defi ciency in a patient when insuffi cient biopsy material was available for the analysis of the MTP protein or RNA. The functional studies of the substitution of Leu435 to a charged amino acid (acid Glu or basic His) highlight the requirement for this hydrophobic uncharged residue for MTP activity. In cases of unusual phenotype, the functional characterization of the MTTP mutants allows a better understanding of the milder ABL phenotype.
The authors thank S. Dumont (Service de Biochimie, CBE, HCL, Lyon), S. Faïna (Service de Biochimie et Génétique The p.Leu435His mutation affects an amino acid in helix 8 of the predicted ␣ -helical domain of MTP ( 43 ). Although this helix is not included in the major binding site for PDI and ApoB (43)(44)(45), the MTP activity of the mutant protein is very low and similar to that of "severe" mutations. The p.Leu435His mutation leads to a nonfunctional protein probably by affecting the MTP folding. Misfolded MTP may be targeted for degradation in the intestine or the liver; however, this recognition of misfolded proteins cannot be observed in heterologous expression systems (transfected COS-1 cells).
The c.619-5_619-2del is probably responsible for the mild phenotype, as it produces two transcripts. One is normal-sized and contains exon 6 of the MTTP . The other transcript is aberrantly spliced and lacks exon 6. The phenotype may result from the correctly spliced RNA, which is able to produce an active MTP. Although there may be some variability in the amounts of correctly spliced RNA between HeLa cells and the liver or the intestine, the marked effect of the intronic deletion observed in HeLa cells most likely refl ects what might be expected in other cell types. A possible molecular mechanism for this splicing anomaly, predicted by the in silico analysis, is the shortening of the polypyrimidine tract by the deletion of one TTTA repeat. This deletion, by changing the number of uridines in the MTTP pre-mRNA from 9 to 6, would decrease the number of interactions between the polypyrimidine tract and the pre-mRNA splicing factor U2AF65. This step is crucial as it is one of the fi rst stages of splice site recognition. Short polypyrimidine tracts have been shown to be associated with variable levels of correctly spliced transcripts in the CFTR gene ( 46 ), thus providing evidence for incomplete penetrance of some splicing mutations in disease.