A New Case of Abetalipoproteinemia Caused by Novel Compound Heterozygote Mutations in the MTTP Gene without Fat or Vitamin Malabsorption

Abstract

Abetalipoproteinemia (ABL) is a rare disease characterized by extremely low apolipoprotein B (apoB)-containing lipoprotein levels, dietary fat, and fat-soluble vitamin malabsorption, leading to gastrointestinal, neuromuscular, and ophthalmological symptoms. We herein report a case of ABL with novel compound heterozygous mutations in the microsomal triglyceride transfer protein gene (c.1686_1687del [p.Ser563TyrfsTer10] and c.1862T>C [p.Ile621Thr]), identified via panel sequencing. Although the patient had extremely reduced low-density lipoprotein cholesterol levels and a fatty liver, he did not exhibit other typical complications. Furthermore, unlike typical ABL, this patient had a preserved apoB-48 secretion and increased concentrations of high-density lipoprotein cholesterol, which may account for the normal serum fat-soluble vitamin levels.

Introduction

Abetalipoproteinemia (ABL, OMIM #200100) is an autosomal recessive disorder caused by mutations in microsomal triglyceride transfer protein (MTTP) gene¹⁾. MTTP is essential for transferring triglyceride (TG) and cholesterol esters to apolipoprotein B (apoB)-containing lipoproteins. ApoB is essential for the formation of chylomicrons (CMs), very low-density lipoproteins (VLDLs), and low-density lipoproteins (LDLs)²⁾. In patients with ABL, the secretion of apoB-containing lipoproteins is abrogated, resulting in various clinical manifestations, such as neuromuscular, gastrointestinal, and ophthalmological symptoms due to malabsorption of dietary fat and fat-soluble vitamin malabsorption.

ApoB is classified into two major isoforms of apoB-48 and apoB-100 ²⁾, with the former secreted into the intestinal epithelium to form CMs and the latter produced in the liver to form VLDLs and LDLs. MTTP physically interacts with apoB for lipid transfer³⁾. To date, several case reports have presented ABL with MTTP gene mutation that lacked apoB-100 but had normal plasma apoB-48 concentrations^{4, 5)}. The authors discussed possible mechanisms for the different levels of apoB-100 and apoB-48 as follows: an increased affinity of shorter apoB peptides for MTTP and the secretion of apoB-100 being more sensitive to MTTP activity than apoB-48. Interestingly, lipid malabsorption and fat-soluble vitamin deficiency occurred in these patients despite the presence of apoB-48.

We herein report a novel compound heterozygous mutation in MTTP without lipid malabsorption or fat-soluble vitamin deficiency.

Methods

Study Subjects

A 40-year-old Japanese man was referred to our hospital because of extremely low LDL-cholesterol (LDL-C) levels of 4 mg/dL and fatty liver. He was an official of the Japan Ground Self-Defense Forces. Based on annual health checkup data, his LDL-C levels had been continuously low since his first health checkup at 24 years old. This study also included his parents, older sisters, younger brothers, and children.

Biochemical Analyses

Using fasting blood samples, the total serum cholesterol (TC), TG, high-density lipoprotein cholesterol (HDL-C), and LDL-C concentrations were determined enzymatically (Qualigent; Sekisui Medical, Tokyo, Japan). L-aspartate (2-oxoglutarate aminotransferase) and L-alanine (2-oxoglutarate aminotransferase, γ-glutamyl transferase) levels were measured enzymatically (Qualigent; FUJIFILM Wako Pure Chemical, Osaka, Japan). The apolipoprotein E (Apo-E) phenotype was determined by isoelectric focusing with Western blotting using an apo-E polyclonal antibody (Phenotyping Apo-E IEF System; JOKOH, Tokyo, Japan). Serum apolipoprotein, cholesteryl ester transfer protein, vitamin A, and 25-OH vitamin D levels were measured using a turbid metric immunoassay, enzymatic method, high-performance liquid chromatography, and chemiluminescent enzyme immunoassay (BML, Tokyo, Japan), respectively. The serum vitamin E concentration was measured using the fluorescence method (LSI Medience Corp., Tokyo, Japan). The apolipoprotein B-100 and apolipoprotein B-48 levels were measured using enzyme-linked immunosorbent assays (IBL, Fujioka, Japan; and FUJIFILM Wako Shibayagi, Shibukawa, Japan).

Genetic Analyses

The DNA from each participant was isolated from peripheral white blood cells using a standard DNA extraction protocol. The DNA was pooled, selected according to size, ligated to sequencing adapters, and amplified to enrich the sequenced targets using the Kapa DNA Library Preparation. A custom NimbleGen in-solution DNA capture library (Roche NimbleGen Inc., Madison, WI, USA) was designed to capture all coding lesions in 21 dyslipidemia-related Mendelian genes, including 4 genes associated with primary hypobetalipoproteinemia (ANGPTL3, APOB, MTTP, and PCSK9) and 5 associated with HDL (ABCA1, APOA1, CETP, LCAT, and LIPG). These details were described in a previous study⁶⁾. Target-enriched products were sequenced using an Illumina iSeq (San Diego, CA, USA).

Ethical Considerations

The genetic analyses were approved by the Ethics Committee of Kanazawa University (2016-021 [313]). All procedures were performed in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and the 1975 Declaration of Helsinki, as revised in 2008. Written informed consent for genetic analyses was obtained from all participants.

Case Reports

·Characteristics of the Study Subjects

Table 1 and Fig.1 show the biochemical profiles of the family and the family tree, respectively. They did not exhibit any secondary causes of hypobetalipoproteinemia, including hyperthyroidism, bleeding, or malignancy. There were no consanguineous marriages in the family.

Table 1.Characteristics of the family

Subject (gender)	I.1 (male)＊	I.2 (female)	II.1 (female)	II.2 (male)	II.3 (male)	III.1 (male)	III.2 (male)
MTTP c.1686_1687del/p.Ser563TyrfsTer10 carrier	+	-	+	+	-	-	+
MTTP c.1862T>C/p.Ile621Thr carrier	-	+	-	+	+	+	-
Age (yr)	71	68	42	40	38	14	12
Total cholesterol (mg/dL) (142 - 219)	228	185	231	115	259	173	186
Triglyceride (mg/dL) (40 - 149)	97	61	82	32	86	66	55
HDL cholesterol (mg/dL) (40 - 90)	59	61	83	91	51	75	76
LDL cholesterol (mg/dL) (65 - 139)	137	102	128	4	186	83	99
Apolipoprotein AI (mg/dL) (119 - 155)	141	133	189	217	126	124	169
Apolipoprotein AII (mg/dL) (25.9 - 35.7)	31.4	40.1	29.4	26.9	34.3	40.8	33.5
Apolipoprotein B (mg/dL) (73 - 109)	98	72	82	＜7.0	126	71	60
Apolipoprotein B100 (mg/dL)	96.1	73.6	N/D	0.0	N/D	N/D	N/D
Apolipoprotein B48 (ug/mL)	8.1	6.5	N/D	1.7	N/D	N/D	N/D
Apolipoprotein CII (mg/dL) (1.8 - 4.6)	5.4	4.2	3.9	3.9	6.4	4.1	2.9
Apolipoprotein CIII (mg/dL) (5.8 - 10.0)	13.7	10.1	11.6	6.9	10.0	9.2	11.8
Apolipoprotein E (mg/dL) (2.7 - 4.3)	5.7	5.2	4.3	3.1	4.5	5.0	4.2
Apolipoprotein E phenotype	E3/E3	E3/E3	E3/E3	E3/E3	E3/E3	E3/E3	E3/E3
CETP (µg/mL)	3.0	2.2	3.3	2.9	2.8	1.6	1.4
Vitamin A (IU/L) (27.2 - 102.7)	33.0	43.2	39.7	41.9	60.8	35.7	36.0
25-OH Vitamin D (ng/mL) (＞30)	12.5	29.3	14.5	35.4	28.4	19.8	21.6
Vitamin E (IU/L) (0.75 - 1.41)	1.40	0.98	1.09	0.85	1.05	1.03	0.87
AST (IU/L) (13 - 30)	39	26	45＊＊	74	28	17	26
ALT (IU/L) (10 - 30)	38	17	14	69	50	12	14
γ-GTP (IU/L) (13 - 64)	14	13	10	33	36	16	13

MTTP, microsomal triglyceride transfer protein; HDL, high-density lipoprotein; LDL, low-density lipoprotein; CETP, cholesteryl ester transfer protein; AST, aspartate transaminase; ALT, alanine transaminase; γ-GTP, γ-glutamyl transpeptidase. ^＊Atorvastatin 10 mg/day. ^＊＊Hemolyzed sample.

Fig.1. Family tree

The arrow indicates the proband. The black color represents a carrier of the MTTP mutation c.1686_1687del (p.Ser563Tyr fsTer10) (MTTP). The gray color indicates a carrier of the MTTP mutation c.1862T>C (p.Ile621Thr) (MTTP).

The proband (II-2) was physically normal with respect to his height (172.6 cm), weight (65.2 kg), body mass index (22.0 kg/m²), and blood pressure (106/66 mmHg) but had extremely low LDL-C and apoB levels (4 and ＜7 mg/dL, respectively). Although the apoB-100 level was initially undetectable, it was identified using an enzyme-linked immunosorbent assay. The patient did not exhibit gastrointestinal symptoms, even after fat intake. An endoscopic examination showed no abnormalities in the duodenum or ileal mucosa. His serum fat-soluble vitamin levels, including vitamins A, 25-OH, vitamin D, and vitamin E, were within the normal range without any supplementation. Furthermore, the patient did not have any neurological, ophthalmological, or hematological symptoms. The liver enzyme levels were two to three times higher than the normal upper limits. Computed tomography (CT) revealed severe fatty liver (CT attenuation values liver/spleen ratio was Liver; 32.0 Hounsfield unit/Spleen; 56.1 Hounsfield unit, 0.57). Atherosclerotic changes were not evident on carotid artery ultrasonography.

The patient’s father was on statin medications because of hypercholesterolemia, but the other family members had normal lipid and liver enzyme levels. None of the family members exhibited other ABL-related complications, including liver dysfunction, neurocognitive disorders, or cerebral hemorrhaging.

·Genetic Analyses

The proband was a compound heterozygote carrying a c.1686_1687del (p.Ser563TyrfsTer10) MTTP mutation in exon 12 and c.1862T>C (p.Ile621Thr) MTTP mutation in exon 14. Genetic mutations were not found in other genes associated with dyslipidemia-related Mendelian mutations (including HDL-associated mutations). c.1686_1687del was inherited from the proband’s father (I-1), his sister (II-1), and his second son (III-2). p.Ile621Thr was inherited from the proband’s mother (I-1), his brother (II-3), and oldest son (III-1) (Fig.1). The p.Ile621Thr missense mutation could be “possibly damaging” by PolyPhen-2 (score: 0.535(HumVar)) and “damaging” according to SIFT (score: 0.02). According to the ACMG guidelines⁷⁾. It is classified as a Variant of Uncertain Significance because this mutation is extremely rare.

Discussion

We herein report a case of compound heterozygosity for c.1686_1687del and p.Ile621Thr missense mutations in MTTP gene. The proband exhibited extremely low LDL-C levels and fatty liver due to the absence of apoB-100 in the plasma. However, the presence of apoB-48 in the plasma may be associated with the absence of impaired fat absorption and the typical neurological symptoms observed in ABL. Notably, this case was characterized by markedly elevated HDL-C level.

This case is classified as familial hypobetalipoproteinemia due to lipoprotein assembly and secretion defect 1 (FHBL-SD1), commonly known as ABL⁸⁾. However, the criteria for diagnosing ABL in Japan require clinical manifestations and/or acanthocytosis¹⁾. Our case, which lacked fat and vitamin malabsorption, did not meet these criteria. Although the reasons for the lack of typical manifestations of ABL in our case are unknown, we consider this aspect a novel feature of ABL.

The MTTP protein comprises two subunits forming a heterodimer: a larger subunit, encoded by the MTTP gene, with a size of 97 kDa, and a protein disulfide isomerase (PDI) unit, with a size of 55 kDa, encoded by the P4HB gene⁹⁾. The c.1686_1687del mutation is a frameshift mutation in the α-helical domain, which interacts with PDI. Both the c.1389del frameshift mutation¹⁰⁾ and c.1820del frameshift mutation⁹⁾ have been documented in homozygous cases, thereby showing the classical features of ABL accompanied by lipid absorption disorders. Therefore, the c.1686_1687del mutation in this case is believed to disrupt PDI binding sites, resulting in loss of TG transfer activity. Conversely, the p.Ile621Thr missense mutation occurred in the C-terminal domain, which plays a role in lipid transport. In the p.Arg623Leu mutation, which was observed in a specific ABL case, 65% of MTTP activity was preserved. Furthermore, in a compound heterozygous case with the c.1067+1217_1141del mutation⁴⁾, apoB-48, but not apoB-100, was secreted. Since the p.Ile621Thr missense mutation in this case is similar to the above mutation, this may be a reason for the preserved residual TG transport capacity of MTTP and subsequent apoB-48 secretion. Indeed, the ability to secrete apoB-48 is retained in some cases with MTTP mutations. Nonetheless, only a preserved apoB-48 secretion and fat/fat-soluble vitamin absorption were observed in this case^{4, 5)}. Although the reasons for this discrepancy are unknown, we need to conduct a functional analysis assessing the MTTP functionality, in particular apoB-48 secretion capability.

In the family, only the proband showed elevated HDL-C and apoA-I levels. No mutations were found in CETP or LIPG. In typical ABL cases, plasma levels of HDL are decreased¹⁾. A previous kinetic study showed increased catabolism of apoA-I, which is consistent with other low HDL-C cases¹¹⁾. Therefore, it is unlikely that the increased HDL levels were due to an MTTP mutation.

Enterocytes synthesize apoB-containing CM and apoA-I-containing HDL, and fat-soluble vitamins are absorbed via these two pathways^{12, 13)}. Although it is considered that CM receives fat-soluble vitamins more dominantly than HDL in healthy humans¹⁴⁾, HDL transports the majority of vitamins in patients with impaired CM and apoB-100 production¹⁵⁾. In our previous case of familial hypobetalipoproteinemia with homozygous apoB-87.77 mutations without fat absorption impairment or neurological abnormalities¹⁶⁾, vitamin E was found in HDL fractions isolated by ultracentrifugation (unpublished data). Taken together, these findings suggest that increased HDL levels might contribute to the compensatory transport of fat-soluble vitamins.

Regarding cirrhosis in ABL, the details have been previously reported¹⁷⁾. Therefore, the status of non-alcoholic fatty liver disease should be monitored. The patient’s liver enzyme levels remained stable or even recovered after discontinuing habitual alcohol consumption (Supplementary Table 1).

Supplemental Table 1.Annual health check-up results of proband (II.2)

Age		24Y11M	34Y8M	36Y2M	38Y10M	40Y3M	40Y10M
Body weight	Kg	N/D	69	73	69	65	65
Lipid metabolism parameters
Total cholesterol	mg/dL	93	125	107	119	115	132
HDL-cholesterol	mg/dL	76	104	82	108	91	97
LDL-cholesterol	mg/dL	N/D	18	14	14	4	4
Triglyceride	mg/dL	66	17	79	35	32	21
Liver function parameters
AST	U/L	50	50	66	47	74	36
ALT	U/L	55	55	52	38	69	27
g-GTP	U/L	20	20	27	29	33	19

HDL: high density lipoprotein, LDL: low density lipoprotein, AST: aspartate transaminase, ALT: alanine transaminase, γ-GTP: γ-glutamyl transpeptidas

The patient did not exhibit any growth disorders and was fertile. Thus, this is a relatively healthy and rare case compared with other ABL cases. However, similar to other ABL cases, extremely low LDL-C and apoB levels are associated with a reduced risk of atherosclerotic cardiovascular disease. MTP inhibitors implement “pharmacological” ABL conditions. Although our patient with ABL showed a favorable clinical phenotype, this does not change the fact that a cautious nature should be practiced when MTP inhibitors are administered to FH homozygotes.

As a limitation of this report, the genetic analysis was confined to 21 genes, and a genetic analysis of P4HB, associated with the structure of MTTP, and SCARB1, which is involved in HDL metabolism, was not conducted. Nevertheless, the probability of harboring mutations in these genes is exceedingly low.

We herein report a case of compound heterozygosity for c.1686_1687del and p.Ile621Thr missense mutations in MTTP. Although the patient had extremely low LDL-C levels, his fat-soluble vitamin concentrations were within the normal range, which may be associated with the preserved secretion of apoB-48 and/or increased HDL-C concentrations. Therefore, this case provides novel insights into the roles of MTTP in lipoprotein metabolism, intestinal fat, and fat-soluble vitamin absorption, and further analyses including next-generation genetic sequencing should be conducted.

Acknowledgments

We thank Ms. Kazuko Honda and Ms. Sachio Yamamoto for their technical assistance.

Sources of Funding

This work was supported by JSPS KAKENHI (no. 21K08066) and a grant from the Ministry of Health, Labor, and Welfare of Japan (Sciences Research Grant for Research on Rare and Intractable Diseases).

Conflicts of Interest

None declared.

References

• 1)  Takahashi M, Okazaki H, Ohashi K, Ogura M, Ishibashi S, Okazaki S, Hirayama S, Hori M, Matsuki K, Yokoyama S, Harada-Shiba M. Current Diagnosis and Management of Abetalipoproteinemia. J Atheroscler Thromb, 2021; 28: 1009-1019
• 2)  Young SG. Recent progress in understanding apolipoprotein B. Circulation, 1990; 82: 1574-1594
• 3)  Hussain MM, Bakillah A, Jamil H. Apolipoprotein B binding to microsomal triglyceride transfer protein decreases with increases in length and lipidation: implications in lipoprotein biosynthesis. Biochemistry, 1997; 36: 13060-13067
• 4)  Di Filippo M, Collardeau Frachon S, Janin A, Rajan S, Marmontel O, Decourt C, Rubio A, Nony S, Dumont S, Cuerq C, Charrière S, Moulin P, Lachaux A, Hussain MM, Bozon D, Peretti N. Normal serum ApoB48 and red cells vitamin E concentrations after supplementation in a novel compound heterozygous case of abetalipoproteinemia. Atherosclerosis, 2019; 284: 75-82
• 5)  Takahashi M, Ozaki N, Nagashima S, Wakabayashi T, Iwamoto S, Ishibashi S. Normal plasma apoB48 despite the virtual absence of apoB100 in a compound heterozygote with novel mutations in the MTTP gene. J Clin Lipidol, 2021; 15: 569-573
• 6)  Tada H, Kawashiri MA, Nomura A, Teramoto R, Hosomichi K, Nohara A, Inazu A, Mabuchi H, Tajima A, Yamagishi M. Oligogenic familial hypercholesterolemia, LDL cholesterol, and coronary artery disease. J Clin Lipidol, 2018; 12: 1436-1444
• 7)  Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, Grody WW, Hegde M, Lyon E, Spector E, Voelkerding K, Rehm HL; ACMG Laboratory Quality Assurance Committee. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med, 2015; 17: 405-424
• 8)  Bredefeld C, Hussain MM, Averna M, Black DD, Brin MF, Burnett JR, Charrière S, Cuerq C, Davidson NO, Deckelbaum RJ, Goldberg IJ, Granot E, Hegele RA, Ishibashi S, Karmally W, Levy E, Moulin P, Okazaki H, Poinsot P, Rader DJ, Takahashi M, Tarugi P, Traber MG, Di Filippo M, Peretti N. Guidance for the diagnosis and treatment of hypolipidemia disorders. J Clin Lipidol, 2022; 16: 797-812
• 9)  Ohashi K, Ishibashi S, Osuga J, Tozawa R, Harada K, Yahagi N, Shionoiri F, Iizuka Y, Tamura Y, Nagai R, Illingworth DR, Gotoda T, Yamada N. Novel mutations in the microsomal triglyceride transfer protein gene causing abetalipoproteinemia. J Lipid Res, 2000; 41: 1199-1204
• 10)  Wang J, Hegele RA. Microsomal triglyceride transfer protein (MTP) gene mutations in Canadian subjects with abetalipoproteinemia. Hum Mutat, 2000; 15: 294-295
• 11)  Ikewaki K, Rader DJ, Zech LA, Brewer HB Jr. In vivo metabolism of apolipoproteins A-I and E in patients with abetalipoproteinemia: implications for the roles of apolipoproteins B and E in HDL metabolism. J Lipid Res, 1994; 35: 1809-1819
• 12)  Anwar K, Kayden HJ, Hussain MM. Transport of vitamin E by differentiated Caco-2 cells. J Lipid Res, 2006; 47: 1261-1273
• 13)  Anwar K, Iqbal J, Hussain MM. Mechanisms involved in vitamin E transport by primary enterocytes and in vivo absorption. J Lipid Res, 2007; 48: 2028-2038
• 14)  Traber MG, Leonard SW, Ebenuwa I, Violet PC, Wang Y, Niyyati M, Padayatty S, Tu H, Courville A, Bernstein S, Choi J, Shamburek R, Smith S, Head B, Bobe G, Ramakrishnan R, Levine M. Vitamin E absorption and kinetics in healthy women, as modulated by food and by fat, studied using 2 deuterium-labeled α-tocopherols in a 3-phase crossover design. Am J Clin Nutr, 2019; 110: 1148-1167
• 15)  Traber MG, Burton GW, Hughes L, Ingold KU, Hidaka H, Malloy M, Kane J, Hyams J, Kayden HJ. Discrimination between forms of vitamin E by humans with and without genetic abnormalities of lipoprotein metabolism. J Lipid Res, 1992; 33: 1171-1182
• 16)  Kawashiri MA, Tada H, Hashimoto M, Taniyama M, Nakano T, Nakajima K, Inoue T, Mori M, Nakanishi C, Konno T, Hayashi K, Nohara A, Inazu A, Koizumi J, Ishihara H, Kobayashi J, Hirano T, Mabuchi H, Yamagishi M. Extreme Contrast of Postprandial Remnant-Like Particles Formed in Abetalipoproteinemia and Homozygous Familial Hypobetalipoproteinemia. JIMD Rep, 2015; 22: 85-94
• 17)  Di Filippo M, Moulin P, Roy P, Samson-Bouma ME, Collardeau-Frachon S, Chebel-Dumont S, Peretti N, Dumortier J, Zoulim F, Fontanges T, Parini R, Rigoldi M, Furlan F, Mancini G, Bonnefont-Rousselot D, Bruckert E, Schmitz J, Scoazec JY, Charrière S, Villar-Fimbel S, Gottrand F, Dubern B, Doummar D, Joly F, Liard-Meillon ME, Lachaux A, Sassolas A. Homozygous MTTP and APOB mutations may lead to hepatic steatosis and fibrosis despite metabolic differences in congenital hypocholesterolemia. J Hepatol, 2014; 61: 891-902