Mutations of the Microsomal Triglyceride-Transfer–Protein Gene in Abetalipoproteinemia

Elevated plasma levels of apolipoprotein B (apoB)–containing lipoproteins constitute a major risk factor for the development of coronary heart disease. In the rare recessively inherited disorder abetalipoproteinemia (ABL) the production of apoB-containing lipoproteins is abolished, despite no abnormality of the apoB gene. In the current study we have characterized the gene encoding a microsomal triglyceride-transfer protein (MTP), localized to chromosome 4q22-24, and have identified a mutation of the MTP gene in both alleles of all individuals in a cohort of eight patients with classical ABL. Each mutant allele is predicted to encode a truncated form of MTP with a variable number of aberrant amino acids at its C-terminal end. Expression of genetically engineered forms of MTP in Cos-1 cells indicates that the C-terminal portion of MTP is necessary for triglyceride-transfer activity. Deletion of 20 amino acids from the carboxyl terminus of the 894-amino-acid protein and a missense mutation of cysteine 878 to serine both abolished activity. These results establish that defects of the MTP gene are the predominant, if not sole, cause of hereditary ABL and that an intact carboxyl terminus is necessary for activity.     

Structure-function analyses of microsomal triglyceride transfer protein missense mutations in abetalipoproteinemia and hypobetalipoproteinemia subjects

We describe two new hypolipidemic patients with very low plasma triglyceride and apolipoprotein B (apoB) levels with plasma lipid profiles similar to abetalipoproteinemia (ABL) patients. In these patients, we identified two previously uncharacterized missense mutations in the microsomal triglyceride transfer protein (MTP) gene, R46G and D361Y, and studied their functional effects. We also characterized three missense mutations (H297Q, D384A, and G661A) reported earlier in a familial hypobetalipoproteinemia patient. R46G had no effect on MTP expression or function and supported apoB secretion. H297Q, D384A, and G661A mutants also supported apoB secretion similarly to WT MTP. Contrary to these four missense mutations, D361Y was unable to support apoB secretion. Functional analysis revealed that this mutant was unable to bind protein disulfide isomerase (PDI) or transfer lipids. The negative charge at residue 361 was critical for MTP function as D361E was able to support apoB secretion and transfer lipids. D361Y most likely disrupts the tightly packed middle α-helical region of MTP, mitigates PDI binding, abolishes lipid transfer activity, and causes ABL. On the other hand, the hypolipidemia in the other two patients was not due to MTP dysfunction. Thus, in this study of five missense mutations spread throughout MTP's three structural domains found in three hypolipidemic patients, we found that four of the mutations did not affect MTP function. Thus, novel mutations that cause severe hypolipidemia probably exist in other genes in these patients, and their recognition may identify novel proteins involved in the synthesis and/or catabolism of plasma lipoproteins.     

Known mutations of apoB account for only a small minority of hypobetalipoproteinemia

Low LDL cholesterol and apoB levels in plasma cosegregate with mutations of apoB in some kindreds with familial hypobetalipoproteinemia. Approximately 35 apoB mutations, many specifying apoB truncations, have been described. Based on the centile nomenclature where the full-length nature apoB consisting of 4536 amino acids is designated as apoB-100, only those truncations of apoB >25% of normal length are detectable in plasma. Previously, we reported on five unrelated kindreds with familial hypobetalipoproteinemia in whom although no apoB truncations were detectable in plasma, low apoB levels were nevertheless linked to the apoB gene. In one of those kindreds, we reported a donor splice site mutation in intron 5 (specifying apoB- 4). We now describe a nonsense mutation in exon 10 (apoB-9) in two of the other unrelated families. Both the apoB-4 and apoB-9 mutations have been reported by others in unrelated families. Recurrent mutations of apoB-40 and apoB-55 also have been reported, suggesting that recurrent mutations of apoB may account for an appreciable proportion of familial hypobetalipoproteinemia kindreds. To test this hypothesis, we searched for four apoB mutations whose products are not detected in plasma including the apoB-4, apoB-9, and two other previously reported mutations in exons 21 and 25. We studied three groups with plasma cholesterols <130 mg/dl in whom no apoB truncations were detected in plasma: a) 28 FHBL probands from St. Louis, b) 151 individual St. Louisians, and c) 28 individual Sicilians. One subject from the 28 kindreds and two subjects among 151 hypobeta individuals from St. Louis harbored the exon 10 mutation. None of the other mutations were detected. Thus, among hypobeta lipoproteinemic subjects without any detectable apoB truncations in plasma, <5% had an apoB truncation-producing mutation. As only about 0.5% of hypobeta lipoproteinemic subjects have plasma-detectable apoB truncations, our data suggest that the known apoB truncations account for only a small proportion of hypocholesterolemia.     

Hypobetalipoproteinemia with an apparently recessive inheritance due to a "de novo" mutation of apolipoprotein B

Familial hypobetalipoproteinemia (FHBL) is a co-dominant disorder either linked or not linked to apolipoprotein (apo) B gene. Abetalipoproteinemia (ABL) is a recessive disorder due to mutations of microsomal triglyceride transfer protein (MTP) gene. We investigated a patient with apparently recessive hypobetalipoproteinemia consistent with symptomatic heterozygous FHBL or a "mild" form of ABL. The proband had fatty liver associated with LDL-cholesterol (LDL-C) and apo B levels <5th percentile but no truncated apo B forms detectable in plasma. MTP gene sequence revealed that he was a carrier of the I128T polymorphism and an unreported amino acid substitution (V168I) unlikely to be the cause of hypobetalipoproteinemia. Apo B gene sequence showed that he was heterozygous for two single base substitutions in exon 9 and 22 resulting in a nonsense (Q294X) and a missense (R1101H) mutation, respectively. Neither of his parents carried the Q294X; his father and paternal grandmother carried the R1101H mutation. Analysis of polymorphic genetic markers excluded non-paternity. In conclusion, the proband has a "de novo" mutation of apo B gene resulting in a short truncated apo B form (apo B-6.46). Sporadic cases of FHBL with an apparently recessive transmission may be caused by "de novo" mutations of apo B gene.     

Two novel mutations in the lipoprotein lipase gene in a family with marked hypertriglyceridemia in heterozygous carriers. Potential interaction with the polymorphic marker D1S104 on chromosome 1q21-q23

Two novel mutations in the lipoprotein lipase (LPL) gene are described in an Austrian family: a splice site mutation in intron 1 (3 bp deletion of nucleotides -2 to -4) which results in skipping of exon 2, and a missense mutation in exon 5 which causes an asparagine for histidine substitution in codon 183 and complete loss of enzyme activity. A 5-year-old boy who exhibited all the clinical features of primary hyperchylomicronemia was a compound heterozygote for these two mutations. Nine other family members were investigated: seven were heterozygotes for the splice site mutation, one was a heterozygote for the missense mutation, and one had two wild-type alleles of the LPL gene. LPL activity in the post-heparin plasma of the heterozygotes was reduced to 49;-79% of the mean observed in normal individuals. Two of the heterozygotes had extremely high plasma triglyceride levels; in three of the other heterozygotes the plasma triglycerides were also elevated. As plasma triglycerides in carriers of one defective LPL allele can be normal or elevated, the heterozygotes of this family have been studied for a possible additional cause of the expression of hypertriglyceridemia in these subjects. Body mass index, insulin resistance, mutations in other candidate genes (Asn291Ser and Asp9Asn in the LPL gene, apoE isoforms, polymorphisms in the apoA-II gene and in the apoAI-CIII-AIV gene cluster, and in the IRS-1 gene) could be ruled out as possible factors contributing to the expression of hypertriglyceridemia in this family. A linkage analysis using the allelic marker D1S104 on chromosome 1q21;-q23 suggested that a gene in this region could play a role in the expression of hypertriglyceridemia in the heterozygous carriers of this family, but the evidence was not sufficiently strong to prove this assumption. Nevertheless, this polymorphic marker seems to be a good candidate for further studies.     

A Tyrosinase missense mutation causes albinism in the Wistar rat

Tyrosinase serves as a key enzyme in the synthesis of melanin. In humans mutations in the TYR gene are associated with type 1 oculocutaneous albinism (OCA1) that leads to reduced or absent pigmentation of skin, hair and eye. Various mutations causing OCA in man, mouse, rabbit and cattle have been identified throughout the Tyrosinase gene including nonsense, missense, frameshift and splice site alterations. Here we report a missense substitution at codon R299H in exon 2 of the Tyr gene in the albino Wistar rat. As this very exchange has already been described in OCA patients, our findings reinforce the significance of this region for normal catalytic activity of tyrosinase protein.     

A deficiency of microsomal triglyceride transfer protein reduces apolipoprotein B secretion

Microsomal triglyceride transfer protein (MTP) transfers lipids to apolipoprotein B (apoB) within the endoplasmic reticulum, a process that involves direct interactions between apoB and the large subunit of MTP. Recent studies with heterozygous MTP knockout mice have suggested that half-normal levels of MTP in the liver reduce apoB secretion. We hypothesized that reduced apoB secretion in the setting of half-normal MTP levels might be caused by a reduced MTP:apoB ratio in the endoplasmic reticulum, which would reduce the number of apoB-MTP interactions. If this hypothesis were true, half-normal levels of MTP might have little impact on lipoprotein secretion in the setting of half-normal levels of apoB synthesis (since the ratio of MTP to apoB would not be abnormally low) and might cause an exaggerated reduction in lipoprotein secretion in the setting of apoB overexpression (since the MTP:apoB ratio would be even lower). To test this hypothesis, we examined the effects of heterozygous MTP deficiency on apoB metabolism in the setting of normal levels of apoB synthesis, half-normal levels of apoB synthesis (heterozygous Apob deficiency), and increased levels of apoB synthesis (transgenic overexpression of human apoB). Contrary to our expectations, half-normal levels of MTP reduced the plasma apoB100 levels to the same extent ( approximately 25-35%) at each level of apoB synthesis. In addition, apoB secretion from primary hepatocytes was reduced to a comparable extent at each level of apoB synthesis. Thus, these results indicate that the concentration of MTP within the endoplasmic reticulum rather than the MTP:apoB ratio is the critical determinant of lipoprotein secretion. Finally, we found that heterozygosity for an apoB knockout mutation lowered plasma apoB100 levels more than heterozygosity for an MTP knockout allele. Consistent with that result, hepatic triglyceride accumulation was greater in heterozygous apoB knockout mice than in heterozygous MTP knockout mice.     

Application of dHPLC for mutation detection of the fibrillin-1 gene for the diagnosis of Marfan syndrome in a National Health Service Laboratory

Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder caused by mutations in the fibrillin-1 gene FBN1. Mutation detection of this 65-exon gene presents a particular challenge for the diagnostic service in cost, time constraints, and the need to maintain a stringently optimized assay procedure. Using denaturing high-performance liquid chromatography (dHPLC), we have designed a procedure for rapid mutation scanning, redesigning 50% of published primer sets, screening by Ensembl to avoid inclusion of polymorphic variations and employing a limited set of PCR conditions to allow for a high-throughput 96-well format. We have screened 262 unrelated patients with MFS or Marfan-like phenotypes and detected 103 (39.3%) mutations including 93 different mutations, 72 of which are novel. The mutations include 55 missense (53.4%) 19 splice site (18.5%), 17 frameshift (16.5%), 11 nonsense (10.7%) and 1 in-frame deletion/insertion.     

Molecular characterization of seven beta-thalassemia mutations in Asian Indians

To characterize systematically the mutations which produce beta-thalassemia in Asian Indians, we first determined the DNA polymorphism haplotype in the beta-globin gene cluster of 44 beta-thalassemia chromosomes in the ethnic group. Nine different haplotypes were observed. Upon molecular cloning and partial DNA sequencing of one beta-gene from each of eight haplotypes and two from the ninth, seven different mutations were found. None of these have been identified in Mediterranean patients, even among the five haplotypes which appeared identical in the two groups. Asian Indian mutations included one nonsense and three frameshift mutations, one deletion affecting an acceptor splice site, and two mutations affecting a donor splice site. The correlation of a specific mutation with a specific haplotype was high but not invariant. Two mutations were associated with more than one haplotype but, in each instance, the mutation spread to a new haplotype could be explained most simply by recombination 5' to the beta-globin gene. In addition, four mutations, one reported here and three others previously reported, have been observed on two chromosome backgrounds that are identical except for the status of a polymorphic HinfI site 5' to the beta gene. This HinfI site does not show significant linkage disequilibrium with markers both 5' and 3' to it, suggesting that it lies within a region of relative sequence randomization.     

ApoB gene nonsense and splicing mutations in a compound heterozygote for familial hypobetalipoproteinemia.

Two novel apoB gene mutations were identified in a patient (CM) with phenotypic homozygous hypobetalipoproteinemia. Haplotype analysis of the apoB alleles from this patient and his family members revealed him to be a genetic compound for the disease. In contrast to previous studies of other hypobetalipoproteinemic patients, no clues existed as to where in the apoB gene the molecular defects resided. Therefore, it was necessary to characterize the apoB genes of the patient by sequence analysis. The apoB gene contains 29 exons and is 43 kb in length. The gene encodes a 14.1 kb mRNA and a 4563 amino acid protein. Both apoB alleles from the patient were cloned via 26 sets of polymerase chain reactions (PCR). These clones contained a total of approximately 24 kb of apoB gene sequence, including regions 5' and 3' to the coding region, 29 exons, and the intron/exon junctions. Complete DNA sequence analysis of these clones showed that each apoB allele had a mutation. In the paternal apoB allele, there was a splicing mutation. The first base of the dinucleotide consensus sequence (GT) in the 5' splice donor site in intron 5 was replaced by a T. It is likely that this base substitution interferes with proper splicing and results in the observed absence of plasma apoB. In the maternal apoB allele, there was a nonsense mutation. The first base of the Arg codon (CGA) at residue 412 in exon 10 was replaced by a T, resulting in a termination codon (TGA). The nonsense mutation is likely to terminate translation after residue 411 resulting in a severely truncated protein only 9% of the length of B-100.(ABSTRACT TRUNCATED AT 250 WORDS)     

Novel mutations in the transmembrane natriuretic peptide receptor <Emphasis Type="Italic">NPR-B</Emphasis> gene in four Indian families with acromesomelic dysplasia,type Maroteaux

Acromesomelic dysplasia, type Maroteaux is a disorder characterized by disproportionate short stature predominantly affecting the middle and distal segments of the upper and lower limbs. It is an autosomal recessive disorder due to mutation in NPR2 gene which impairs skeletal growth. To screen the mutations in the gene NPR2, all of its coding exons and splice junction sites were PCR amplified from genomic DNA of affected individuals of four families and sequenced. Four homozygous mutations in four different families were identified. These include three novel mutations including a deletion frameshift mutation (p.Cys586Ter), one nonsense mutation (p.Arg479Ter), one missense mutation (p.Val187Asp) and one reported missense mutation (p.Tyr338Cys). The study describes phenotypes of Indian patients and expands the mutation spectrum of the disorder.     

Molecular analysis of eight mutations in FBN1

Mutations in the gene encoding extracellular glycoprotein fibrillin-1 (FBN1) cause Marfan syndrome (MFS) and other related connective tissue disorders. In this study, eight mutations have been detected in MFS patients by heteroduplex analysis. These comprise two missense mutations, C1835Y and C2258Y in calcium-binding epidermal growth factor-like domains, two nonsense mutations, R1541X and R2394X in transforming growth factor beta1-binding protein-like domains, one splice site mutation, which has been detected previously, and three small insertions or deletions resulting in a frameshift. Fibroblast cells have been established from seven of the MFS patients and the biochemical effects of the mutations on fibrillin-1 synthesis and secretion assessed by pulse-chase analysis. Each cysteine mutation resulted in the delayed secretion of fibrillin-1 and both nonsense and frameshift mutations caused reduced levels of synthesis and/or deposition of fibrillin-1. Indirect immunofluorescence and rotary shadowing electron microscopy analysis of fibrillin microfibrils revealed no major differences between normal and patient samples. We discuss the relative merits of the biochemical techniques used in this study.     

Mutations in the GlyT2 Gene (SLC6A5) Are a Second Major Cause of Startle Disease

Hereditary hyperekplexia or startle disease is characterized by an exaggerated startle response, evoked by tactile or auditory stimuli, leading to hypertonia and apnea episodes. Missense, nonsense, frameshift, splice site mutations, and large deletions in the human glycine receptor α1 subunit gene (GLRA1) are the major known cause of this disorder. However, mutations are also found in the genes encoding the glycine receptor β subunit (GLRB) and the presynaptic Na(+)/Cl(-)-dependent glycine transporter GlyT2 (SLC6A5). In this study, systematic DNA sequencing of SLC6A5 in 93 new unrelated human hyperekplexia patients revealed 20 sequence variants in 17 index cases presenting with homozygous or compound heterozygous recessive inheritance. Five apparently unrelated cases had the truncating mutation R439X. Genotype-phenotype analysis revealed a high rate of neonatal apneas and learning difficulties associated with SLC6A5 mutations. From the 20 SLC6A5 sequence variants, we investigated glycine uptake for 16 novel mutations, confirming that all were defective in glycine transport. Although the most common mechanism of disrupting GlyT2 function is protein truncation, new pathogenic mechanisms included splice site mutations and missense mutations affecting residues implicated in Cl(-) binding, conformational changes mediated by extracellular loop 4, and cation-π interactions. Detailed electrophysiology of mutation A275T revealed that this substitution results in a voltage-sensitive decrease in glycine transport caused by lower Na(+) affinity. This study firmly establishes the combination of missense, nonsense, frameshift, and splice site mutations in the GlyT2 gene as the second major cause of startle disease.     

Two novel mutations in exons 19a and 20 and a BsaI polymorphism in a newly characterized intron of the neurofibromatosis type 1 gene

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder. It is caused by mutations in the NF1 gene, which comprises 60 exons and is located on chromosome 17q11.2. A total of 170 unrelated NF1 patients were screened for mutations in four exons by temperature-gradient gel electrophoresis. Preparatory work revealed the presence of a previously uncharacterized intron (19a) in what was previously designated exon 19; this allowed us to develop assays for genomic mutation screening in the newly defined exons 19a and 19b. Two novel NF1 mutations were detected: a single-base insertion in exon 19a creating a frameshift, and a second mutation affecting the splice donor site of intron 20 and leading to skipping of exon 20. A novel BsaBI polymorphism was identified in intron 19a. Received: 11 August 1997 / Accepted: 13 November 1997     

Novel missense MTTP gene mutations causing abetalipoproteinemia

Objective

The microsomal triglyceride transfer protein (MTTP) plays a critical role in the formation of hepatic very low density lipoprotein. Abetalipoproteinemia (ABL) is a rare, naturally occurring extreme form of MTTP inhibition, which is characterized by the virtual absence of apolipoprotein (apo) B-containing lipoproteins in blood. The goal of this study was to examine the effect that four novel MTTP missense mutations had on protein interactions, expression and lipid-transfer activity, and to determine which mutations were responsible for the ABL phenotype observed in two patients.

Approach and results

In two patients with ABL, we identified in MTTP a novel frameshift mutation (K35Ffs*37), and four novel missense mutations, namely, G264R, Y528H, R540C, and N649S. When transiently expressed in COS-7 cells, all missense MTTP mutations interacted with apoB17, apoB48, and protein disulfide isomerase. Mutations Y528H and R540C, however, displayed negligible levels of MTTP activity and N649S displayed a partial reduction relative to the wild-type MTTP. In contrast, G264R retained full lipid-transfer activity.

Conclusions

These studies indicate that missense mutations Y528H, R540C, and N649S appear to cause ABL by reducing MTTP activity rather than by reducing binding of MTTP with protein disulfide isomerase or apoB. The region of MTTP containing amino acids 528 and 540 constitutes a critical domain for its lipid-transfer activity.     

Generation of hepatoma cell lines deficient in microsomal triglyceride transfer protein

The microsomal triglyceride transfer protein (MTP) is essential for the secretion of apolipoprotein B (apoB)48- and apoB100-containing lipoproteins in the intestine and liver, respectively. Loss of function mutations in MTP cause abetalipoproteinemia. Heterologous cells are used to evaluate the function of MTP in apoB secretion to avoid background MTP activity in liver and intestine-derived cells. However, these systems are not suitable to study the role of MTP in the secretion of apoB100-containing lipoproteins, as expression of a large apoB100 peptide using plasmids is difficult. Here, we report a new cell culture model amenable for studying the role of different MTP mutations on apoB100 secretion. The endogenous MTTP gene was ablated in human hepatoma Huh-7 cells using single guide RNA and RNA-guided clustered regularly interspaced short palindromic repeats-associated sequence 9 ribonucleoprotein complexes. We successfully established three different clones that did not express any detectable MTTP mRNA or MTP protein or activity. These cells were defective in secreting apoB-containing lipoproteins and accumulated lipids. Furthermore, we show that transfection of these cells with plasmids expressing human MTTP cDNA resulted in the expression of MTP protein, restoration of triglyceride transfer activity, and secretion of apoB100. Thus, these new cells can be valuable tools for studying structure-function of MTP, roles of different missense mutations in various lipid transfer activities of MTP, and their ability to support apoB100 secretion, compensatory changes associated with loss of MTP, and in the identification of novel proteins that may require MTP for their synthesis and secretion.     

An intron 1 splice mutation and a nonsense mutation (W23X) in CYP21 causing severe congenital adrenal hyperplasia

Direct DNA sequencing of the steroid 21-hydroxylase gene (CYP21) revealed two novel mutations in two patients with severe congenital adrenal hyperplasia. The nonsense mutation Trp23Stop (TGG → TGA) was found in a woman with the simple virilizing form of the disease. She was a compound heterozygote, with the previously described Ile173Asn mutation on her other allele. A boy, who developed salt-wasting in the neonatal period, carried an allele with a novel mutation of the canonical splice acceptor site in intron 1 (AG→GG). He was also a compound heterozygote, with the well-known splice mutation in intron 2 on his other allele. Received: 26 February 1996     

X-linked hypophosphatemia in Polish patients. 1. Mutations in the PHEX gene

We present twenty-nine PHEX gene mutations extending our previous work, giving it to a total of 37 different mutations identified in Polish patients with familial or sporadic X-linked hypophosphatemia. Deletions, insertions and nucleotide substitutions leading to frameshift (27%), stop codon (29%), splice site (24%), and missense mutations (20%) were found. The mutations are distributed along the gene; exons 3, 4, 11, 12, 14, 15, 17, 20 and 22 are regions with the most frequent mutation events. Four mutations, P534L, G579R, R549X and IVS15+1nt, recurred in three, four, two and three unrelated patients, respectively. They have also been detected in affected persons from other countries. Twenty-eight mutations are specific for Polish population and almost all of them are unique. Most of the identified mutations are expected to result in major changes in protein structure and/or function.