Genetic heterogeneity in adenosine deaminase (ADA) deficiency: five different mutations in five new patients with partial ADA deficiency.

Complete genetic deficiency of adenosine deaminase (ADA) results in a fatal syndrome of severe combined immunodeficiency (SCID). Genetic partial deficiency of ADA, with no detectable enzyme activity in erythrocytes but with variable amounts of enzyme activity detectable in other cells, is usually associated with normal immunologic function but can give rise to a late-onset, cellular immunodeficiency syndrome. We have previously described four different mutant alleles in four such partially ADA-deficient children. We have now examined ADA in lymphoid cells from five additional newly ascertained children with partial ADA deficiency with respect to electrophoretic mobility in starch gel, isoelectric point, heat-stability, and apparent Km and Vmax. These techniques identify at least five different abnormal alleles in these five additional unrelated subjects. Three of these abnormal alleles result in expression of abnormal allelic isozymes (allozymes) different from those previously described. These are: (1) an acidic allozyme that is less acidic than the acidic allozyme we have previously reported; (2) an allozyme that is even less acidic than (1); and (3) an allozyme with apparently normal charge but which is so heat sensitive that the lability to heat can easily be detected at physiologic to febrile temperatures. Two abnormal alleles detected in these five children could correspond with previously reported mutants. These are (4) a basic allozyme that could (but probably doesn't) correspond to the basic allozyme we have previously reported and (5) a "null" allele that cannot be differentiated by these methods from any other "null" allele seen in complete ADA- -SCIDs. Three of the five new patients are genetic compounds, identified either by the presence of two electrophoretically distinguishable allozymes or by family studies that demonstrate presence of a "null" allele in addition to an electrophoretically abnormal allozyme. In three patients, one or both allozymes are phenotypically indistinguishable from an abnormal allozyme also seen in a different individual. Determination of the nucleotide sequence will be required to determine whether or not the phenotypically indistinguishable mutations are indeed genotypically identical. The newly ascertained individuals appear to share a common ethnic West Indian background, out of proportion to the frequency of this ethnic background in the newborn population from which they were ascertained, suggesting that partial ADA deficiency may confer a selective advantage to the homozygous or heterozygous phenotype.     

Establishment and characterization of adenosine deaminase-deficient human T cell lines

We have established long term cell lines from a patient with adenosine deaminase (ADA)-deficient severe combined immunodeficiency by stimulation of blood and bone marrow cells with PHA and IL-2 followed by transformation of the activated cells with the human retrovirus HTLV-I. Despite the absence of detectable T cells in the patients blood, cell lines grew that carried the phenotype of mature activated T cells. TJF-2, the line established from blood, was characterized in detail. The concentration of ADA in TJF-2 cells was less than 1% of normal (3.2 U vs 413.0 U). Studies with pharmacologic inhibitors of ADA suggest that the residual adenosine deaminating activity of TJF-2 is from an enzyme distinct from true ADA, a nonspecific aminohydrolyase. Growth of TJF-2 cells was hypersensitive to inhibition by 2'-deoxyadenosine compared to normal T cells (ID50, 55 microM vs greater than 1000 microM). Analysis of 2'-deoxyadenosine-challenged cells showed that TJF-2 cells accumulated significant levels of deoxyadenosine triphosphate, whereas normal T cells did not unless they were also incubated with the ADA inhibitor deoxycoformycin. Southern and Northern blot analysis of these cells revealed a grossly intact ADA gene that produced a normal size ADA mRNA. Yet, despite ADA deficiency, cells of the TJF-2 line were otherwise indistinguishable from HTLV-I-transformed T cells derived from normal donors with respect to dependence on exogenous IL-2 for growth, clonal rearrangement patterns of TCR beta-chain genes, response to PHA, and rapid restoration of cellular volume after hypotonic challenge. The TJF-2 line thus represents a unique HTLV-I-transformed human T cell line exhibiting ADA deficiency and its expected metabolic consequences.     

Adenosine deaminase deficiency: genotype-phenotype correlations based on expressed activity of 29 mutant alleles.

Adenosine deaminase (ADA) deficiency causes lymphopenia and immunodeficiency due to toxic effects of its substrates. Most patients are infants with severe combined immunodeficiency disease (SCID), but others are diagnosed later in childhood (delayed onset) or as adults (late onset); healthy individuals with "partial" ADA deficiency have been identified. More than 50 ADA mutations are known; most patients are heteroallelic, and most alleles are rare. To analyze the relationship of genotype to phenotype, we quantitated the expression of 29 amino acid sequence-altering alleles in the ADA-deleted Escherichia coli strain SO3834. Expressed ADA activity of wild-type and mutant alleles ranged over five orders of magnitude. The 26 disease-associated alleles expressed 0.001%-0.6% of wild-type activity, versus 5%-28% for 3 alleles from "partials." We related these data to the clinical phenotypes and erythrocyte deoxyadenosine nucleotide (dAXP) levels of 52 patients (49 immunodeficient and 3 with partial deficiency) who had 43 genotypes derived from 42 different mutations, including 28 of the expressed alleles. We reduced this complexity to 13 "genotype categories," ranked according to the potential of their constituent alleles to provide ADA activity. Of 31 SCID patients, 28 fell into 3 genotype categories that could express <=0.05% of wild-type ADA activity. Only 2 of 21 patients with delayed, late-onset, or partial phenotypes had one of these "severe" genotypes. Among 37 patients for whom pretreatment metabolic data were available, we found a strong inverse correlation between red-cell dAXP level and total ADA activity expressed by each patient's alleles in SO3834. Our system provides a quantitative framework and ranking system for relating genotype to phenotype.     

Two newly identified mutations (Thr233Ile and Leu152Met) in partially adenosine deaminase-deficient (ADA–) individuals that result in differing biochemical and metabolic phenotypes

Deficiency of adenosine deaminase (ADA^–) results in autosomal recessive immunodeficiency disease of varying severity. Partial ADA^– [ADA deficiency in erythrocytes (RBCs) but substantial ADA in non-RBCs] has also been identified, primarily by population screening of healthy adults in Africa and newborns in New York State. Normal immune function and/or minimal elevations of toxic metabolites in childhood suggested that partial ADA deficiency was benign and therefore that six mutations identified in partially ADA-deficient newborns and expressing 8–80% of normal ADA in non-RBCs were not pathogenic. However, the lowest activity mutation (Arg211Cys) has now been reported in patients with adult-onset immunodeficiency. We have now molecularly and biochemically studied two additional individuals whom we found to represent opposite ends of the spectrum of partial ADA deficiency as to biochemical abnormalities and age of ascertainment. Homozygosity for a newly identified Leu152Met mutation expressing considerably less activity than the pathogenic Arg211Cys mutation was found in a currently healthy 10-year-old Afghanistani child (ascertained at birth). He had the highest accumulation of the metabolite dATP among 13 partially ADA-deficient patients studied, but considerably lower than in those with immunodeficiency. Homozygosity for a newly identified Thr233Ile mutation expressing somewhat greater ADA activity than Arg211Cys was found in a healthy young adult Kung individual, associated with very low metabolite concentrations. Biochemical findings and a family history suggestive of immunodeficiency in prior offspring support the idea that the Leu152Met mutation could result in disease in homozygous individuals challenged by severe environmental insult or in heterozygosity with a null mutation. The pathogenicity of the Thr233Ile mutation, as well as a previously described Ala215Thr mutation with relatively lower activity is less likely but will only be determined by long-term observation of individuals carrying these mutations. Although, in contrast to other partial mutations, neither of these two mutations are at CpG hot spots, the frequency of CpG mutations remains high for partial mutations but is also similarly high in ADA^– immunodeficient patients (5/8 vs 12/21). Received: 7 August 1996 / Accepted: 29 November 1996     

Analysis of normal and mutant forms of human adenosine deaminase — A review

Summary A deficiency of the enzyme adenosine deaminase is associated with an autosomal recessive form of severe combined immunodeficiency disease in man. The molecular forms of the normal human enzyme have now been well characterized in an effort to better understand the nature of the enzyme defect in affected patients.In some human tissues adenosine deaminase exists predominantly as a small molecular form while in other tissues a large form composed of adenosine deaminase (small form) and an adenosine deaminase-binding protein predominates. The small form of the enzyme purified to homogeneity by antibody affinity chromatography is a monomer of native molecular weight of 37,600. The adenosine deaminase-binding protein, purified by adenosine deaminase affinity chromatography, appears to be a dimer of native molecular weight 213,000 and contains carbohydrate. Based on direct binding measurements, chemical cross-linking studies and sedimentation equilibrium analyses, small form adenosine deaminase has been shown to combine with purified binding protein in a molar ratio of 2:1 respectively to produce the large form adenosine deaminase.Reduced, but widely ranging levels of adenosine deaminating activity, have been reported in various tissues of adenosine deaminase deficient patients. Further, the characteristics of this residual enzyme activity have been analyzed immunochemically to substantiate genetic heterogeneity in this disorder.While many types of immunodeficiency are currently recognized in man, in most cases the molecular defect is unknown. The discovery of a deficiency of the enzyme, adenosine deaminase, ADA, (EC 3.5.4.4), in some patients with severe combined immunodeficiency disease represented an early clue to the pathogenesis of immune dysfunction at the molecular level^1-4. Affected patients with markedly reduced levels of ADA exhibit a defect of both cellular and humoral immunity characterized clinically by severe recurrent infections with a fatal outcome if untreated. Attempts to elucidate the nature of the genetic mutation(s) leading to the reduction of ADA activity in these immunodeficient patients have been complicated in part by an incomplete understanding of the nature of ADA in normal tissues. In this review we will consider the structural characteristics of the normal and mutant forms of ADA as they are currently understood.     

Heterogeneity for adenosine deaminase deficiency: Expression of the enzyme in cultured skin fibroblasts and amniotic fluid cells.

Adenosine deaminase (ADA) could be quantitated and the isozyme pattern characterized in cultured amniotic fluid cells. In 20 amniotic fluid cell cultures the mean specific activity was 14.3 U/g protein +/- 6.7 (SD) and compared favorably with values of 14.6 U/g protein +/- 6.8 (SD) observed in 26 cultures of skin fibroblasts. In cultures of skin fibroblasts established from two obligate heterozygotes for ADA deficiency, the specific activity of ADA was 7.0 and 7.7 U/g protein. The ADA isozyme pattern that existed in cultures of amniotic fluid cells was the same as that observed in cultured skin fibroblasts. This identification of the same apparent enzyme may permit the prenatal diagnosis of that form of combined immunodeficiency disease caused by ADA deficiency. Residual enzyme activity of less than 1% and 10% of the mean of normal fibroblasts could be measured in cultured fibroblasts from two unrelated children with ADA deficiency and combined immunodeficiency disease. The tissue-specific enzyme from cultured skin fibroblasts from the child with 10% residual activity had a faster electrophoretic mobility and greater heat stability than normal ADA. This enzymatic evidence indicates that at least two mutant alleles exist at the locus for ADA which predispose to combined immunodeficiency disease when present in the homozygous state.     

Characterization of normal and mutant adenosine deaminase messenger RNAs by translation and hybridization to a cDNA probe

Summary Using both in vitro translation and hybridization to an adenosine deaminase (ADA) cDNA probe, ADA mRNA has been characterized in B lymphoblast lines established from seven ADA-deficient children, two parents of an ADA-deficient child, and three normal people. All ADA-deficient lines except GM-2825A, including those with less than 1% of normal catalytic activity, had normal or greater amounts of hybridizable, 1.6 kilobase in size, ADA mRNA. Immunoreactive ADA protein of normal size was produced by in vitro translation of the mRNAs. Deficiency of ADA activity in these lines appears secondary to synthesis of structurally altered proteins rather than to a quantitative deficiency in ADA mRNA. The GM-2825A line contains electrophoretically abnormal species of RNA which hybridize to the cDNA probe. Deficiency of ADA activity in this line appears at least in part secondary to a structural defect in the ADA mRNA or its precursors.     

Deoxyadenosine metabolism and cytotoxicity in cultured mouse T lymphoma cells: a model for immunodeficiency disease.

The inherited absence of either adenosine deaminase (ADA) or purine nucleoside phosphorylase is associated with severe immunological impairment. We have developed a cell culture model using a mouse T cell lymphoma to simulate ADA deficiency and to study the relationship between purine salvage enzymes and immune function. 2′-deoxyadenosine triphosphate (deoxyATP) levels have been shown to be greatly elevated in erythrocytes of immunodeficient, ADA-deficient patients, suggesting that deoxyadenosine is the potentially toxic substrate in ADA deficiency. Using a potent ADA inhibitor, we have demonstrated that deoxyadenosine is growth-inhibitory and cytotoxic to S49 cells, and that deoxyATP accumulates in these cells. Cell variants, unable to transport or phosphorylate deoxyadenosine, are much less sensitive to deoxyadenosine, indicating that intracellular phosphorylation of deoxyadenosine is required for the lethal effects.We have partially reversed the cytotoxic effects of deoxyadenosine with deoxycytidine in wild-type cells, but we cannot show any reversal in cell lines lacking deoxycytidine kinase. Adenosine (ado) kinase-deficient cells are extremely resistant to deoxyadenosine in the presence of deoxycytidine. This deoxycytidine reversal of deoxyadenosine toxicity is consistent with an inhibition of ribonucleotide reductase by deoxyATP, and we have shown that incubation of S49 cells with deoxyadenosine markedly reduces intracellular levels of deoxyCTP, deoxyGTP and TTP.Kinetics data in wild-type cells and in cell variants are consistent with the presence of two deoxyadenosine-phosphorylating activities — one associated with ado kinase and another associated with deoxycytidine kinase.The S49 cells appear to be a valid model for the simulation of ADA deficiency in cell culture, and from our results, we can suggest administration of deoxycytidine as a pharmacological regimen to circumvent the clinicopathologic symptoms in ADA deficiency.     

Correct splicing despite mutation of the invariant first nucleotide of a 5' splice site: a possible basis for disparate clinical phenotypes in siblings with adenosine deaminase deficiency.

Adenosine deaminase (ADA) deficiency usually causes severe combined immune deficiency in infancy. Milder phenotypes, with delayed or late onset and gradual decline in immune function, also occur and are associated with less severely impaired deoxyadenosine (dAdo) catabolism. We have characterized the mutations responsible for ADA deficiency in siblings with striking disparity in clinical phenotype. Erythrocyte dAdo nucleotide pool size, which reflects total residual ADA activity, was lower in the older, more mildly affected sib (RG) than in her younger, more severely affected sister (EG). Cultured T cells, fibroblasts, and B lymphoblasts of RG had detectable residual ADA activity, while cells of EG did not. ADA mRNA was undetectable by northern analysis in these cells of both patients. Both sibs were found to be compound heterozygotes for the following novel splicing defects: (1) a G+1-->A substitution at the 5' splice site of IVS 2 and (2) a complex 17-bp rearrangement of the 3' splice site of IVS 8, which inserted a run of seven purines into the polypyrimidine tract and altered the reading frame of exon 9. PCR-amplified ADA cDNA clones with premature translation stop codons arising from aberrant pre-mRNA splicing were identified, which were consistent with these mutations. However, some cDNA clones from T cells of both patients and from fibroblasts and Epstein-Barr virus (EBV)-transformed B cells of RG, were normally spliced at both the exon 2/3 and exon 8/9 junctions. A normal coding sequence was documented for clones from both sibs. The normal cDNA clones did not appear to arise from either contamination or PCR artifact, and mosaicism seems unlikely to have been involved. These findings suggest (1) that a low level of normal pre-mRNA splicing may occur despite mutation of the invariant first nucleotide of the 5' splice donor sequence and (2) that differences in efficiency of such splicing may account for the difference in residual ADA activity, immune dysfunction, and clinical severity in these siblings.     

A high proportion of ADA point mutations associated with a specific alanine-to-valine substitution.

In 15%-20% of children with severe combined immunodeficiency (SCID), the underlying defect is adenosine deaminase (ADA) deficiency. The overall goal of our research has been to identify the precise molecular defects in patients with ADA-deficient SCID. In this study, we focused on a patient whom we found to have normal sized ADA mRNA by Northern analysis and an intact ADA structural gene by Southern analysis. By cloning and sequencing this patient's ADA cDNA, we found a C-to-T point mutation in exon 11. This resulted in the amino acid substitution of a valine for an alanine at position 329 of the ADA protein. Sequence analysis revealed that this mutation created a new BalI restriction site. Using Southern analyses, we were able to directly screen individuals to determine the frequency of this mutation. By combining data on eight families followed at our institution with data on five other families reported in the literature, we established that five of 13 patients (seven of 22 alleles) with known or suspected point mutations have this defect. This mutation was found to be associated with three different ADA haplotypes. This argues against a founder effect and suggests that the mutation is very old. In summary, a conservative amino acid substitution is found in a high proportion of patients with ADA deficiency; this can easily be detected by Southern analysis.     

Human T cell receptor-gamma delta-expressing T-cell lines recognize MHC-controlled elements on autologous EBV-LCL that are not HLA-A, -B, -C, -DR, -DQ, or -DP.

HLA-loss variants of an EBV-transformed B lymphoblastoid cell line (EBV-LCL) 721 were used to investigate whether human MHC molecules other than known class I or class II were involved in autologous T cell responses. Bulk lymphocyte cultures of purified T cells primed to an autologous variant EBV-LCL that fails to express HLA-class II and has reduced cell surface HLA-class I expression, and oligoclonal TCR-gamma delta-bearing lines derived from them, could lyse both this EBV-LCL and an independently derived, class II expressing autologous variant EBV-LCL that bears no HLA-A, -B, or -C, suggesting the presence of additional HLA-like restriction elements. Cold target inhibition of cytolysis mediated by these lines indicated that a shared or cross-reactive MHC controlled restriction element other than the known MHC determinants was retained by the EBV-LCL variants. Single-cell derived clones from these T cell lines which expressed only the TCR-gamma delta showed this same target cell specificity pattern, proving recognition of MHC-controlled determinants by autologous gamma delta T cells. Anti-gamma delta antibody could inhibit cytolysis by the gamma delta-expressing lines, suggesting that the TCR-gamma delta was involved in recognition of the EBV-LCL targets. Flow cytometric analysis with separate HLA-reactive antibodies indicated that the restriction element for these cytolytic responses is a molecule serologically cross-reactive with HLA-B and -C Ag, yet is a determinant that cannot be HLA-A, -B, -C, -DR, -DQ or -DP.     

Severe combined immune deficiency due to a homozygous 3.2-kb deletion spanning the promoter and first exon of the adenosine deaminase gene

We have investigated the structural gene for adenosine deaminase (ADA) in a female infant with ADA deficiency associated severe combined immune deficiency (ADA-SCID) disease and her family by DNA restriction-fragment-length analysis. In this family a new ADA-specific restriction-fragment-length variant was detected, which involves a 3.2-kb deletion spanning the ADA promoter as well as the first exon. It was found that the patient, who was born to a consanguineous couple, was homozygous and both her parents and her brother were heterozygous for the deletion. No ADA-specific mRNA could be detected by hybridization in fibroblasts derived from this patient. Thus the patient was established to be homozygous for a true null ADA allele. In the light of the apparently normal development of most tissues except the lymphoid tissue the above finding directly questions the classification of ADA as a 'housekeeping' enzyme.     

Interaction between ABO blood groups and ADA genetic polymorphism during intrauterine life

A total of 203 couples with unexplained habitual abortions and 364 consecutive normal puerperae along with their live-born babies were studied. The analysis of wife-husband joint ABO blood group distribution in couples with habitual abortion showed an excess of A incompatible mating type and a defect of B incompatible type as compared with expected proportions assuming random mating. The joint wife-husband ABO blood group distribution was further analysed in relation to the adenosine deaminase (ADA) genotype. A defect of O-A and A-O couples when the wife carries the ADA*1/*1 genotype and the husband carries the ADA*2 allele, and a defect of O-O and A-A when the wife carries the ADA*2 allele were observed. In the sample of normal puerperae, analysis of the joint mother-newborn ABO distribution in relation to the ADA genotype showed a pattern similar to that observed in couples with habitual abortion, i.e. there is a defect of O-A and A-O when the mother carries the ADA*1/*1 genotype and the newborn carries the ADA*2 allele and a defect of O-O and A-A types when the mother carries the ADA*2 allele. Altogether the data suggest an early loss of O-A and A-O zygotes when they carry the ADA*2 allele and an early loss of O-O and A-A zygotes when the mother carries the ADA*2 allele resulting in a deficit of these zygotic classes among both spontaneously aborted fetuses and live-born infants. The pattern of association observed in the mother-fetus type O-A (incompatible according to conventional terminology) appears similar to that observed for the reciprocal A-O type (compatible according to conventional terminolgy). Therefore strictly conventional immunological mechanisms cannot explain the whole pattern of associations. Cell to cell interactions involving ABO antigens may have an important role at implantation: ADA, through the control of local adenosine concentration, could modulate these interactions influencing the probability of successful implantation.     

Adenosine deaminase deficiency due to heterozygous abnormality consisting of a deletion of exon 7 and the absence of enzyme mRNA

An adenosine deaminase (ADA;EC 3.5.4.4)-deficient B lymphoblastoid cell line BADO5 derived from a Japanese patient with severe combined immunodeficiency disease and two B lymphoblastoid cell lines, BAMO5 from his mother and BAFO5 from his father, were characterized. To identify mutations affecting ADA activity, we prepared cDNAs to ADA mRNAs of the BADO5 cell line for nucleotide sequencing. Sequence analysis of one of the BADO5 ADA cDNA clones revealed deletion of exon 7, and one point mutation of base 629 from G to A that did not affect the amino acid sequence. All clones of the BADO5 cell line so far examined showed the absence of exon 7 by Southern blotting analysis. Ribonuclease protection assay with an RNA probe spanning from exon 5 to exon 11 showed that the BADO5 ADA mRNA had a deletion of exon 7, the BAMO5 mRNA had normal length, and the BAFO5 mRNA had two species with a deletion of exon 7 and with normal length. Consequently, the patient's ADA genes resulted from one allele of the BAMO5 ADA gene that did not produce a detectable mRNA, and the other allele of the BAFO5 ADA gene producing an aberrant mRNA without exon 7.     

Lack of adenosine deaminase deficiency in the mutant mouse wasted

The possibility that the mutant mouse wasted (wst/wst) may serve as an animal model for studies of severe combined immunodeficiency disease (SCID) and the role of adenosine deaminase (ADA, EC 3.5.4.4) in adenosine metabolism were investigated. The specific activity of ADA in wst/wst compared with control mice was significantly lower by 26% in thymus, but significantly higher by 18% in spleen and 32% in cerebellum. Vmax values of ADA in spleens were 43% higher in wst/wst mice and no changes were observed in Km values. In contrast, the Vmax of ADA was unchanged in erythrocytes from wst/wst mice, but the Km for adenosine was significantly elevated. Thus, based on ADA measurements alone, it may be premature to consider wst/wst mice as a model for ADA deficiency and SCID in humans.     

Identification and characterization of nine RFLPs at the adenosine deaminase (ADA) locus.

We have identified and/or characterized at least nine RFLPs at the adenosine deaminase (ADA) locus, detected by digestion of DNA with MspI, BanII, PstI, BalI, and PvuII. The RFLPs were distributed over approximately 15 kb of the gene, from IVS 2 to IVS 10. They exhibited Mendelian inheritance and were in Hardy-Weinberg equilibrium. For seven fully characterized RFLPs, the gene frequencies of the rare alleles in 90 chromosomes examined ranged from .33 to .04, the PIC from .34 to .07, and the heterozygosity from .09 to .58. In kindreds examined (58 independent chromosomes), a total of nine haplotypes could be defined on the basis of seven fully characterized RFLPs with a heterozygosity of .62 and PIC of .53. Because there was considerable linkage disequilibrium, only three haplotypes accounted for 90% of individuals. Similar heterozygosity and PIC values (.59 and .51, respectively) could be obtained on the basis of haplotypes defined by the two sites that were the most polymorphic and that were in the least degree of linkage disequilibrium. A strategy for use of the RFLPs in linkage studies is suggested. We have also examined DNA from 17 patients with complete genetic deficiency of ADA (resulting in severe combined immunodeficiency [ADA-SCID] and from 10 patients with partial ADA deficiency (deficient in erythrocytes, with varying levels of ADA in other cells and normal immune function). Although the RFLPs detected genetic compounds among both types of patients, there was, as expected, a decreased incidence of heterozygosity (ADA-SCIDs, .29; partial ADA deficients, .20). Two additional haplotypes not found in the normal population were identified in homozygous form in patients. This information should be useful in developing a rational approach to delineation of mutations at the ADA locus as well as in distinguishing recurrent mutations of independent origin from those derived from a common progenitor.     

A newly identified lipoprotein lipase (LPL) gene mutation (F270L) in a Japanese patient with familial LPL deficiency

We have systematically investigated the molecular defects resulting in a primary lipoprotein lipase (LPL) deficiency in a Japanese male infant (proband SH) with fasting hyperchylomicronemia. Neither LPL activity nor immunoreactive LPL mass was detected in pre- or postheparin plasma from proband SH. DNA sequence analysis of the LPL gene of proband SH revealed homozygosity for a novel missense mutation of F270L (Phe(270)-->Leu/TTT(1065)-->TTG) in exon 6. The function of the mutant F270L LPL was determined by both biochemical and immunocytochemical studies. In vitro expression experiments on the mutant F270L LPL cDNA in COS-1 cells demonstrated that the mutant LPL protein was synthesized as a catalytically inactive form and its total amount was almost equal to that of the normal LPL. Moreover, the synthesized mutant LPL was non-releasable by heparin because the intracellular transport of the mutant LPL to the cell surface - by which normal LPL becomes heparin-releasable - was impaired due to the abnormal structure of the mutant LPL protein. These findings explain the failure to detect LPL activities and masses in pre- and postheparin plasma of the proband. The mutant F270L allele generated an XcmI restriction enzyme site in exon 6 of the LPL gene. The carrier status of F270L in the proband's family members was examined by digestion with XcmI. The proband was ascertained to be homozygous for the F270L mutation and his parents and sister were all heterozygous. The LPL activities and masses of the parents and the sister (carriers) were half or less than half of the control values. Regarding the phenotype of the carriers, the mother with a sign of hyperinsulinemia manifested hypertriglyceridemia (type IV hyperlipoproteinemia), whereas the healthy father and the sister were normolipidemic. Hyperinsulinemia may be a strong determinant of hypertriglyceridemia in subjects with heterozygous LPL deficiency.