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.     

A missense mutation Pro157 Arg in lipoprotein lipase (LPLNijmegen) resulting in loss of catalytic activity.

Here we report on the molecular defect that leads to a deficiency of lipoprotein lipase (LPL) activity in a proband of Dutch descent. Southern-blot analysis of the LPL gene from the patient did not reveal any major DNA rearrangements. Sequencing of polymerase-chain-reaction-amplified DNA revealed that the proband is a homozygote for G725C, resulting in a substitution of Pro157 for Arg. This substitution alters a restriction site for PvuII, which allowed rapid identification of the mutant allele in family members. Site-directed mutagenesis and transient expression of the mutant LPL in COS cells produced an enzymatically inactive protein, establishing the functional significance of this mutation. This naturally occurring mutation which alters the Pro157 adjacent to Asp156 of the proposed catalytic triad, indicates that this region of the protein is indeed crucial for LPL catalytic activity.     

A missense mutation (Trp86----Arg) in exon 3 of the lipoprotein lipase gene: a cause of familial chylomicronemia.

We have investigated a patient of English ancestry with familial chylomicronemia caused by lipoprotein lipase (LPL) deficiency. DNA sequence analysis of all exons and intron-exon boundaries of the LPL gene identified two single-base mutations, a T----C transition for codon 86 (TGG) at nucleotide 511, resulting in a Trp86----Arg substitution, and a C----T transition at nucleotide 571, involving the codon CAG encoding Gln106 and producing Gln106----Stop, a mutation described by Emi et al. The functional significance of the two mutations was confirmed by in vitro expression and enzyme activity assays of the mutant LPL. Linkage analysis established that the patient is a compound heterozygote for the two mutations. The Trp86----Arg mutation in exon 3 is the first natural mutation identified outside exons 4-6, which encompass the catalytic triad residues.     

Catalytic triad residue mutation (Asp156----Gly) causing familial lipoprotein lipase deficiency. Co-inheritance with a nonsense mutation (Ser447----Ter) in a Turkish family

We studied the molecular basis of familial Type I hyperlipoproteinemia in two brothers of Turkish descent who had normal plasma apolipoprotein C-II levels and undetectable plasma post-heparin lipoprotein lipase (LPL) activity. We cloned the cDNAs of LPL mRNA from adipose tissue biopsies obtained from these individuals by the polymerase chain reaction and directional cloning into M13 vectors. Direct sequencing of pools of greater than 2000 cDNA clones indicates that their LPL mRNA contains two mutations: a missense mutation changing codon 156 from GAU to GGU predicting an Asp156----Gly substitution and a nonsense mutation changing the codon for Ser447 from UCA to UGA, a stop codon, predicting a truncated LPL protein that contains 446 instead of 448 amino acid residues. Both patients were homozygous for both mutations. Analysis of genomic DNAs of the patients and their family members by the polymerase chain reaction, restriction enzyme digestion (the GAT----GGT mutation abolishes a TaqI restriction site), and allele-specific oligonucleotide hybridization confirms that the patients were homozygous for these mutations at the chromosomal level, and the clinically unaffected parents and sibling were true obligate heterozygotes for both mutations. In order to examine the functional significance of the mutations in this family, we expressed wild type and mutant LPLs in vitro using a eukaryotic expression vector. Five types of LPL proteins were produced in COS cells by transient transfection: (i) wild type LPL, (ii) Asp156----Gly mutant, (iii) Ser447----Ter mutant, (iv) Gly448----Ter mutant, and (v) Asp156----Gly/Ser447----Ter double mutant. Both LPL immunoreactive mass and enzyme activity were determined in the culture media and intracellularly. Immunoreactive LPLs were produced in all cases. The mutant LPLs, Asp156----Gly and Asp156----Gly/Ser447----Ter, were devoid of enzyme activity, indicating that the Asp156----Gly mutation is the underlying defect for the LPL deficiency in the two patients. The two mutant LPLs missing a single residue (Gly448) or a dipeptide (Ser447-Gly448) from its carboxyl terminus had normal enzyme activity. Thus, despite its conservation among all mammalian LPLs examined to date, the carboxyl terminus of LPL is not essential for enzyme activity. We further screened 224 unrelated normal Caucasians for the Ser447----Ter mutation and found 36 individuals who were heterozygous and one individual who was homozygous for this mutation, indicating that it is a sequence polymorphism of no functional significance. Human LPL shows high homology to hepatic triglyceride lipase and pancreatic lipase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of two separate allelic mutations in the lipoprotein lipase gene of a patient with the familial hyperchylomicronemia syndrome.

The molecular defects resulting in a deficiency of lipoprotein lipase activity in a patient with the familial hyperchylomicronemia syndrome have been identified. Increased lipoprotein lipase mass but undetectable lipoprotein lipase activity in the patient's post-heparin plasma indicate the presence of an inactive enzyme. No major gene rearrangements were identified by Southern blot analysis of the patient's lipoprotein lipase gene and Northern blot hybridization revealed an lipoprotein lipase mRNA of normal size. Sequence analysis of polymerase chain reaction-amplified lipoprotein lipase cDNA identified two separate allelic mutations. A T to C transition at nucleotide 836 results in the substitution of Ile194, located near the putative interfacial recognition site of lipoprotein lipase, to a Thr. A G to A mutation at base 983 leads to the substitution of a His for Arg243 and the loss of a HhaI restriction enzyme site. Arg243 is near His241, which has been postulated to be part of the catalytic triad of lipoprotein lipase. Direct sequencing of amplified cDNA and digestion with HhaI established that the proband is a compound heterozygote for each base substitution. Transient expression of each of the mutant lipoprotein lipase cDNAs in human embryonal kidney-293 cells resulted in the synthesis of enzymically inactive proteins, establishing the functional significance of the mutations. We conclude that the Ile194 to Thr194 and Arg243 to His243 substitutions occur in lipoprotein lipase regions essential for normal enzyme activity and each mutation results in the expression of a nonfunctional enzyme leading to the hyperchylomicronemia syndrome manifested in the proband.     

In vitro expression and site-specific mutagenesis of the cloned human lipoprotein lipase gene. Potential N-linked glycosylation site asparagine 43 is important for both enzyme activity and secretion

Detailed structure-function information about human lipoprotein lipase (LPL) is unavailable because it is difficult to purify large amounts of the enzyme for study. To circumvent this problem, we constructed an in vitro LPL expression vector. Human LPL cDNA was cloned and inserted into the expression vector p91023(B). After transfection of COS M-6 cells with the human LPL cDNA construct, LPL enzyme activity was detected in cell extracts and culture medium. Purified human apolipoprotein C-II caused a 5-fold stimulation of the recombinant human LPL expressed in vitro. Using site-specific mutagenesis, Ala residues were substituted for Asn residues at two potential N-linked glycosylation sites (positions 43 and 359) and at a third unrelated Asn (position 257) in the LPL cDNA. RNA blot analysis demonstrated the presence of a single mRNA species in COS cells transfected with wild-type and mutant LPL expression vectors. Intracellular and secreted LPL activity was absent in the construct containing an Ala for Asn mutation at position 43, whereas the same substitutions at positions 257 and 359 did not appreciably affect activity. LPL activity was also absent in another construct containing a Gln for Asn mutation at position 43. Quantitation of LPL protein mass concomitant with measurement of enzyme activity showed that substitution of Ala or Gln for Asn at position 43 resulted in the production of an enzymatically inactive protein which accumulated intracellularly but was not secreted into the culture medium. Our report represents an initial documentation of the expression of cloned human LPL in vitro and of the importance of Asn-43 for both enzyme activity and secretion.     

A novel substitution at the translation initiator codon (ATG-->ATC) of the lipoprotein lipase gene is mainly responsible for lipoprotein lipase deficiency in a patient with severe hypertriglyceridemia and recurrent pancreatitis

A patient with severe hypertriglyceridemia and recurrent pancreatitis was found to have significantly decreased lipoprotein lipase (LPL) activity and normal apolipoprotein C-II concentration in post-heparin plasma. DNA analysis of the LPL gene revealed two mutations, one of which was a novel homozygous G-->C substitution, resulting in the conversion of a translation initiation codon methionine to isoleucine (LPL-1). The second was the previously reported heterozygous substitution of glutamic acid at residue 242 with lysine (LPL-242). In vitro expression of both mutations separately or in combination demonstrated that LPL-1 had approximately 3% protein mass and 2% activity, whereas LPL-242 had undetectable activity but normal mass. The combined mutation LPL-1-242 exhibited similar changes as for LPL-1, with markedly reduced mass, and for LPL-242, with undetectable activity. These results suggest that the homozygous initiator codon mutation rather than the heterozygous LPL-242 alteration was mainly responsible for the patient phenotypes.     

Structural and functional consequences of missense mutations in exon 5 of the lipoprotein lipase gene

Missense mutations in exon 5 of the LPL gene are the most common reported cause of LPL deficiency. Exon 5 is also the region with the strongest homology to pancreatic and hepatic lipase, and is conserved in LPL from different species. Mutant LPL proteins from post-heparin plasma from patients homozygous for missense mutations at amino acid positions 176, 188, 194, 205, and 207, and from COS cells transiently transfected with the corresponding cDNAs were quantified and characterized, in an attempt to determine which aspect of enzyme function was affected by each specific mutation. All but one of the mutant proteins were present, mainly as partially denatured LPL monomer, rendering further detailed assessment of their catalytic activity, affinity to heparin, and binding to lipoprotein particles difficult. However, the fresh unstable Gly(188)-->Glu LPL and the stable Ile(194)-->Thr LPL, although in native conformation, did not express lipase activity. It is proposed that many of the exon 5 mutant proteins are unable to achieve or maintain native dimer conformation, and that the Ile(194)-->Thr substitution interferes with access of lipid substrate to the catalytic pocket. These results stress the importance of conformational evaluation of mutant LPL. Absence of catalytic activity does not necessarily imply that the substituted amino acid plays a specific direct role in catalysis.     

Novel compound heterozygous mutations for lipoprotein lipase deficiency. A G-to-T transversion at the first position of exon 5 causing G154V missense mutation and a 5' splice site mutation of intron 8.

We systematically investigated the molecular defects causing a primary LPL deficiency in a Japanese male infant (patient DI) with fasting hyperchylomicronemia (type I hyperlipoproteinemia) and in his parents. Patient DI had neither LPL activity nor immunoreactive LPL mass in the pre- and post-heparin plasma. The patient was a compound heterozygote for novel mutations consisting of a G-to-T transversion at the first nucleotide of exon 5 [+1 position of 3' acceptor splice site (3'-ass) of intron 4] and a T-to-C transition in the invariant GT at position +2 of the 5' donor splice site (5'-dss) of intron 8 (Int8/5'-dss/t(+2)c). The G-to-T transversion, although affecting the 11 nucleotide of the 3'-consensus acceptor splice site, resulted in a substitution of Gly(154) to Val (G154V; GG(716)C(-->)GTC). The mutant G154V LPL expressed in COS-1 cells was catalytically inactive and hardly released from the cells by heparin. The Int8/5'-dss/t(+2)c mutation inactivated the authentic 5' splice site of intron 8 and led to the utilization of a cryptic 5'-dss in exon 8 as an alternative splice site 133 basepairs upstream from the authentic splice site, thereby causing joining of a part of exon 8 to exon 9 with skipping of a 134-bp fragment of exon 8 and intron 8. These additional mutations in the consensus sequences of the 3' and 5' splice sites might be useful for better understanding the factors that are involved in splice site selection in vivo.     

A missense (Asp250----Asn) mutation in the lipoprotein lipase gene in two unrelated families with familial lipoprotein lipase deficiency.

We have identified the molecular basis for familial lipoprotein lipase (LPL) deficiency in two unrelated families with the syndrome of familial hyperchylomicronemia. All 10 exons of the LPL gene were amplified from the two probands' genomic DNA by polymerase chain reaction. In family 1 of French descent, direct sequencing of the amplification products revealed that the patient was heterozygous for two missense mutations, Gly188----Glu (in exon 5) and Asp250----Asn (in exon 6). In family 2 of Italian descent, sequencing of multiple amplification products cloned in plasmids indicated that the patient was a compound heterozygote harboring two mutations, Arg243----His and Asp250----Asn, both in exon 6. Studies using polymerase chain reaction, restriction enzyme digestion (the Gly188----Glu mutation disrupts an Ava II site, the Arg243----His mutation, a Hha I site, and the Asp250----Asn mutation, a Taq I site), and allele-specific oligonucleotide hybridization confirmed that the patients were indeed compound heterozygous for the respective mutations. LPL constructs carrying the three mutations were expressed individually in Cos cells. All three mutant LPLs were synthesized and secreted efficiently; one (Asp250----Asn) had minimal (approximately 5%) catalytic activity and the other two were totally inactive. The three mutations occurred in highly conserved regions of the LPL gene. The fact that the newly identified Asp250----Asn mutation produced an almost totally inactive LPL and the location of this residue with respect to the three-dimensional structure of the highly homologous human pancreatic lipase suggest that Asp250 may be involved in a charge interaction with an alpha-helix in the amino terminal region of LPL. The occurrence of this mutation in two unrelated families of different ancestries (French and Italian) indicates either two independent mutational events affecting unrelated individuals or a common shared ancestral allele. Screening for the Asp250----Asn mutation should be included in future genetic epidemiology studies on LPL deficiency and familial combined hyperlipidemia.     

Human lipoprotein lipase. Analysis of the catalytic triad by site-directed mutagenesis of Ser-132, Asp-156, and His-241.

Lipoprotein lipase (LPL) plays a central role in normal lipid metabolism as the key enzyme involved in the hydrolysis of triglycerides present in chylomicrons and very low density lipoproteins. LPL is a member of a family of hydrolytic enzymes that include hepatic lipase and pancreatic lipase. Based on primary sequence homology of LPL to pancreatic lipase, Ser-132, Asp-156, and His-241 have been proposed to be part of a domain required for normal enzymic activity. We have analyzed the role of these potential catalytic residues by site-directed mutagenesis and expression of the mutant LPL in human embryonic kidney-293 cells. Substitution of Ser-132, Asp-156, and His-241 by several different residues resulted in the expression of an enzyme that lacked both triolein and tributyrin esterase activities. Mutation of other conserved residues, including Ser-97, Ser-307, Asp-78, Asp-371, Asp-440, His-93, and His-439 resulted in the expression of active enzymes. Despite their effect on LPL activity, substitutions of Ser-132, Asp-156, and His-241 did not change either the heparin affinity or lipid binding properties of the mutant LPL. In summary, mutation of Ser-132, Asp-156, and His-241 specifically abolishes total hydrolytic activity without disrupting other important functional domains of LPL. These combined results strongly support the conclusion that Ser-132, Asp-156, and His-241 form the catalytic triad of LPL and are essential for LPL hydrolytic activity.     

An amino acid substitution in the pyruvate dehydrogenase E1 alpha gene, affecting mitochondrial import of the precursor protein.

A mutation in the mitochondrial targeting sequence was characterized in a male patient with X chromosome-linked pyruvate dehydrogenase E1 alpha deficiency. The mutation was a base substitution of G by C at nucleotide 134 in the mitochondrial targeting sequence of the PDHA1 gene, resulting in an arginine-to-proline substitution at codon 10 (R10P). Pyruvate dehydrogenase activity in cultured skin fibroblasts was 28% of the control value, and immunoblot analysis revealed a decreased level of pyruvate dehydrogenase E1 alpha immunoreactivity. Chimeric constructs in which the normal and mutant pyruvate dehydrogenase E1 alpha targeting sequences were attached to the mitochondrial matrix protein ornithine transcarbamylase were synthesized in a cell free translation system, and mitochondrial import of normal and mutant proteins was compared in vitro. The results show that ornithine transcarbamylase targeted by the mutant pyruvate dehydrogenase E1 alpha sequence was translocated into the mitochondrial matrix at a reduced rate, suggesting that defective import is responsible for the reduced pyruvate dehydrogenase level in mitochondria. The mutation was also present in an affected brother and the mildly affected mother. The clinical presentations of this X chromosome-linked disorder in affected family members are discussed. To our knowledge, this is the first report of an amino acid substitution in a mitochondrial targeting sequence resulting in a human genetic disease.     

A missense mutation (Asp250----Asn) in exon 6 of the human lipoprotein lipase gene causes chylomicronemia in patients of different ancestries.

We have previously reported two common lipoprotein lipase (LPL) gene mutations underlying LPL deficiency in the majority of 37 French Canadians (Monsalve et al., 1990. J. Clin. Invest. 86: 728-734; Ma et al., 1991. N. Engl. J. Med. 324: 1761-1766). By examining the 10 coding exons of the LPL gene in another French Canadian patient, we have identified a third missense mutation that is found in two of the three remaining patients for whom mutations are undefined. This is a G to A transition in exon 6 that results in a substitution of asparagine for aspartic acid at residue 250. Using in vitro site-directed mutagenesis, we have confirmed that this mutation causes a catalytically defective LPL protein. In addition, the Asp250----Asn mutation was also found on the same haplotype in an LPL-deficient patient of Dutch ancestry, suggesting a common origin. This mutation alters a TaqI restriction site in exon 6 and will allow for rapid screening in patients with LPL deficiency.     

Hyperlipoproteinemia type I in a patient with active lipoprotein lipase in adipose tissue and indications of defective transport of the enzyme

This paper presents a case of typical hyperlipoproteinemia type I in a young woman. Her serum triglycerides varied between 2 and 90 mmol/l and she had substantial amounts of apolipoprotein B-48 in fasting plasma. She had no detectable lipoprotein lipase (LPL) activity in post-heparin plasma (less than 0.2 percent of normal). Southern blot analysis suggested no major defect in her LPL gene and Northern blot analysis of adipose tissue RNA showed normal-sized LPL-mRNA. A 2-h [35S]methionine incorporation experiment with adipose tissue pieces in vitro showed that she produced normal-sized LPL and had LPL catalytic activity in the tissue. The amounts were, however, only 5-10% of control. No detectable LPL radioactivity or catalytic activity was released from patient tissue even in the presence of heparin in the incubations. Immunofluorescent staining of adipose tissue biopsies from the patient showed LPL immunoreactivity only in adipocytes and little or none within the capillaries. Treatment of immunoprecipitated labeled LPL with endoglycosidase H showed that the oligosaccharide chains on her enzyme were of the high-mannose type and not processed as in controls. Taken together the data suggest that the patient synthesizes a relatively normal LPL protein which is core-glycosylated and folded into active enzyme as in normal subjects, but is not effectively transported via the Golgi to the cell surface.     

Comparative in vitro expression study of four Fabry disease causing mutations at glutamine 279 of the alpha-galactosidase A protein

Fabry disease is an X-linked lysosomal storage disorder caused by the deficiency of alpha-galactosidase A that results in the accumulation of neutral sphingolipids. We report a novel point mutation in exon 6, Q279K, carried by an asymptomatic child with a family history of classic Fabry disease. Moreover, we comparatively study the in vitro expression and enzyme activity of Q279K and three other already described mutants in glutamine 279. The Q279K, Q279H and Q279R mutants transfected in COS-1 cells expressed no activity while the residual enzyme activity of the Q279E mutant represented 10% of wild type value. Western blot analysis demonstrated a differential behavior of the mutant proteins: Q279K and Q279H persisted as the inactive 50-kD precursor, indicating that these mutations may affect the normal processing of the enzyme, while the Q279R mutant was not detected probably due to an unstable protein which is rapidly degraded. The in vitro expression studies of the novel Q279K mutation were confirmed by Western blot analysis performed in the patient's lymphocytes which revealed the alpha-galactosidase A precursor of 50 kD but not the processed form.     

Homozygosity for a mutation in the lipoprotein lipase gene (Gly139→Ser) causes chylomicronaemia in a boy of Spanish descent

The enzyme lipoprotein lipase (LPL) plays a crucial role in triglyceride metabolism through catalysis of triglyceride-rich chylomicrons and very low density lipoproteins. Primary LPL deficiency manifests with chylomicronaemia and is caused by mutations in the LPL gene. In this paper we report a novel molecular defect (G₆₇₀A) in exon 4 of the LPL gene, resulting in a substitution of serine for glycine at position 139 in the mature protein. We identified homozygosity for this mutation in a boy of Spanish descent. In vitro mutagenesis provided formal proof that this missense mutation completely abolishes LPL function and therefore is the cause of LPL deficiency.     

Sex- and age-related variations in the in vitro heparin-releasable lipoprotein lipase from mononuclear leukocytes in blood

An in vitro heparin release of lipoprotein lipase (LPL) from whole blood, mainly from monocytes, was demonstrated by (1) the time-course of lipolytic activity with the presence of 10 U/ml heparin at 37 degrees C, (2) the distribution of LPL activity in monocyte and lymphocyte fractions, (3) an immuno-inactivation with anti-LPL immunoglobulin (IgG) and (4) responses to various compounds such as NaCl, protamine sulfate, heparin, and serum activator. The in vitro heparin-releasable LPL activity from blood correlated well with the LPL activity of postheparin plasma obtained from both normolipidemic and hyperlipidemic rabbits. Studies in humans revealed sex- and age-related variations in the in vitro heparin-releasable LPL from monocytes in the blood of 134 normal subjects and 24 hypertriglyceridemic subjects: The mean LPL activity was significantly higher in normal females over the age of 30, than in the corresponding males. In the hypertriglyceridemic group, the LPL activity was also higher in females than in males, but it was not significant. The in vitro heparin-releasable LPL activity from monocytes in blood was comparable to the LPL activity derived from adipose tissue and postheparin plasma, and thus it reflects lipoprotein metabolism.     

cld and lec23 are disparate mutations that affect maturation of lipoprotein lipase in the endoplasmic reticulum.

The mutations cld (combined lipase deficiency) and lec23 disrupt in a similar manner the expression of lipoprotein lipase (LPL). Whereas cld affects an unknown gene, lec23 abolishes the activity of alpha-glucosidase I, an enzyme essential for proper folding and assembly of nascent glycoproteins. The hypothesis that cld, like lec23, affects the folding/assembly of nascent LPL was confirmed by showing that in cell lines homozygous for these mutations (Cld and Lec23, respectively), the majority of LPL was inactive, displayed heterogeneous aggregation, and had a decreased affinity for heparin. While inactive LPL was retained in the ER, a small amount of LPL that had attained a native conformation was transported through the Golgi and secreted. Thus, Cld and Lec23 cells recognized and retained the majority of LPL as misfolded, maintaining the standard of quality control. Examination of candidate factors affecting protein maturation, such as glucose addition and trimming, proteins involved in lectin chaperone cycling, and other abundant ER chaperones, revealed that calnexin levels were dramatically reduced in livers from cld/cld mice; this finding was also confirmed in Cld cells.We conclude that cld may affect components in the ER, such as calnexin, that play a role in protein maturation. Whether the reduced calnexin levels per se contribute to the LPL deficiency awaits confirmation.     

Identification of a missense mutation in one allele of a patient with Pompe disease, and use of endonuclease digestion of PCR-amplified RNA to demonstrate lack of mRNA expression from the second allele.

Infantile-onset glycogen storage disease type II, or Pompe disease, results from a genetic deficiency of the lysosomal enzyme acid alpha glucosidase (GAA). Sequencing of the cDNA from a cell line (GM 244) derived from a patient with Pompe disease demonstrated a T953-to-C transition that predicted a methionine-to-threonine substitution at codon 318. The basepair substitution resulted in loss of restriction-endonuclease sites for NcoI and StyI. Analysis of genomic DNA revealed both a normal and an abnormal NcoI fragment, indicating that the patient was a genetic compound. NcoI and StyI digestion of cDNA, amplified by PCR from reverse-transcribed RNA, demonstrated that greater than 95% of the GAA mRNA in GM 244 was derived from the allele carrying the missense mutation. The missense mutation was uncommon, since it was not detected in 37 additional GAA-deficient chromosomes, as determined by digestion of genomic DNA with NcoI and hybridization. The amino acid substitution predicts a new potential site for N-linked glycosylation, as well as major changes in secondary structure of the protein. We could confirm that the mutation was responsible for the enzyme deficiency by demonstrating that a hybrid minigene containing the mutation did not express GAA enzyme activity after transient gene expression. We have therefore now provided the first identification of a single-basepair missense mutation in a patient with Pompe disease and furthermore have demonstrated that the patient is a genetic compound with the second allele barely expressing mRNA.