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.     

Molecular defects in erythropoietic protoporphyria with terminal liver failure

We identified two additional mutations in the ferrochelatase gene in two Swiss patients with erythropoietic protoporphyria (EPP). Ferrochelatase cDNA from patients was amplified by the polymerase chain reaction (PCR) and subjected to mutation analysis by sequencing PCR products either directly or after subcloning. The first patient, who underwent liver transplantation because of terminal liver failure, was identified as having a single point mutation (C to T) at nucleotide 175 that resulted in a Gln to stop codon conversion in one allele of the gene. In the second case, in which the patient has so far no liver involvement, a two-base deletion (T899G900) was found in one allele. Frameshift as a result of the deletion creates a stop codon. This study presents two new genotypes of EPP, including one with liver failure, a rare and fatal form of EPP.     

LDL-R and Apo-B-100 gene mutations in Polish familial hypercholesterolemias

A group of 30 Polish families with clinical signs of familial hypercholesterolemia was studied for the presence of germ-line mutations in the LDL-R and ApoB-100 genes. Screening of the LDL-R gene was performed at the genomic DNA level by single-strand conformation polymorphism analysis of all 18 exons and extended by sequencing of polymerase chain reaction (PCR) products showing abnormalities. The occurrence of large LDL-R gene alterations was evaluated by analysis of restriction enzyme patterns on Southern blots and using the long-PCR technique. The ApoB-100 gene was studied by combined allele-specific and asymmetric PCR for the occurrence of the common B-3500 missense mutation G to A at nucleotide position 10,708. Germ-line mutations were found in 17 families. In 12 of them LDL-R gene mutations were detected. Three of 11 different mutations had previously been described in other populations (3-bp deletion of codon 197; Ser156Leu; Gly571Glu). Of the mutations not previously recognized and identified in Polish families, there were three small deletions (2-bp deletion AG at codon 291; 4-bp deletion CCCT at codons 661–662; 1-bp deletion A at codon 830), and four point mutations (Arg239Stop, Cys331Stop, Asn543Ser, Gln665Stop). Additionally, one large (∼1-kb) LDL-R gene deletion between exons 6 and 9 was identified. In five families, the B-3500 mutation within the ApoB-100 gene was revealed. Received: 15 September 1997 / Accepted: 10 February 1998     

Molecular basis of familial chylomicronemia: mutations in the lipoprotein lipase and apolipoprotein C-II genes.

The molecular basis of familial chylomicronemia (type I hyperlipoproteinemia), a rare autosomal recessive trait, was investigated in six unrelated individuals (five of Spanish descent and one of Northern European extraction). DNA amplification by polymerase chain reaction (PCR) followed by single strand conformation polymorphism (SSCP) analysis allowed rapid identification of the underlying mutations. Six different mutant alleles (three of which are previously undescribed) of the gene encoding lipoprotein lipase (LPL) were discovered in the five LPL-deficient patients. These included an 11 bp deletion in exon 2, and five missense mutations: Trp 86 Arg (exon 3), His 136 Arg (exon 4), Gly 188 Glu (exon 5), Ile 194 Thr (exon 5), and Ile 205 Ser (exon 5). The Trp 86 Arg mutation is the only known missense mutation in exon 3. The other missense mutations lie in the highly conserved "central homology region" in close proximity with the catalytic site of LPL. These and other previously reported missense mutations provide insight into structure/function relationships in the lipase family. The missense mutations point to the important role of particular highly conserved helices and beta-strands in proper folding of the LPL molecule, and of certain connecting loops in the catalytic process. A nonsense mutation (Arg 19 Term) in the gene encoding apolipoprotein C-II (apoC-II), the cofactor of LPL, was found to underlie chylomicronemia in the sixth patient who had normal LPL but was apoC-II-deficient.     

Lipoprotein lipase (LPL) deficiency: a new patient homozygote for the preponderant mutation Gly188Glu in the human LPL gene and review of reported mutations: 75 % are clustered in exons 5 and 6

We have investigated the lipoprotein lipase (LPL) gene of a 2-year-old patient presenting classical features of the familial LPL deficiency including undetectable LPL activity. DNA sequence analysis of exon 5 identified the patient as a homozygote for the Gly188Glu mutation, frequently involved in this disease. A review of cases of LPL deficiency with molecular study of the LPL gene showed a total number of 221 reported mutations involved in this disease. Gly188Glu was involved in 23.5 % of cases and 74.6 % of mutations were clustered in exons 5 and 6. Based on these observations, we propose a method of screening for mutations in this gene.     

Recurrence of a nonsense mutation in the NF1 gene causing classical neurofibromatosis type 1

Summary The gene responsible for von Recklinghausen neurofibromatosis (NF1) has recently been identified, and several point mutations and deletions have been described. The availability of intron-exon boundaries of several exons of the NF1 gene facilitates the search for mutations in affected patients. We have analysed 38 patients for mutations in exon 4 of the NF1 gene, and found one patient with a CT transition at base position 1087 of the cDNA, changing an arginine codon to a stop codon, at amino acid position 365. Sequencing of other members of the family, including both parents, did not show the mutation, confirming that this mutation is responsible for this sporadic NF1 case. As the mutation described here was previously identified in an independent case by others, this case represents a recurrence of this mutation and suggests that codon 365 might be a hot spot for mutations in the NF1 gene. Thus, a specific search for this mutation should be performed when studying NF1 sporadic or familiar cases for genetic analysis.     

Three nonsense mutations responsible for group A xeroderma pigmentosum.

The molecular basis of xeroderma pigmentosum (XP) group A was studied and 3 nonsense mutations of the XP-A complementing gene (XPAC) were identified. One was a nucleotide transition altering the Arg-228 codon (CGA) to a nonsense codon (TGA). This transition creates a new cleavage site for the restriction endonuclease HphI. Of 21 unrelated Japanese XP-A patients examined, 1 (XP39OS) was a homozygote for this mutation and 3 were compound heterozygotes for this mutation and for the splicing mutation of intron 3 reported previously which is the most common mutation in Japanese patients and creates a new cleavage site for the restriction endonuclease AlwNI. The second mutation was a nucleotide transition altering the Arg-207 codon (CGA) to a nonsense codon (TGA). A Palestinian patient (XP12RO) who had severe symptoms of XP was homozygous for this mutation. The third mutation was a nucleotide transversion altering the Tyr-116 codon (TAT) to a nonsense codon (TAA). This transversion creates a new cleavage site for the restriction endonuclease MseI. Of the Japanese patients, 2 with severe clinical symptoms had this mutant allele. One was a compound heterozygote for this mutation and for the splicing mutation, and the other was heterozygous for this mutation and homozygous for the splicing mutation. Although most XP-A patients such as XP12RO have severe skin symptoms and neurological abnormalities of the de Sanctis-Cacchione syndrome, patient XP39OS was an atypical XP-A patient who had mild skin symptoms and minimal neurological abnormalities. Our results suggest that the clinical heterogeneity in XP-A is due to different mutations in the XPAC gene. Moreover, our data indicate that almost all Japanese cases of XP-A are caused by one or more of the 3 mutations, i.e., the splicing mutation of intron 3 and the 2 nonsense mutations of codons 116 and 228. Therefore, by restriction fragment length polymorphism analysis of PCR-amplified DNA sequences using the 3 restriction enzymes described above, rapid and reliable diagnosis of XP-A can be achieved in almost all Japanese subjects including prenatal cases and carriers.     

Two naturally occurring mutations at the first and second bases of codon aspartic acid 156 in the proposed catalytic triad of human lipoprotein lipase. In vivo evidence that aspartic acid 156 is essential for catalysis.

We are studying naturally occurring mutations in the gene for lipoprotein lipase (LPL) to advance our knowledge about the structure/function relationships for this enzyme. We and others have previously described 11 mutations in human LPL gene and until now none of these directly involves any of the residues in the proposed Asp156-His241-Ser132 catalytic triad. Here we report two separate probands who are deficient in LPL activity and have three different LPL gene haplotypes, suggesting three distinct mutations. Using polymerase chain reaction cloning and DNA sequencing we have identified that proband 1 is a compound heterozygote for a G----A transition at nucleotide 721, resulting in a substitution of asparagine for aspartic acid at residue 156, and a T----A transversion, resulting in a substitution of serine for cysteine at residues 216. Proband 2 is homozygous for an A----G base change at nucleotide 722, leading to a substitution of glycine for aspartic acid at residue 156. The presence of these mutations in the patients and available family members was confirmed by restriction analysis of polymerase chain reaction-amplified DNA. In vitro site-directed mutagenesis and subsequent expression in COS cells have confirmed that all three mutations result in catalytically defective LPL. The two naturally occurring mutations, which both alter the same aspartic acid residue in the proposed Asp156-His241-Ser132 catalytic triad of human LPL, indicate that Asp156 plays a significant role in LPL catalysis. The Cys216----Ser mutation destroys a conserved disulfide bridge that is apparently critical for maintaining LPL structure and function.     

The molecular genetic basis of muscle phosphoglycerate mutase (PGAM) deficiency.

The glycolytic enzyme phosphoglycerate mutase (PGAM) is a dimer, and mature human skeletal muscle contains almost exclusively the MM form of the enzyme, PGAM-M. In 1981, we identified a patient with PGAM-M deficiency, and three additional patients have since been described. All presented with exercise intolerance, cramps, and myoglobinuria. We report two new patients with PGAM-M deficiency and describe the molecular lesions in five patients--four African-Americans and one Caucasian. Three patients were homozygous for an identical G-to-A transition converting an encoded Trp to an in-frame stop codon (codon 78). A fourth patient was heterozygous for this mutation and also carried an A-to-C mutation converting Glu to Ala (codon 89). The fifth patient, the only Caucasian, was homozygous for a different point mutation, a C-to-T mutation, converting Arg to Trp (codon 90).     

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.     

Characterization of mutations in the factor VIII gene by direct sequencing of amplified genomic DNA

In order to search for mutations resulting in hemophilia A that are not detectable by restriction analysis, three regions of the factor VIII gene were chosen for direct sequence analysis. Short segments of genomic DNA of 127 unrelated patients with hemophilia A were amplified by polymerase chain reaction. A total of 136,017 nucleotides were sequenced, and four mutations leading to the disease were found: a frameshift at codon 360 due to deletion of two nucleotides (GA), a nonsense codon 1705 due to a C----T transition, and two missense codons at positions 1699 and 1708. The first missense mutation (A----T) results in a Tyr----Phe substitution at a putative von Willebrand factor binding site. The second results in an Arg----Cys substitution at a thrombin cleavage site. In addition, we identified three rare sequence variants: a silent C----T transition at codon 34 which does not result in an amino acid change, a G----C change at codon 345 (Val----Leu), and an A----G change at the third nucleotide of intron 14. Direct sequence analysis of amplified DNA is a powerful but labor-intensive method of identifying mutations in large genes such as the human factor VIII gene.     

Identification of splicing mutations of the last nucleotides of exons, a nonsense mutation, and a missense mutation of the XPAC gene as causes of group A xeroderma pigmentosum.

Four mutations of the XPAC gene were identified as molecular bases of different UV-sensitive subgroups of xeroderma pigmentosum (XP) group A. One was a G to C transversion at the last nucleotide of exon 4 in GM1630/GM2062, a little less hypersensitive subgroup than the most sensitive XP2OS/XP12RO. The second mutation was a G to A transition at the last nucleotide of exon 3 in GM2033/GM2090, an intermediate subgroup. Both mutations caused almost complete inactivation of the canonical 5' splice donor site and aberrant RNA splicing. The third mutation was a nucleotide transition altering the Arg-211 codon (CGA) to a nonsense codon (TGA) in another allele of GM2062. The fourth mutation was a nucleotide transversion altering the His-244 codon (CAT) to an Arg codon (CGT) in XP8LO, an intermediate subgroup. Our results strongly suggest that the clinical heterogeneity in XP-A is due to different mutations in the XPAC gene.     

Two novel mutations of SURF1 in Leigh syndrome with cytochrome c oxidase deficiency

Cytochrome c oxidase (COX) deficiency is the most common cause of Leigh syndrome (LS). COX consists of ten nuclear-encoded and three mtDNA-encoded structural subunits. Although the nucleotide sequences of all 13 genes are known, no mutation was found in nuclear-encoded subunit genes of COX-deficiency patients. Zhu et al. (1998) and Tiranti et al. (1998) found nine mutations in the surfeit 1 (SURF1) gene in LS families with COX deficiency. The mouse surfeit gene cluster consists of six closely spaced housekeeping genes unrelated by sequence homology. Except for the Surf3 gene, the function is still not known. The juxtaposition of at least five of the surfeit genes is conserved between birds and mammals. We identified two novel mutations of SURF1 in a Japanese LS patient with COX deficiency using direct sequencing analysis. Firstly, a 2-bp deletion at nucleotide position 790 (790delAG) in exon 8 was found, which shifts the reading frame such that the mutant protein has a completely different amino acid sequence from codon 264 to the premature stop codon at 290. Secondly, we found a T-to-G transversion at nucleotide 820, resulting in the substitution of tyrosine by aspartic acid at codon 274 (Y274D). We also studied the parents' genes, and found that the Y274D mutation was in his father and the 790delAG mutation was in his mother heterozygously. Therefore, we concluded that the patient was a compound heterozygote with these mutations. These are the first pathogenetic SURF1 mutations identified in a Japanese family.     

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.     

Genetic polymorphisms and haplotype structures of the CYP4A22 gene in a Japanese population

The CYP4A fatty acid monooxygenases oxidize endogenous arachidonic acid to 20-hydroxyeicosatetraenoic acid that acts as a regulator of blood pressure. Among the isoforms of the CYP4A subfamily, the human CYP4A22 was recently identified. In this study, we report the comprehensive investigation of polymorphisms in the CYP4A22 gene. To investigate genetic variation in CYP4A22 in 191 Japanese subjects, we used denaturing HPLC (DHPLC) and direct sequencing. Our investigation has enabled the identification of 13 sequence variations in the CYP4A22 coding region, thereby demonstrating for the first time that this gene is subject to polymorphism. Two of these sequence variations correspond to silent mutations located in exons 8 (His323His) and 9 (Gly390Gly). Nine of these sequence variations correspond to missense mutations located in exons 1 (Arg11Cys), 3 (Arg126Trp), 4 (Gly130Ser and Asn152Tyr), 5 (Val185Phe), 6 (Cys231Arg), 7 (Lys276Thr), 10 (Leu428Pro), and 12 (Leu509Phe). One of these sequence variations corresponds to nonsense mutations located in exon 9 (Gln368stop). The 13th mutation corresponds to a nucleotide deletion (G7067del) that causes a frameshift and consequently results in a stop codon 80 nucleotides downstream. In addition to the wild-type CYP4A22*1 allele, 20 variants, namely CYP4A22*2-15, were characterized by haplotype analysis. Based on these data, we concluded that allelic variants of the human CYP4A22 gene exist and speculated that some of these variants may be functionally relevant.     

A mutant allele common to the type I adenine phosphoribosyltransferase deficiency in Japanese subjects

Adenine phosphoribosyltransferase (APRT) deficiency is a genetic disorder which causes 2,8-dihydroxy-adenine urolithiasis. The estimated incidence of heterozygosity in Caucasian and Japanese populations is 1%. Mutant alleles responsible for the disease have been classified as APRT*Q0 (type I) and APRT* (type II). In our previous study, we demonstrated in APRT*J a single common base change which accounts for 70% of the Japanese mutants. The present report describes the analysis of an APRT*Q0 mutation in Japanese subjects. Two nucleotide substitutions common to all seven affected alleles from four unrelated subjects (three homozygotes and a heterozygote) were identified: G----A at nucleotide position 1453 and C----T at 1456. The G----A altered the amino acid Trp98 to a stop codon. The C----T did not alter Ala99. These point mutations were demonstrated by sequence analysis of polymerase chain reaction (PCR)-amplified genomic DNA and cDNA. The G----A change at 1453 results in the elimination of a PflMI site in the APRT gene. PflMI digests, which were used to confirm the G----A transition, can be useful in screening for this specific mutation.     

Analysis of naturally occurring and site-directed mutations in the argininosuccinate lyase gene.

Argininosuccinic aciduria is an inborn error of metabolism due to the genetic deficiency of argininosuccinate lyase. In order to determine the molecular basis for the disease, RNA isolated from cultured skin fibroblasts derived from four unrelated patients was reverse-transcribed and amplified using the polymerase chain reaction and the products were cloned and sequenced. Three single base missense mutations were identified: Arg111----Trp, Gln286----Arg, and Arg193----Gln. One single base amber mutation was identified at Gln454. One mutation involved a 13-base pair deletion within exon 13, and it was noted that the majority of the mature RNA derived from this allele was deleted for the entire exon rather than containing the exon with the 13 bases deleted. A final mutation was observed in which exon 2 was deleted from the mature RNA. The molecular basis for this deletion was not determined. Of the eight potential mutations present in the four cell lines studied, six mutations were identified and further data indicate that the remaining two unidentified mutations were different from those identified. Two site-directed mutations were created in the cDNA, Lys51----Asn and His89----Gln, and these were expressed in yeast. The Lys51 mutation caused an approximate 2-fold reduction in activity and the His89 mutation resulted in an approximate 10-fold reduction in activity. The combination of determination of naturally occurring mutations and the study of the effect of site-directed mutations on the activity of argininosuccinate lyase provide insight into the amino acid residues critical to the function of the enzyme.     

A new ATP7B gene mutation with severe condition in two unrelated Iranian families with Wilson disease

Wilson disease is associated with a defect in copper metabolism and caused by different mutations in ATP7B gene. The aim of this study was to determine mutation frequency of ATP7B exons 8 and 14 in Wilson disease patients from the south of Iran. The exons 8 and 14 of ATP7B gene were analyzed in 65 unrelated Wilson disease patients by Denaturing High Performance Liquid Chromatography, and samples with abnormal peak profile were selected for direct DNA sequencing. Seven out of 65 (10.8%) patients had mutations at exon 14, including c.3061-1G>A in four and c.3207C>A in three patients. In addition, four different mutations were identified at exon 8 of six patients (9.2%). Three of these mutations have been previously reported, including c.2304delC in two patients, c.2293G>A and 2304dupC each in one patient. Furthermore, a novel mutation, c.2335T>G (p.Trp779Gly), was identified in two unrelated patients. The patients with this novel mutation demonstrated severe neuropsychiatric condition. All together, 13 out of 65 (20%) patients had mutations within exons 8 and 14. We also identified a lower frequency of the most common mutations of exons 8 and 14 in the southern Iranian population.     

Ornithine transcarbamylase deficiency: new sites with increased probability of mutation

Ornithine transcarbamylase (OTC) deficiency, the most common inborn error of the urea cycle, shows an X-linked inheritance with frequent new mutations. Investigations of patients with OTC deficiency have indicated an overproportionate share of mutations at CpG dinucleotides. These statistics may, however, be biased because of the easy detection of CpG mutations by screening for TaqI and MspI restriction sites. In the present study, we investigated 30 patients, with diagnosed OTC deficiency, for new sites with an increased probability of mutation by complete DNA sequence analysis of all ten exons of the OTC gene. In six patients, two codons in exons 2 and 5, respectively, contained novel recurrent mutations, all of them affecting CpG dinucleotides. They included C to T and G to A transitions in codon 40, changing an arginine to cysteine and histidine, respectively, and a C to T transition in codon 178 causing the substitution of threonine by methionine. The first two mutations were characterized by a mild clinical course with high risk of sudden death in late childhood or early adulthood, whereas the third mutation showed a more severe phenotypic expression. In addition to these novel mutations, we identified four patients with the known R277W mutation, making it the most common point mutation of the OTC gene.     

Truncating ribosomal protein S19 mutations and variable clinical expression in Diamond-Blackfan anemia

Diamond-Blackfan anemia (DBA) is a rare constitutional erythroblastopenia characterized by a specific defect in erythroid differentiation. Recently, mutations in the gene encoding ribosomal protein (RP) S19 were found in a subset of patients with the disease. To characterize further RPS19 mutations and to investigate genotype-phenotype relationships, we screened this gene for mutations in patients with DBA by direct sequencing and Southern-blot analysis. Four novel mutations were identified. A G120A nonsense mutation resulting in a stop at codon 33, a C302T nonsense mutation introducing a premature stop at codon 84, and a 327delG which results in a frame shift at codon 103. A fourth and more complex mutation (TT157-158AA, 160insCT) resulting in a Leu45Gln and a frame shift from codon 47 was found in three affected family members with variable phenotypes. The different clinical expression for identical mutations suggest the presence of other modulating factors for the disease. The mutations presented here further support the role of RPS19 in erythropoietic differentiation and proliferation.