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Biomedical subjects

P Lohse

Publications and source records attributed to P Lohse.

At least 37 records · Page 2Linked to original sources
PubMed2001

Thrombophilic gene mutations and recurrent spontaneous abortion: prothrombin mutation increases the risk in the first trimester.

PROBLEM: Thrombophilic predisposition may be one of the underlying causes of recurrent spontaneous abortions (RSA). We studied the prevalence of five thrombophilic gene mutations in patients with RSA. METHOD OF STUDY: 102 patients with two or more consecutive abortions and 128 women without miscarriage were analyzed for factor V Leiden mutation (FVL), prothrombin G20210A mutation (PTM), C677T mutation in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene, glycoprotein IIIa (GPIIIa) C1565T polymorphism, and beta-fibrinogen G-455A polymorphism by polymerase chain reaction (PCR) techniques. RESULTS: No differences in the prevalence of FVL, MTHFR T/T, GPIIIa and 1-fibrinogen polymorphism were detected. Heterozygous PTM occurred more often in patients with RSA. This effect was significant in a subgroup with abortions exclusively in the first trimester (6.7%, vs. 0.8%, P = 0.027, OR 8.5). CONCLUSIONS: In contrast to the other mutations and polymorphisms, heterozygous PTM is more common in patients with abortions in the first trimester. This might reflect an influence of PTM on pathogenesis of early pregnancy loss.

Abortion, Habitual↗
PubMed2001

R116C mutation of cationic trypsinogen in a Turkish family with recurrent pancreatitis illustrates genetic microheterogeneity of hereditary pancreatitis.

Hereditary pancreatitis is due to heterozygosity for gain-of-function mutations in the cationic trypsinogen gene which result in increased levels of active trypsin within pancreatic acinar cells and autodigestion of the pancreas. The number of disease-causing defects is generally considered to be low. To gain further insight into the molecular basis of this disorder, DNA sequence analysis of all five exons was performed in 109 unrelated patients with idiopathic chronic pancreatitis in order to determine the variability of the underlying mutations. Two German females and one German male were carriers of the most common N29I and R122H mutations (trypsinogen numbering system). In a Turkish proband, an arginine (CGT) to cysteine (TGT) substitution at amino acid position 116 was identified. Family screening demonstrated that the patient had inherited the mutation from his asymptomatic father and that he had transmitted it to both of his children, his daughter being symptomatic since the age of 3 years. In addition, a German male was found to be a heterozygote for a D100H (GAC-->CAC) amino acid replacement. Our data provide evidence for genetic heterogeneity of hereditary pancreatitis. The growing number of cationic trypsinogen mutations is expected to change current mutation screening practices for this disease.

Adult↗
PubMed2001

Long-term administration of the HMG-CoA reductase inhibitor lovastatin in two patients with cholesteryl ester storage disease.

OBJECTIVE: In order to suppress de novo cholesterol and VLDL biosynthesis, a long-term therapy trial with lovastatin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, was initiated in two patients with cholesteryl ester storage disease (CESD), and concentrations of plasma lipids were monitored over a period of 9 years. METHODS: We studied two male patients with enzymatically confirmed CESD in whom long-term lovastatin therapy (8 and 9 years) was begun at the age of 7 and 19 years. The diagnosis of CESD was confirmed by the measurement of human lysosomal acid lipase (hLAL) activity in cultured skin fibroblasts and leukocytes. Restriction fragment length polymorphism (RFLP) analysis revealed that both subjects are homozygotes for the common CESD splice site mutation. Levels of serum lipids and lipoproteins were measured yearly. RESULTS: During the first year, total serum cholesterol decreased from 317 to 201 mg/dl in Patient A and from 228 to 120 mg/dl in Patient B, due mainly to the reduction of low-density lipoprotein (LDL) cholesterol from 262 to 151 mg/dt in Patient A and from 166 to 66 mg/dl in Patient B. Accordingly, the LDL cholesterol : high density lipoprotein (HDL) cholesterol ratio was markedly reduced in both patients after one year of therapy. The treatment was continued and, after 9 years of further medication, low total cholesterol and LDL cholesterol levels were still maintained. CONCLUSIONS: The study demonstrates that HMG-CoA reductase inhibitors are well tolerated drugs during long-term treatment of CESD patients and may help to prevent the development of premature atherosclerosis.

Adult↗
PubMed2001

Fluorescence-based detection of the CETP TaqIB polymorphism: false positives with the TaqMan-based exonuclease assay attributable to a previously unknown gene variant.

BACKGROUND: Previous studies have shown an association between the TaqIB polymorphism of the cholesteryl ester transfer protein (CETP) gene with plasma CETP and HDL concentrations and the progression of coronary artery disease (CAD). The aim of the present study was to determine the performance of two new fluorescence-based detection systems in the analysis of the TaqIB genotype. METHODS: CAD patients (n = 150) with known TaqIB genotype, as determined by restriction fragment length polymorphism (RFLP) analysis, were selected, including three groups of 50 patients, carrying the B1/1, B1/2, and B2/2 genotypes, respectively. The genotypes were also analyzed by fluorescence-based allele-specific TaqMan PCR and melting curve analysis (LightCycler). In addition, DNA sequencing was applied. RESULTS: The TaqIB genotypes obtained by fluorescence analysis corresponded to those determined by RFLP analysis with the exception of three heterozygous patients (B1/2), who were misclassified as homozygous B2 carriers with the TaqMan system. Melting curve analysis of these samples demonstrated an additional melting point at 59.1 degrees C, which was also found in four patients homozygous for the B1 allele. DNA sequencing revealed a previously unknown C270T nucleotide exchange in intron 1 of the CETP gene, only nine base pairs from the TaqIB site. CONCLUSIONS: Determination of the TaqIB polymorphism with the TaqMan system led to misclassifications because of a previously unknown C270T polymorphism of the CETP gene. The base substitution was detected with the LightCycler because of the occurrence of an additional melting point. Our data indicate the importance of thorough evaluation of new gene analysis systems before using them on a routine basis.

Carrier Proteins↗
PubMed2000

Subclinical course of cholesteryl ester storage disease in an adult with hypercholesterolemia, accelerated atherosclerosis, and liver cancer.

Few cases of asymptomatic cholesteryl ester storage disease (CESD) due to low enzymatic activity of human lysosomal acid lipase/cholesteryl ester hydrolase (hLAL) have been reported thus far in adults Here, we describe a 51-year-old man with a long clinical history of mixed hyperlipoproteinemia and severe premature atherosclerosis, but with no signs of hepatomegaly, liver dysfunction, or splenomegaly. The disease was discovered by chance in a biopsy performed because of suspected liver cancer (proven to be a cholangiocarcinoma). Residual hLAL activity in peripheral leukocytes was determined to be 6% of control values. DNA sequence and restriction fragment length polymorphism analysis demonstrated that the patient was a compound heterozygote for the prevalent CESD exon 8 splice site mutation (G934A) and the deletion of a C (nucleotide 673, 674, or 675) in exon 6 of the hLAL gene, resulting in premature termination of protein translation at residue 195. The patient died of liver failure as a consequence of extensive tumor infiltration at age 52. Lipid analysis revealed moderate cholesteryl ester storage in the liver and in the suprarenal cortex, and massive accumulation in the testicular histiocytes and Leydig cells, resulting in a pronounced secondary atrophy of the seminiferous tubules. Our case study demonstrates that hepatomegaly is an inconstant feature, even in CESD patients compound heterozygous for a Wolman mutation which results in complete loss of hLAL enzymic activity. It also highlights the need to be aware of this condition as it may be underdiagnosed.

Adult↗
PubMed2000

The number of dichorionic twin pregnancies is reduced by the common MTHFR 677C-->T mutation.

In multiple pregnancies, demands for folic acid are considerably increased. The most common inborn error of folate metabolism is mild methylenetetrahydrofolate reductase (MTHFR) deficiency due to the synthesis of a thermolabile variant of the enzyme with impaired catalytic activity which leads to reduced 5-methyltetrahydrofolate (5-methyl-THF) and mildly elevated homocysteine plasma concentrations when folate status is inadequate. To investigate whether the number of offspring is influenced by this mutation, we determined the frequency of the 677C-->T substitution in 156 singleton and 40 twin mothers with dichorionic placentation. The T allele frequency in singleton (0.30) and twin mothers (0.16) was significantly different (P = 0.011). Mothers with the 677C-->T mutation had a 2.28 times lower risk of having a twin pregnancy than those without (95% confidence interval = 1.18-4.66; P = 0.008). Our observation would explain, at least in part, the hereditary trait of multiple gestations and is in agreement with the ethnic distribution pattern of the T allele which has been found to be inversely correlated with the incidence of dichorionic twins. Our findings suggest that the MTHFR 677C-->T mutation interferes with human brood size, probably by influencing the proliferation of rapidly dividing embryonic and maternal cells.

Abortion, Spontaneous↗
PubMed2000

Compound heterozygosity for a Wolman mutation is frequent among patients with cholesteryl ester storage disease.

Cholesteryl ester storage disease and Wolman disease are rare autosomal recessive lipoprotein-processing disorders caused by mutations in the gene encoding human lysosomal acid lipase. Thus far we have elucidated the genetic defects in 15 unrelated CESD patients. Seven were homozygotes for the prevalent hLAL exon 8 splice junction mutation which results in incomplete exon skipping, while eight probands were compound heterozygotes for E8SJM and a rare mutation on the second chromosome. In this report, we describe the molecular basis of CESD in three compound heterozygous subjects of Czech and Irish origin. RFLP and DNA sequence analysis revealed that they were heteroallelic for the common G(934)-->A substitution in exon 8 of the hLAL gene and a mutation which, if inherited on both alleles, would be expected to result in complete loss of enzyme activity and to cause Wolman disease. In patients A. M. and J. J., two nucleotide deletions in exons 7 and 10 were detected, involving a T at position 722, 723, or 724 and a G in a stretch of five guanosines at positions 1064;-1068 of the hLAL cDNA. Both mutations result in premature termination of protein translation at residues 219 and 336, respectively, and in the production of truncated, inactive enzymes. Subject D. H., in contrast, is a compound heterozygote for the Arg(44)-->Stop mutation previously described in a French CESD proband. Combined with data in the literature, our results demonstrate that compound heterozygosity for a mutation causing Wolman disease is common among cholesteryl ester storage disease patients.

Base Sequence↗
PubMed1999

[Lysosomal acid lipase deficiency. Overview of Czech patients].

Lysosomal lipase deficiency is a hereditary autosomal recessive enzymopathy leading to lysosomal storage of triacylglycerols (TAG) and cholesterol esters (CE). In particular cells with a permanently high receptor-mediated LDL endocytosis are affected (liver, kidneys). There are two basic phenotypes. The fatal infantile phenotype (Wolman's disease) with generalized storage of both types of apolar lipids. This form was diagnosed in this country only once. The opposite is the protracted, oligosymptomatic form encountered in all age groups. It is characterized by the storage of CE (which gave this entity the name of cholesteryl storage disease--CESD). Its main sign is affection of the liver (hepatomegaly, hepatopathy), which in some instances may lead to organ failure, directly or after cirrhotic transformation. Furthermore there is permanent hypercholesterolaemia (high LDL cholesterol) due to increased VLDL synthesis by hepatocytes, low HDL cholesterol and variably raised TAG. This constellation of blood lipids is a risk factor for the development of atherosclerosis. In the course of 25 years in the Czech Republic 13 cases of CESD were diagnosed in 11 families. Ten of these cases were characterized by clinically manifest hepatopathy with hepatomegaly, detected incidentally during medical examinations (at the age of 2-14 years). In three adult patients with permanent hypercholesterolaemia the storage process was subclinical and the diagnosis was established quite incidentally by examination of non-specific secondary and tertiary manifestations of the disease. The diagnosis was established in all cases of CESD at the tissue level (liver biopsy), at the biochemical (acid lipase deficiency) and molecular genetic level (mutation in enzyme locus). In all instances mutation of G934A was found leading to reduction and loss of the eighth exon. This mutation was present in five patients in a homozygous state. Six mutations were heterozygous. In one instance for technical reasons only one allele was analyzed. In three instances a point "missense" mutation was found: T323A (Trp74Arg), T4(75)A (Asp124Glu), A210T (Asp36Gl), in one instance a "nonsense" mutation: C233T (Arg44-stop) and twice a deletion mutation delta C673-5 and delta G1068-8 leading to impairment of the reading frame and to premature stop of the codon.

Adult↗
PubMed1999

Molecular defects underlying Wolman disease appear to be more heterogeneous than those resulting in cholesteryl ester storage disease.

Human lysosomal acid lipase/cholesteryl ester hydrolase (hLAL) is essential for the intralysosomal metabolism of cholesteryl esters and triglycerides taken up by receptor-mediated endocytosis of lipoprotein particles. The key role of the enzyme in intracellular lipid homeostasis is illustrated by two lysosomal storage diseases inherited as autosomal recessive traits. Wolman disease, associated with deficient hLAL activity, leads to massive intracellular substrate accumulation and is always fatal in early infancy. Cholesteryl ester storage disease (CESD), in contrast, is characterized by very low levels of enzymic activity sufficient to allow survival of the affected patients into adulthood. In order to elucidate the underlying molecular defects in Wolman disease, we have characterized the hLAL gene in two female Wolman patients of German and Turkish origin by SSCP and DNA sequence analysis. Our results demonstrate that the German proband was compound heterozygous for an 8-bp deletion in exon 3 and a 2-bp deletion in exon 4 of the hLAL gene. These frameshift mutations lead to protein truncation at amino acid positions 24 and 116 and to complete loss of hydrolytic activity. The Turkish proband, in contrast, was homozygous for a G(1064)-->T substitution in exon 10 of the hLAL gene which converts the completely conserved glycine (GGG) residue at position 321 of the mature enzyme to tryptophan (TGG). In vitro expression of the hLAL(Gly(321)-->Trp) cDNA construct revealed that the amino acid replacement results in a more than 99% reduction of neutral lipid hydrolysis. The mutations provide new insights into the molecular basis of Wolman disease which is apparently more heterogeneous at the genetic level than cholesteryl ester storage disease.-Lohse, P., S. Maas, P. Lohse, A. C. Sewell, O. P. van Diggelen, and D. Seidel. Molecular defects underlying Wolman disease appear to be more heterogeneous than those resulting in cholesteryl ester storage disease.

Alleles↗
PubMed1998

Early loss of renal transplants in patients with thrombophilia.

BACKGROUND: We considered the possibility that thrombophilia may propagate graft thrombosis and therefore we evaluated the protein C system, which is a natural anticoagulant. Potential alterations in this system include protein C or protein S deficiency, inhibition through a lupus anticoagulant (LA), or a resistance to activated protein C due to the factor V Leiden (FVL) mutation. METHODS: One hundred thirty-two consecutive renal transplant patients, not known to have abnormal thrombostasis, in whom 1-year graft survival could be assessed, underwent laboratory testing for protein C or S activity, LA, and FVL. Transplant survival and demographic data were extracted from the hospital record. RESULTS: We identified 18 patients with thrombophilia (FVL, 10; LA, 6; protein S, 2) who had received a total of 28 renal transplants. Of these 28 transplant recipients, 11 transplants were lost within the first year, compared with 21 of 155 transplants to 114 patients without thrombophilia (P=0.0003). Median graft survival for patients with thrombophilia was 30 months (range: 0 to 166), compared with 86 months (range: 0 to 212) for patients without thrombophilia (P<0.01). The presence of thrombophilia represented a 3.5-fold (95% confidence interval, 2.3-5.3-fold) risk for 1-year graft loss. CONCLUSION: In this retrospective study, patients with thrombophilia had a significantly higher risk of early transplant failure. These data point toward a potential contribution of thrombophilia to transplant loss, a hypothesis that needs further study.

Adult↗
PubMed1997

A new composite made of polyurethane and glass ceramic in a loaded implant model: a biomechanical and histological analysis.

The biocompatibility and osseous integration of a new composite material made of polyurethane and a calcium silicophosphate ceramic was investigated in a loaded implant model in sheep and compared to that of commercially pure titanium. Six months after implantation, interfacial shear strength was higher between the titanium and bone than between the composite and bone. After 2 years, however, the shear strength was significantly higher in the composite group. Histologically, both implants were surrounded by bone and fibrous tissue and there were no signs of inflammation. Direct contact of bone on the composite surface increased significantly with time, whereas there was no time-dependent increase of bone contact on titanium. It can be concluded that the biocompatibility and osseous integration of the composite was very good in the loaded implant model used. It is therefore suggested that the new composite is a promising biomaterial for orthopaedic implants.

Journal Article↗
PubMed1997

The acid lipase gene family: three enzymes, one highly conserved gene structure.

Human gastric lipase (HGL; triacylglycerol lipase; EC 3.1.1.3) plays an important role in the digestion of dietary triglycerides in the gastrointestinal tract, especially in patients suffering from pancreatic lipase deficiencies. The enzyme is secreted by the fundic mucosa of the stomach and hydrolyzes the ester bonds of triglycerides under acidic pH conditions, while cholesteryl esters are not attacked. The 379-amino acid protein is highly homologous to two other acidic lipases, rat lingual lipase (RLL; triacylglycerol lipase; EC 3.1.1.3) and human lysosomal acid lipase (HLAL; cholesteryl esterase; EC 3.1.1.13). To determine whether this remarkable similarity is also present at the genomic level, we have elucidated the respective gene structures by screening three bacteriophage lambda libraries and by polymerase chain reaction-based intron amplification. The genes encoding HGL, RLL, and HLAL are composed of 10 exons interrupted by nine introns and span about 14 kb, 18.7 kb, and 38.8 kb of genomic DNA, respectively. The HGL and RLL gene organizations are identical, suggesting that RLL is the rat gastric lipase expressed in the serous von Ebner glands of the tongue. The positions of the HLAL intervening sequences are also absolutely conserved, except for the location of intron 1. Our results support the concept that HLAL and HGL/RLL are members of a gene family of lipases that most likely have evolved by duplication of an ancestral gene and subsequently assumed distinct roles in neutral lipid metabolism due to sequence divergence and different expression patterns.

Animals↗
PubMed1997

Human lysosomal acid lipase/cholesteryl ester hydrolase and human gastric lipase: identification of the catalytically active serine, aspartic acid, and histidine residues.

Human lysosomal acid lipase/cholesteryl ester hydrolase (HLAL), human gastric lipase (HGL), and rat lingual lipase (RLL) constitute a family of mammalian lipases characterized by an acidic pH optimum. HGL and RLL are secreted by the chief cells of the stomach and by the serous von Ebner's glands of the tongue, respectively, and hydrolyze dietary longchain triglycerides in the gastrointestinal tract. HLAL, in contrast, catalyzes the intralysosomal degradation of both triglycerides and cholesteryl esters in virtually all cells except erythrocytes. All three enzymes are proposed to be serine esterases with a catalytic Ser-Asp-His triad similar to other lipases, despite their sensitivity towards sulfhydryl modifying reagents. To investigate the role of conserved serine, aspartic acid, and histidine residues in HLAL and HGL, we constructed 24 mutant lipases with single amino acid substitutions using the site-directed mutagenesis approach. Our combined data strongly support the conclusion that Ser153, Asp324, and His355 are components of the catalytic triad of HLAL and HGL. Structural integrity of the conserved His-Gly dipeptide of lipases also appears to be important for neutral lipid hydrolysis, as replacement of His65 by glutamine abolished HLAL and HGL enzymic activity. Substitution of HLAL residues Asp93, Asp130, and Asp328 with glycine, in contrast, had a more pronounced impact on cholesteryl oleate hydrolysis than on triglyceride hydrolysis. These results provide new insights into the structural basis of HLAL and HGL function.

Animals↗
PubMed1997

Human lysosomal acid lipase/cholesteryl ester hydrolase and human gastric lipase: site-directed mutagenesis of Cys227 and Cys236 results in substrate-dependent reduction of enzymatic activity.

Chemical modification studies and site-directed mutagenesis experiments have provided evidence that human lysosomal acid lipase/cholesteryl ester hydrolase (HLAL), human gastric lipase (HGL), and rat lingual lipase (RLL) are serine esterases. Loss of HLAL and HGL activity was also observed in the presence of sulfhydryl-reactive substances, suggesting that cysteines are likewise essential for substrate hydrolysis. To study the functional role of the HLAL and HGL cysteine residues, we replaced these amino acids with alanine by site-directed mutagenesis. Substitutions at positions 227 and 236, alone or together, drastically reduced hydrolytic activity in a substrate-dependent manner while the other mutants were not affected to any great extent. HLAL(Cys227-->Ala), HLAL(Cys236-->Ala), and HLAL(Cys227-->Ala, Cys236-->Ala) were essentially inactive against cholesteryl oleate, but retained about 23-39%, 28-37%, and 13-17% of catalytic activity for both triolein and tributyrin, respectively. The data obtained with the corresponding HGL mutants confirmed the importance of residues 227 and 236 in maintaining enzymatic activity towards long- and short-chain triglycerides. In order to assess the contribution of the eight amino acids delimited by Cys227 and Cys236 to lipolysis, we generated HLAL replacement mutants containing the corresponding residues 228-235 of HGL or RLL. Both HLAL chimeras were catalytically active towards all three substrates analyzed, indicating that these amino acids do not determine HLAL substrate specificity. Deletion of the eight-amino acid alpha-helix as well as disruption of its hydrophobic surface, in contrast, abolished enzymatic activity. Our studies suggest that Cys227, Cys236, and the amphipathic helix formed by residues 228-235 are essential for HLAL- and HGL-mediated neutral lipid catabolism.

Amino Acid Sequence↗
PubMed1996

Homozygosity for two point mutations in the lipoprotein lipase (LPL) gene in a patient with familial LPL deficiency: LPL(Asp9-->Asn, Tyr262-->His).

Familial lipoprotein lipase (LPL) deficiency is an inherited disorder of lipoprotein metabolism characterized by hypertriglyceridemia and recurrent episodes of abdominal pain and pancreatitis. We have studied the genetic basis of LPL deficiency in a 62-year-old black male with undetectable pre- and post-heparin plasma LPL mass and activity, DNA sequence analysis of the patient's LPL cDNA and gene as well as digestion with Bcl I and Asu I revealed that the proband is a homozygote for two separate gene defects. One mutation changed a G to an A, resulting in the conversion of amino acid 9 of the mature protein, aspartic acid (GAC), to asparagine (AAC). The second substitution, a C for a T, replaced tyrosine (TAC) at residue 262 with histidine (CAC). Northern blot analysis of monocyte-derived macrophage RNA demonstrated the presence of LPL mRNA of approximately normal size and quantity when compared to control. Expression of both mutations separately (pCMV-9 and pCMV-262) or in combination (pCMV-9+262) in human embryonal kidney-293 cells demonstrated that LPL-9 had approximately 80% the specific activity of wild type LPL, but LPL-262 and LPL-9+262 had no enzymic activity, thus establishing the functional significance of the LPL-262 defect. Despite an absolute deficiency of LPL mass and activity demonstrated by analysis of patient post-heparin plasma, in vitro expression of both LPL mutants was normal, suggesting that the absence of LPL in patient post-heparin plasma was a result of altered in vivo processing. Analysis of the heparin binding properties of the mutant enzymes by heparin-Sepharose affinity chromatography indicated that most of the LPL-262 mass was present in an inactive peak, which like the normal LPL monomer, eluted at 0.8 M NaCl. Thus, the Tyr262 --> His mutation may alter the stability of the LPL dimer, leading to the formation of inactive LPL-262 monomer which exhibits reduced heparin affinity. Based on these results, we propose that, in vivo, enhanced formation of LPL-9+262 monomer leads to abnormal binding of the mutant lipase to endothelial glycosaminoglycans ultimately resulting in enhanced catabolism of the mutant enzyme and lower enzyme mass in post-heparin plasma.

Amino Acid Sequence↗