Klaus Altland

Apolipoprotein A-I variants. Naturally occurring substitutions of proline residues affect plasma concentration of apolipoprotein A-I.

Six unrelated families with genetically determined structural variants of apo A-I were found in the course of an electrophoretic screening program for apo A-I variants in dried blood samples of newborns. The following structural variations were identified by the combined use of HPLC, time-of-flight secondary ion mass spectrometry (TOF-SIMS), and automated gas phase sequencing: Pro3----Arg (1x), Pro4----Arg (1x), and Pro165----Arg (4x). All variant carriers were heterozygous for their mutant of apo A-I. Subjects heterozygous for apo A-I(Pro165----Arg) (n = 12) were found to exhibit lower mean values for apo A-I (109 +/- 16 mg/dl) and HDL cholesterol (37 +/- 9 mg/dl) than unaffected family members (n = 9): 176 +/- 41 and 64 +/- 18 mg/dl, respectively (P less than 0.001). In 9 of 12 apo A-I(Pro165----Arg) variant carriers the concentrations of apo A-I were below the fifth percentile of sex-matched controls. By two-dimensional immunoelectrophoresis as well as by densitometry the relative concentration of the variant apo A-I in heterozygous carriers of apo A-I(Pro165----Arg) was determined to account for only 30% of the total plasma apo A-I mass instead of the expected 50%. Thus, the observed apo A-I deficiency may be largely a consequence of the decreased concentration of the variant apo A-I. In the case of the apo A-I(Pro3----Arg) mutant, densitometry of HDL apolipoproteins demonstrated a distinctly increased concentration of the variant proapo A-I relative to normal proapo A-I. This phenomenon was not observed in the apo A-I(Pro4----Arg) mutant or in other mutants. This suggests that the interspecies conserved proline residue in position 3 of mature apo A-I is functionally important for the regular enzymatic conversion of proapo A-I to mature apo A-I.

Screening and biochemical characterization of transthyretin variants in the Portuguese population.

The study of pathogenic and nonpathogenic transthyretin (TTR) variants is very important for the understanding of such TTR‐related diseases as hereditary amyloidosis and also to establish a relationship between the structure and function of the molecule. Variants with clinical manifestations can be easily detected, but clinically silent variants can be detected only by population screening programs using specialized techniques. Hybrid isoelectric focusing (HIEF) in extremely flattened immobilized pH gradients (IPG) allows the detection of even neutral aminoacid substitutions and has been used to analyze ∼5,000 samples from the Portuguese population. Comparison with samples from carriers of three known TTR mutations (Met 30 associated with hereditary amyloidosis, Met 119, and Asn 90) was also made. In this study we detected: (1) 8 individuals carriers of TTR Met 30, (2) 35 carriers of TTR Met 119, (3) 12 carriers of TTR Asn 90, (4) 1 compound heterozygote for TTR Met 30/Met 119, and (5) 5 variants that presented a different pattern from the controls used. We also performed DNA sequencing analyses of two of the variants with the different band pattern in HIEF. The individuals were found to be carriers of TTR Ile 122 and TTR Thr 109, respectively. All the mutations detected, except for Asn 90, result from substitutions in CpG hot spots and thus can be rather frequent in the populations. Studies on the clinical evolution of the compound heterozygotes and on the physical‐chemical properties of these hybrid TTRs will help to understand the pathogenicity associated with TTR. Hum Mutat 9:226–233, 1997

Potential treatment of transthyretin-type amyloidoses by sulfite

ABSTRACT Familial amyloidotic polyneuropathy (FAP) and senile systemic amyloidosis (SSA) are characterized by systemic extracellular deposition of insoluble transthyretin (TTR) fibrils. While only normal TTR is found in fibrils from SSA patients who predominantly suffer from cardiomyopathy, autosomal dominant FAP preferentially affects peripheral nerves and heart and is associated with so-called amyloidogenic mutations of this protein, giving rise to TTR forms of decreased stability. Using isoelectric focusing in urea gradients we were able to demonstrate a stabilizing effect of sulfite on TTR monomers and tetramers, as well as an increase in the tetramer/monomer ratio. We demonstrate that this ratio, which is decreased in FAP patients, can be increased to beyond normal levels. We show that doses of sulfite which are tolerable in vivo produce a significant increase in the tetramer/monomer ratio and postulate that sulfite may be a potent drug for delaying the onset and progress of FAP and SSA.

Double one-dimensional electrophoresis of human serum transferrin: A new high-resolution screening method for genetically determined variation

SummaryWith one-dimensional electrophoresis or electrofocusing the selective demonstration of proteins and their genetically determined variants from complex compositions like the physiological body fluids often is possible only with specific staining techniques or immunofixation. By the method described here samples are separated by. PAG electrophoresis and a selected detail of the electropherogram is further resolved by PAG electrofocusing. The resulting pattern of bands after a usual protein stain can easily be interpreted due to the reduction of the number of bands. Informative bands hidden in a one-dimensional pherogram by overlapping with one or more other bands can be selectively visualized by the one-dimensional combination of two electrophoretic methods. With the double one-dimensional technique several hundred samples can be analyzed per person day. Thus the application in electrophoretic screening studies appears to be of special interest. The method is demonstrated by using the polymorphism of human serum transferrin as an example. A new variant of Tf-C with a high incidence in the German population is demonstrated.

Apolipoprotein E type 4 allele and Alzheimer's disease: effect on age at onset and relative risk in different age groups.

The effect of the apolipoprotein E (apoE) genotype on the age at onset of Alzheimers disease (AD) and the relative risk conferred by the apoE ε4 allele were studied in 91 patients and 69 healthy age-matched controls. According to the age of presentation, which varied from 44 to 95 years, subjects were divided into four groups. The inheritance of at least one ɛ4 allele was associated with a significant reduction of the age at onset by 7.7 years among patients who were 83 years or older when examined. A weaker inverse relationship between the ɛ4 allele and the age at onset was also observed among patients who were aged 44–63 years at presentation. The effect of the c4 allele was minimal or absent in the two intermediate age categories. The relative risk of AD conferred by the inheritance of at least one £4 allele showed no consistent age-related pattern. The overall risk expressed as an odds ratio was 5.0 (95% CI 2.4–10.5). With respect to the limitations of the study, we tentatively conclude (1) that the effect of the apoE ɛ4 allele on the age at onset is not restricted to AD patients of a particular age, in accordance with current hypotheses on the role of apoE gene products in the biology of AD; (2) that the relative risk of AD associated with the ɛ4 allele is not significantly modulated by age. Although the apoE ɛ4 allele is an important susceptibility factor for AD occurring in middle age as well as in later life, it is of limited value in routine clinical diagnosis and should not be used for predictive testing in asymptomatic individuals.

Skeletal scintigraphy indicates disease severity of cardiac involvement in patients with senile systemic amyloidosis

BACKGROUND Senile systemic amyloidosis (SSA) is a common aging phenomenon in the elderly population. Nevertheless, pre-mortem diagnosis of SSA is rare. Thus, data on clinical characterization and disease severity are limited. METHODS 36 consecutive SSA patients (71.6 [64.7-82.7]years) were evaluated by electrocardiography, echocardiography, laboratory tests, and (99m)Technetium-3,3-diphosphono-1,2-propanodicarboxylic acid ((99m)Tc-DPD) scintigraphy (n=20). RESULTS In addition to cardiac involvement, amyloid deposition was found in rectum (n=6), peripheral nerves (n=2), and urinary bladder (n=2). Five patients showed low voltage pattern. Thickness of interventricular septum (IVS) was 20 [12-27]mm. LV longitudinal function was diminished (TDI-s 5 [3-11] cm/s; MAPSE 6.5 [2.5-19]mm; TAPSE 12.5 [2-24]mm). LV systolic function (LV-EF<45%) was markedly decreased in 19 patients. Plasma levels of troponin T (0.05 [0.01-0.23]µg/L) and NT-proBNP (4318 [205-16597]ng/L) were elevated. (99m)Tc-DPD heart retention was 7.8 [2.4-11.0]% and correlated with MAPSE (ρ=-0.716; p=0.0018), TAPSE (ρ=-0.491; p<0.05), and IVS (ρ=0.556; p=0.0153). Heart-to-body ratio correlated with MAPSE (ρ=-0.771; p=0.0018), IVS (ρ=0.603; p=0.0086). Twelve patients died during follow-up of 27.4 [0.1-106.2]months. Exclusively (99m)Tc-DPD heart retention, diastolic dysfunction and in trend MAPSE were associated with patients outcome. Interestingly, risk predictors that were well established in patients with AL amyloidosis were not predictive for survival in patients with SSA. CONCLUSIONS This study gave first evidence that (99m)Tc-DPD HR may be capable to display the extent of cardiac amyloid deposition. Moreover, this study suggested that (99m)Tc-DPD HR, diastolic dysfunction and in trend MAPSE are associated with poor outcome. Nevertheless, these findings need to be established in a larger prospective trial.

Electrically neutral microheterogeneity of human plasma transthyretin (prealbumin) detected by isoelectric focusing in urea gradients

Mutants of the human plasma transthyretin (TTR, prealbumin) have attracted interest due to their rather frequent association with the autosomal dominant disease familial amyloidotic polyneuropathy (FAP). Some three quarters of known TTR mutations produce electrically neutral amino acid substitutions undetectable via separation by charge. We have developed an electrophoretic procedure sensitive to differences in the stability of tetramers and monomers under partially denaturing conditions. The differential folding states were found to be fully reversible. Applying the procedure we found 14 electrically silent mutants of TTR among 2 000 plasma samples from German donors. We demonstrate that the normal TTR monomer exists in different forms of variable stability and/or charge due to binding of sulfhydryls from plasma to the unique cysteine at position 10 of the primary structure as well as due to modification by treatment with an oxidant. We found that reduction of Cys10 increases the stability of the folded monomeric and tetrameric conformations. The conformational changes of TTR induced by isoelectric focusing in a urea gradient were found to be associated by a gain of three positive charge units. Using published crystallographic data we present structural sites in the TTR molecule which could explain the observed effects.

Transthyretin Leu 68 in a form of cardiac amyloidosis

SummaryA form of transthyretin (TTR)-related cardiac amyloidosis was previously described in a German patient. Electrophoretic analysis of plasma TTR showed the presence of an electrically neutral variant. We have now characterized the variant transthyretin by comparative peptide mapping, aminoacid and DNA sequencing procedures. A new mutation in TTR with a substitution of leucine for isoleucine at position 68 of the monomer is described.

Homozygosity and heterozygosity for the transthyretin Leu64 mutation: clinical, biochemical and molecular findings.

Transthyretin gene point mutations cause hereditary amyloidosis with an autosomal dominant pattern of inheritance. The disease usually manifests itself in heterozygous patients, although a few homozygotes have been reported. We describe two unrelated patients carrying the Leu64 mutation, one of whom presents a homozygous genotype (Family B). Homozygosity was confirmed by sequence analysis, RG‐PCR and double one‐dimensional electrophoresis of the plasma protein. Although the clinical picture of the homozygous patient of Family B was more severe than that shown by the heterozygous members of Family A, the variability often displayed by FAP patients does not allow any firm conclusion about the role of homozygosity in the seriousness of the disease.

Feto-maternal transfusion after chorionic villus sampling

SummaryThe alphafetoprotein (AFP) concentration in maternal serum was determined before and after chorionic villus sampling (CVS). A significant increase of 20% or more in the pre-CVS level was noted immediately after sampling in 59% of 837 pregnancies indicating some degree of feto-maternal haemorrhage. The increase in the AFP concentration in maternal serum was correlated with the weight of the tissue sample but not with the number of sampling attempts. A correlation of AFP increase and frequency of spontaneous abortions following CVS was suggested only in the group with an AFP increase of more than 100% or with a continuing rise in the first hour following CVS. CVS in early pregnancy obviously did not interfere with maternal serum AFP screening for neural tube defects in the second trimester. Although AFP measurement before and after CVS seems to have no immediate diagnostic application, in the research phase of CVS it may help to identify those procedures that are the least traumatic.