The short tandem repeat loci hTPO, THO1 and FGA.

This paper focuses on the three well-established short tandem repeats HumhTPO (human thyroid peroxidase gene), HumTHO1 (human tyrosine hydroxylase gene) and HumFGA (human alpha fibrinogen gene). We present sequencing data which include the sequences of all the different primer pairs used so far, as well as results from a population study which was carried out on an American Black and a southwestern German population. All data are compared with previously published studies. A test for homogeneity confirmed that non-European populations possess different genetic pools for each of the three loci under investigation.

The short tandem repeat locus D3S1359.

Short tandem repeats (STRs) are used by many laboratories throughout the world performing paternity testing or criminal casework. Nevertheless, many of the established STRs have obvious disadvantages such as low number of common alleles (e.g., hTPO, THO1) or alleles with frequencies of nearly 50% (e.g., hTPO, FES). In this paper the new STR locus D3S1359 is described. In a population study which was carried out on 136 unrelated individuals from southwestern Germany, we have detected 17 different alleles. The most common allele was allele 13 (204 bp) with a frequency of 18.8%. Eight further alleles have frequencies higher than 5%. With a heterozygozity index of 90% and 60 different genotypes, D3S1359 has shown to be a highly polymorphic and informative marker. Sequencing data of this STR locus revealed further variation.

D3S1358: sequence analysis and gene frequency in a German population.

Eight alleles of the STR system D3S1358 were observed and sequenced. The alleles ranged in size from 119 bp (13 repeats) to 147 bp (20 repeats) and consist of two diverse tetranucleotides: [AGAT] and [AGAC]. Alleles 16 and 17 show basic repeats which are different in one base. The allele frequency of 499 unrelated persons from SW-Germany yielded no deviation from Hardy-Weinberg equilibrium. A comparison with a Portuguese population showed some small differences, i.e. one allele has not been found in the Portuguese sample until now. The mutation rate was estimated with 0.8% analysing 65 families.

Typing of the short tandem repeat D8S347 locus with different fluorescence markers.

The short tandem repeat (STR) locus D8S347 was analyzed by capillary electrophoresis. Sequencing data and a population study of 203 individuals from a southwestern German population are presented. We detected 12 different alleles, 340-388 bp in length, and found 40 different genotypes. The heterozygosity index was 85.7%. Futhermore, we investigated the consequences of different fluorescent dyes, namely 6-FAM, HEX, and ROX, on the ABI-calculated fragment sizes of defined (i.e., sequenced) alleles (348-376 bp in length). 6-FAM-labeled fragments appear to be smaller than the corresponding HEX- or ROX-labeled fragments. On average, 6-FAM-labeled fragments differ by 3.52 bp from the sequencing data, HEX-labeled ones by 2.04 bp, and ROX-labeled ones by 1.42 bp. Generally, small alleles differ less from the expected sequencing data than larger ones.

Comparison of German population data on the apoB-HVR locus with other Caucasian, Asian and black populations.

A population study of 505 unrelated individuals from Southwestern Germany was carried out on the 3'-apoB hypervariable region (HVR). After amplification via polymerase chain reaction (PCR) and agarose gel electrophoresis, 15 different alleles and 47 genotypes were observed. The most common alleles were hypervariable elements (HVE) 37 and 35 with an allele frequency of 0.374 and 0.244, respectively. The heterozygosity index was calculated to be 78.4%. Allele frequencies of this study are compared with results from other databases obtained from a French, a Spanish, an Asian and an American (Black) population.

Detection of two hypervariable (ATTTT)n loci in the human genome.

Object of this investigation was the isolation of a single-locus probe from a multi-locus fingerprint. Individual specific multi-locus fingerprints in man were generated by using the oligonucleotide probe (ATTTT)5. An isolated (ATTTT)5-positive DNA fragment was analyzed using polymerase chain reaction (PCR) cycle-sequencing and nonradioactive direct-blotting electrophoresis. A digoxigenated oligonucleotide synthesized according to this sequence was used as a single-locus probe. Two hypervariable loci were detected on Southern blots. Formal genetic investigations for the two loci were performed in order to estimate the allele frequencies. Locus 1 shows an individual-specific banding pattern with an autosomal-codominant inheritance and can be used for forensic investigations. Locus 2 also represents a polymorphic pattern, but the inheritance is not according to the Mendelian rules. Probably we have detected a highly mutagenic locus in the human genome.

TGGE and HIEF: a comparison of two methods in the detection of carriers of the Z mutation in the alpha-1-antitrypsin gene.

Alpha-1-antitrypsin (alpha-1-AT) deficiency can lead to juvenile liver cirrhosis and lung emphysema in adulthood. The deficiency Z allele is caused by a base transition. Temperature gradient gel electrophoresis (TGGE) and hybrid isoelectric focusing (HIEF) were used to detect carriers of the Z mutation of the alpha-1-AT gene. The resulting data were compared. To verify carriers at the sequence level, a manual nonradioactive sequencing strategy was established. Among our sample of carriers of the Z mutation, two were not detected by HIEF that could be identified by TGGE. DNA of all TGGE identified individuals harboring the Z mutation of the alpha-1-AT gene were sequenced nonradioactively. All carriers harbored a G to A transition at position 11.940. This mutation is described to cause the altered protein.

Restriction fragment length polymorphism: molecular weight analysis and calculation with a scanner-based computer system.

For an exact and reproducible molecular weight calculation, an essential requirement in all applications of DNA profiling, we present a new system, called RFLP-MAC (restriction fragment length polymorphism-molecular weight analysis and calculation). The system is composed of a sensitive digital gray-scale scanner unit and an Apple Macintosh computer supported by a powerful combination of a shareware and self-developed software package. Molecular weights are calculated by profiling the optical density and band particle processing parameters to identify the unknown DNA fragments. Mathematical models were used to interpolate the transverse and horizontal migration directions of the molecular weight standards, insuring consistent results from gel to gel. All data are stored in a relational multi-user database system. Descriptive statistics are performed and allele frequencies are analyzed. Procedures for image-analysis, molecular weight calculation, statistical evaluations and database management were developed and run within the multitasking environment of 4th Dimension. Two sets of data, intragel and intergel measurements, are investigated in order to check the precision of the RFLP-MAC system. All these data are subjected to a standard analysis of variance resulting in a maximal 3 SD value of 0.005 (0.288%) for the intragel and of 0.091 (1.654%) for the intergel variation.

Subtyping of alkylated human orosomucoid: evidence for a duplicated gene, ORM1*F2S.

Isoelectric focusing of human orosomucoid (ORM) was studied following different sample treatment. It is shown that: (i) alkylation with iodoacetamide leads to a drastic change in the isoelectric point (pI) of both ORM1 F2 and ORM2 A gene products and greatly improves the discrimination between ORM1 F1 and ORM1 F2; (ii) previous reduction of the molecule with dithiothreitol partially inhibits the pI transitions with resultant artifactual ORM1 F1F2S patterns that correspond in most cases to F2S phenotypes. With the technique now described, the persistence of three ORM1 gene products was found in only one individual and the segregation analysis is consistent with the existence of a rare ORM1*F2S haplotype.

Genetic polymorphism of the single locus probes pL159-1 and pL355-8.

The genetic polymorphism of the single-locus probes pL159-1 (D18S17) and pL355-8 (D20S15) was investigated in 445 unrelated individuals using PstI as restriction enzyme. Fragment size calculations were obtained using the molecular weight size marker MW-SBH. The basic relationship between migration distance and molecular weight was transformed using an exponential function. Fragment size frequency data show 2 peaks for pL159-1 at 4.36kb (2.36%) and 4.67 kb (6.29%) and one peak for pL355-8 at 6.04 kb (5.73%). The rate of heterozygosity exceeded 70% for both probes.

Genetic studies of antithrombin III with IEF and ASO hybridization.

Antithrombin III (AT III) was analyzed by two different methods. Isoelectric focusing was used to screen 3 different populations (southwest Germans, Portuguese, Xavante Indians). The same variant was detected both in the German and the Portuguese populations with frequencies of 0.007 and 0.00024, respectively. Further characterization of this variant was performed by allele specific oligonucleotides. By this means, it was possible to identify the variant as AT III Dublin, originally found in 4 Irish families.

Isoelectric focussing of human thyroxine binding globulin (thyropexin) and human prealbumin (transthyretin).

Two batches of the highly purified thyroid hormone-binding plasma proteins, human thyropexin and transthyretin, which were prepared in gram quantities for use in animal experiments, were subjected to analysis by isoelectric focussing. Under these conditions, it was observed that human transthyretin was composed of two components. This was presumably due to the use of 8 mol/l urea. The preparations of both human transthyretin and human thyropexin contained some products of decomposition which probably arose in the course of the purification processes and, in addition, possibly also contained some normal genetic variants of human thyropexin. In spite of the alterations, both protein preparations largely retained their thyroid hormone-binding capacity, which is essential for in vivo studies on the re-entry of thyroid hormones from the extravascular space into the circulation. For therapeutic use in thyrotoxicosis, human transthyretin seems to be preferable to human thyropexin.

Temperature gradient gel electrophoresis: rapid detection of alpha-1-antitrypsin deficiency carriers.

The homozygous state of the alpha-1-antitrypsin (alpha 1AT) deficiency variant Z is associated with severe liver damage in early childhood and progressive lung emphysema in adulthood. A single base transition (G to A in codon 342) in exon V is causing the severe disease. The Glu342 to Lys342 mutation can be detected conventionally by isoelectric focusing (IEF) or on the DNA level by the newly developed method of temperature gradient gel electrophoresis (TGGE). It is the aim of this study to describe the TGGE technique, to compare the results with conventional IEF, and to discuss its efficiency for different diagnostic applications.

Orosomucoid (ORM 1) subtyping and formal genetics.

Orosomucoid (ORM) phenotyping has been performed in 141 families with 407 children from southwest Germany. Eight families were observed in which duplicated ORM1 genes, F1F2, F1F3, segregated. The family data gave no information about the presence of tandemly duplicated ORM1 F1F4 and ORM1 F1F5 genes. To date, the segregation of the phenotypes of the children agrees with the extended formal model: two ORM1 loci with two common (*F1, *S) and several rare (*F1F2, *F1F3, *F4, *F5) alleles. The parental allele frequencies were calculated by gene counting as ORM1 *F1 = 0.5781, *S = 0.3901, *F1F2 = 0.0195, *F4 = 0.0053, *F1F3 = 00.0035, *F5 = 0.0035.

Genetic polymorphism of inter-alpha-trypsin-inhibitor (ITI): formal genetic and linkage analyses.

The polymorphism of inter-alpha-trypsin-inhibitor, ITI, was demonstrated by isoelectric focusing in agarose gels (pH 5-8) followed by protein blotting and immunoassay. Segregation in 239 families with 677 children is consistent with the formal hypothesis that there are two common codominant alleles, ITI*1, ITI*2, and one rare codominant allele, ITI*3, at an autosomal locus ITI. Allele frequencies were calculated as ITI*1 = 0.600, ITI*2 = 0.393, ITI*3 = 0.007. Linkage analysis with 36 markers is presented. Slightly positive lod scores were obtained for PGM3 (zeta = 1.35, theta = 0.10) and AK1 (zeta = 1.34, theta = 0.10).

Two different multi-locus probes MZ1.3 and (CAC)5 show nearly the same RFLP pattern.

The RFLP patterns revealed by 2 different multi-locus probes MZ1.3 and (CAC)5 were compared using 4 different restriction enzymes AluI, MboI, HaeIII, HinfI. Irrespective of the restriction enzyme the fingerprints obtained with MZ1.3 and (CAC)5 were almost identical. The MZ1.3 RFLP pattern showed some extra bands which were absent in the (CAC)5 fingerprint.

Genetic studies on human thyroxine-binding globulin (TBG).

An enzyme immunoassay technique combined with Western blotting is described to demonstrate thyroxine-binding globulin (TBG) by isoelectric focusing in thin-layer polyacrylamide gels with 8 mol/l urea. Quantitative evaluation was by laser densitometry. No genetic charge variants of TBG were encountered in a sample of 840 unrelated individuals from southwestern Germany. There was no correlation between structural and quantitative variations in the TBG protein. Results from a family with quantitative TBG deficiency strongly support the postulated X-linked mode of inheritance. The method described can be considered as an additional diagnostic tool in thyroid evaluation.

Linkage analyses of human peptidase C (PEPC), human factor H (HF), and coagulation factor XIIIB (F13B).

Linkage data on human peptidase C (PEPC), human factor H (HF), and coagulation factor XIIIB (F13B) are presented. The results confirm linkage between HF and F13B (lod = 5.32 at theta = 0.10 in males), and give strong evidence for linkage between PEPC and HF (lod = 5.14 at theta = 0.10 in males) and between PEPC and F13B (lod = 3.55 at theta = 0.10 in males). The claim that PEPA is linked with HF must be withdrawn.

Electrophoretic and isoelectric focusing studies in Brazilian Indians: data on four systems.

Three-hundred ninety-nine individuals living in seven populations of two Brazilian Indian tribes (Macushi and Içana River Indians) were tested for the phosphoglucomutase 1 (PGM1), properdin factor B (BF), haptoglobin (HP), and alpha-1-antitrypsin (PI) systems. We observed significant internal heterogeneity in the two tribes for the PGM1 alleles and in the Macushi for the HP markers. Frequencies in three of the four systems (the exception being BF) also show clear differences in the Macushi and Içana River Indians. Compared with other ethnic groups, South American Indians generally present high frequencies of PGM1*1B, BF*S, HP*1S, and PI*M3. On the other hand, PGM1*1A, PI*M1, and PI*M2 are reduced, and HP*1F is absent or rare. This is the first report about HP subtypes among American Indians.