Seven patients with unexplained anemia and mild thalassemic features were ascertained during a survey of hemoglobinopathies in the Sekong Province in South Laos. These patients belong to the Austroasiatic (Mon-Khmer) population of South Laos (official designation Lao Theung). Hemoglobin electrophoresis on cellulose acetate showed absence of Hb A and two bands in the positions of Hb E and Hb F respectively. Sequencing of DNA isolated from venous blood revealed the codon 26 G-->A mutation characteristic of the HBB*E gene, but none of the common Southeast Asian beta-thalassemia mutations were found. Detailed studies in four of the seven subjects identified a 12.5 kb deletion encompassing part of the delta-globin gene and the entire beta-globin gene. We conclude that this deletion is a common, and possibly the predominant beta-thalassemia mutation of the Austroasiatic Lao Theung population. Similar deletions reported in single individuals in Laos, Thailand and Vietnam are probably due to migrational spreading to areas adjacent to South Laos.

AbstractBackground: Some of the genetic variants of hemoglobin (Hb) and their chemically modified species are known to affect the measurement of Hb A1c. The purpose of this study was to characterize Hb species in the blood sample of a 74-year-old German male with an exceptionally low Hb A1c value.Methods: Hemolysates from the propositus and a healthy individual were analyzed by electrophoresis, cation-exchange HPLC, boronate affinity chromatography, and electrospray ionization-mass spectrometry (ESMS). Genomic DNA was amplified by PCR, and the sequencing was performed on an ABI 310 sequencer. Functional properties of Hb were determined by oxygen equilibrium studies and CO recombination kinetics after flash photodissociation. Glycohemoglobin species were synthesized by incubating hemolysates with glucose.Results: A novel, electrophoretically silent β chain, β5(A2)Pro→Ala or Hb Görwihl, was detected by cation-exchange HPLC. It accounted for ∼44% of the total Hb and had functional properties similar to those of normal Hb A and a mild degree of heat instability. During incubation with glucose, glycation of the β chains (assessed by ESMS) in the hemolysate of a healthy volunteer was twice as fast as in hemolysate from the propositus.Conclusions: The substitution β5(A2)Pro→Ala seems to affect neither the functional properties nor the heterotropic interactions of Hb, but slows glycation of the N-terminal valine by an unknown mechanism.

A deletion/insertion in the human factor VIII gene was found in a patient with severe hemophilia A; 316 bp were removed, viz., those enclosing part of intron 15 and the first 7 bp of exon 16. In addition to the deletion, 6 bp were added to the deletion breakpoints; this resulted in the duplication of an existing 13-bp unit. Thus, an overlapping 13-bp direct repeat was generated at the deletion junction. Moreover, the deleted fragment itself was flanked by two homologous 6-bp sequences, one unit being lost by the deletion. A combination of slipped mispairing during replication and an intragenic recombination is discussed to describe this deletion/insertion process.

The β‐thalassemia mutations of 13 unrelated heterozygous Germans who remained unidentified in a previous study of 40 subjects were investigated at the DNA level. Two Mediterranean, one Asian and three novel mutations (CD6 ‐G, CDs 108 /112‐12nt, CDs 130/131 +GCCT) were identified. Altogether, in 30 of the 35 subjects (86%) in which a mutation in the β‐globin gene was identified, the mutation was of Mediterranean origin. The geographical distribution suggests recent migration from the Mediterranean region as cause of the high proportion of frequent Mediterranean β‐thalassemia mutations in the German population. Our results support the notion that the majority of β‐thalassemia genes in the western and central European population are of Mediterranean origin. © 1999 Wiley‐Liss, Inc.

Allelic frequencies for up to five short tandem repeat systems (HumTH01, HumVWA, HumF13B, HumCD4, HumD21S11) were analyzed in seven population samples from Asia using the polymerase chain reaction and gel electrophoresis. No deviations from Hardy-Weinberg equilibrium were observed. Two new alleles of the CD4 and TH01 loci were detected, and sequenced and their molecular structure is presented. A phylogenetic tree based on Thai, Han Chinese (from the northeast of China), Japanese, German and Ovambo allelic frequencies was constructed and demonstrates the close relationship of the Asian populations. Additionally, allelic frequency data for the VWA and TH01 systems were determined for the south Chinese minorities Bai, Dai and Qiang and for Koreans and compared with the above data. The Bai and Dai populations were clear outliers of the cluster of all other Asians, indicating an unexpected pattern of genetic heterogeneity of the Chinese nation. Two clusters of Asian populations could be established: the Koreans and Japanese together with the Han and Qiang Chinese, and, forming a separate cluster, the Bai and Dai populations.

Mutational analysis of the gene for clotting factor VIII is complicated by its large size, the high frequency of de novo mutations and its tissue-specific expression. In order to facilitate the search for mutations, we have used a combination of reverse transcription-polymerase chain reaction (RT-PCR) of ectopic factor VIII transcripts, PCR of genomic DNA, single-strand conformation polymorphism analysis and direct sequencing. Here we describe the characterization of seven potentially pathogenic mutations: five of them are novel and the reason for the pathogenicity of the sixth could be determined. Here cDNA analysis revealed the absence of the first 47 bp of exon 16 in approximately 80% of the processed factor VIII mRNA, likely due to activation of a cryptic acceptor splice site within exon 16. The other novel mutations reported here include the skipping of exon 19, which predicts the removal of the corresponding 39 amino acids from the A3 domain, and four missense mutations: W14G, Y620C, W1889L, and Q2087R.

A 28 year old man and his 27 year old wife were investigated for infertility of 3 1/2 years' duration. There was azoospermia caused by bilateral aplasia of the vas deferens, and therefore it was planned to aspirate spermatozoa from the epididymis for the purpose of in-vitro fertilisation. As part of the diagnostic workup the man was investigated for those mutations of the cystic fibrosis transmembrane regulator (CFTR) gene which occur with undue frequency in association with aplasia of the vas deferens. Deoxyribonucleic acid (DNA) analysis revealed a typical three base deletion (DELTAF 508). In the wife, CFTR gene mutations were excluded with 80% probability. The likelihood of cystic fibrosis in this couple's children was accordingly estimated to be about 0.2% - an acceptable risk. Assisted fertilization in patients with bilateral aplasia of the vas deferens should not be undertaken until they have been thoroughly investigated and informed of the risks.

To investigate the molecular basis in phenylketonuria (PKU) in the population of southern Poland we characterized the restriction lenght polymorphism (RFLP) haplotypes at the phenylalanine hydroxylase gene locus, screened for mutations with allele-specific oligonucleotides by amplification of the genomic DNA with the polymerase chain reaction and sequenced the exons of the PAH gene from 23 PKU families. 71.7% of all PKU alleles are characterized by the following five mutations: codon R408W, R261Q, R158Q, R252W and the splicing defect in intron 10. The splice mutation in intron 10 was detected by DdeI restriction analysis. In the investigated families the overall frequency for predictions in regard to the genotype of additional offspring was about 96%. With an overall frequency of about 98% the DNA diagnostic approach could be used to predict a phenotypically normal child (genotype: either normal/normal or normal/mutant).

In order to investigate the molecular basis of phenylketonuria (PKU) in the Polish population, we screened 44 mutant chromosomes from PKU probands for six known mutations, frequently occurring in western European countries, by polymerase chain reaction amplification of their genomic DNA and hybridization with allele-specific oligonucleotides. Our results show that the majority (66%) of all PKU alleles are characterized by three different mutations: in codon 408 (56.8%), codon 158 (6.8%) and codon 261 (2.27%). Of the mutant haplotype 2 alleles, 96% were linked to the mutation in codon 408. Out of five mutant haplotype 4 alleles, three showed the codon 158 mutation, and out of four mutant haplotype 1 alleles, one had the codon 261 mutation. In two families, MspI digests revealed an additional 13.5-kb band similar in length to that previously reported. However, analysis of exon 9 excluded the presence of the T to C transition originally described, indicating a new MspI variant in the Polish population.

Out of a population of 138598 infants born in Southern Poland between 1987 through 1989, and screend for PKU, 22 cases were ascertained. DNA from 22 probands and their parents was isolated from peripheral blood leukocytes. Eight restriction enzymes were used (Bgl II, Pvu II, Eco RI, Msp I, Xmn I, Hind III and Eco RV). Polymorphic restriction sites were analyzed with the cDNA probe pH PAH 247. The mutation of codon 408 (exon 12, C … T, Arg … Trp) was tested on amplified DNA by dot-blot hybridization with 32P - labeled allele -specific oligonucleotides. Eight RFLP sites of the PAH gene were defined in 88 parental chromosomes, revealing 21 different haplotypes (HT). Among those, 16 have been already described, 5 - represented the new ones. The most common HT among those carrying normal alleles were: HT1 (27.3%) and HT4 (11.4%). Within the group of HT with mutant allele the most frequent was HT2 (56.8%) whereas the frequency of the same HT in French, Danish and German populations ranged from 12% to 24%. HT3, being the most common in Danish (38%) and relatively frequent in other western European populations (13-14%) appeared to be very rare in our sample (2.3%). The mutation in codon 408 was found in 25 out of 44 probands' chromosomes. Only one mutation was carried by HT5, the remaining 24 were associated with HT2. Our results confirm molecular heterogeneity of PKU haplotypes, as well as their significant interpopulation variation.

AbstractAcrylamid (1) und p‐Nitrophenylacrylat (2) lassen sich mit Azoisobuttersäuredinitril in Dimethylsulfoxid in jedem molaren Verhältnis polymerisieren. Je nach Reaktionsbedingungen entstehen Copolymere, deren mittlerer Polymerisationsgrad zwischen 200 und 500 liegt. Dabei wird 2 zu Beginn der Polymerisation etwas bevorzugt eingebaut. Die Copolymeren sind in Dimethylsulfoxid sehr gut löslich und zeichnen sich durch ihre hervorragende Reaktivität gegenüber primären aliphatischen und aromatischen Aminen aus. So werden sie durch Behandlung mit trockenem Ammoniak in wenigen Minuten glatt in die entsprechenden Polyacrylamide überführt.

Background: Some of the genetic variants of hemoglo- bin (Hb) and their chemically modified species are known to affect the measurement of Hb A1c. The pur- pose of this study was to characterize Hb species in the blood sample of a 74-year-old German male with an exceptionally low Hb A1c value. Methods: Hemolysates from the propositus and a healthy individual were analyzed by electrophoresis, cation-exchange HPLC, boronate affinity chromatogra- phy, and electrospray ionization-mass spectrometry (ESMS). Genomic DNA was amplified by PCR, and the sequencing was performed on an ABI 310 sequencer. Functional properties of Hb were determined by oxygen equilibrium studies and CO recombination kinetics after flash photodissociation. Glycohemoglobin species were synthesized by incubating hemolysates with glu- cose. Results: A novel, electrophoretically silent chain, 5(A2)Pro3 Ala or Hb Gorwihl, was detected by cation- exchange HPLC. It accounted for 44% of the total Hb and had functional properties similar to those of normal Hb A and a mild degree of heat instability. During incubation with glucose, glycation of the chains (as- sessed by ESMS) in the hemolysate of a healthy volun- teer was twice as fast as in hemolysate from the propos- itus. Conclusions: The substitution 5(A2)Pro3 Ala seems to affect neither the functional properties nor the het- erotropic interactions of Hb, but slows glycation of the N-terminal valine by an unknown mechanism.