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Pyruvate kinase deficiency

Pyruvate Kinase Deficiency is an inherited autosomal recessive disorder which affects the survival of red blood cells, causing them to break down easily. This leads to anemia and liver failure among affected dogs. more...

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Pyruvate kinase deficiency

This inherited disease is characterised by signs of anemia, low energy, exercise intolerance, jaundice, yellowing of the white part of the eye, and persistent gallstones. Treatment can include a blood transfusion or removal of the spleen. Treatment is usually effective in reducing the severity of the symptoms, but most canines affected with PK are likely to die between ages 1 and 4.


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Population genetics of four PKLR intragenic polymorphisms in Portugal and Sao Tome e Principe (Gulf of Guinea)
From Human Biology, 6/1/01 by Manco, Licinio

Abstract Four intragenic PKLR polymorphisms [1705A/C, 1738C/T, T10/19, and (ATT)n microsatellite] were studied in normal population samples of Central Portugal and Sdo Tome Principe, a small archipelago located in the Gulf of Guinea, West Africa. For all loci, the observed genotype distributions do not deviate from Hardy-Weinberg equilibrium. The allele frequencies found in the Portuguese population are similar to those previously described in Caucasian populations. Mother-child pair analysis for the (ATT)n microsatellite does not show deviations to the Mendelian rules. In Sao Tome Principe the biallelic polymorphisms 1705A/C, 1738C/T, and T10/19 presented inverse allelic frequencies when compared with the Portuguese population. Two new alleles were found at the (ATT)n microsatellite. Significant statistical differences were found between both populations. The results showed that Sao Tomeans had higher haplotype diversity and lower linkage disequilibrium among the polymorphic sites. The PKLR intragenic polymorphisms, commonly used in haplotype analysis with the gene mutations in PK-deficient patients, can thus be successfully employed in anthropological genetics.

Pyruvate kinase (PK; EC catalyzes the conversion of phosphoenolpyruvate (PEP) to pyruvate in the glycolytic pathway enabling the synthesis of ATP. In man, four tissue-specific isoenzymes exist (M1, M2; L and R) encoded by two genes: PKM, which codes for M1 and M2 isoenzymes (muscle), and PKLR, which codes for L (liver) and R (erythrocyte) isoenzymes using two different tissue-specific promoters (Noguchi et al. 1987). Mutations on the PKLR gene, located on the long arm of chromosome 1 (1q21), are responsible for erythrocyte PK deficiency. The clinical consequence of the enzyme deficiency is chronic nonspherocytic hemolytic anemia with heterogeneous phenotype and autosomal recessive inheritance (Valentine et al. 1989).

The complete genomic sequence of the human PKLR gene has been determined (Lenzner et al. 1997). It has a total length of 8409 nucleotides organized into 12 exons and 11 introns. Several PKLR intragenic polymorphisms have been described: IVS5+51C/T (Fujii et al. 1995; Baronciani et al. 1995; van Solinge et al. 1997); 1705A/C (Kanno et al. 1992); 1738C/T (Zannela et al. 1997; van Solinge et al. 1996); 1992C/T (Lenzner et al. 1997); the oligo T10/19 in intron 10 (Lenzner et al. 1997); and the (ATT)n microsatellite in intron 11 (Lenzner et al. 1994a). These polymorphisms are currently used in haplotype analysis with the PKLR gene mutations in PK-deficient patients. The aim of this work was to estimate allele frequencies of four PKLR intragenic polymorphic sites [1705AIC; 1738C/T; TJO/19; (ATT)n] in Portugal and in Sdo Tome Principe archipelago, a former Portuguese colony in the Gulf of Guinea (West Africa). The Sdo Tome Principe islands were not inhabited permanently up to the late 15th century. The first settlers were European, but sub-Saharan Africans of various geographic origins provided the main demographic input, since Sao Tome was very important in the trade and transshipment of slaves. Present population size is around 100,000 inhabitants. European admixture is very low when judged by conventional (Santos et al. 1996) or molecular autosomal markers (Gusmao et al. 1997; Miranda et al. 1998; Pereira et al. 1999) or from mitochondrial DNA (Mateu et al. 1997).

Along with population and haplotype data analysis, we present the first results on the segregation analysis of the (ATT)n microsatellite. A brief comparative study with other European and sub-Saharan African populations was also performed.

Material and Methods

Samples from unrelated normal individuals and mother-child pairs from Central Portugal and unrelated individuals born and living in Sao Tome Principe were examined for the PKLR intragenic polymorphic sites T10/19 in intron 10, (ATT)n in intron 11, and 1705A/C and 1738C/T in exon 12.

The T10/19 polymorphic marker was studied by polymerase chain reaction (PCR) amplification with the sense oligonucleotide 5'-CTGTGCCTGGCCAGCTTTTA-3' and the antisense 5'-GAGACCAGCCTAGGCAACAT-3'. PCR products were analyzed in 10% horizontal polyacrylamide gels (Luis and Caeiro 1995). After electrophoresis, DNA fragments were silver-stained according to the method of Budowle et al. (1991).

For the study of the (ATT)n microsatellite, PCR amplification was performed using the sense oligonucleotide described by Lenzner et al. (1994a) and the antisense from Baronciani and Beutler (1995). The PCR products were digested with Hinfl and separated by horizontal electrophoresis on polyacrylamide gels (Luis and Caeiro 1995), using samples with a known number of ATT repeats as controls. Bands were visualized by silver staining (Budowle et al. 1991).

The polymorphic sites 1705A/C and 1738C/T were studied by PCR amplification of exon 12, using oligonucleotides described by Lenzner et al. (1994b). DNA fragments were submitted to restriction enzyme analysis with BspHI for 1705A/C polymorphism (Kanno et al. 1992) and BseRI for 1738C/T polymorphism (Zannela et al. 1997) and separated in an ethidium bromide agarose gel (2%).

Allele frequencies were estimated by gene counting. Haplotype frequencies, linkage disequilibrium, expected heterozygosity, and statistical analysis to test conformity with Hardy-Weinberg expectations were performed using the statistical software package ARLEQUIN (Schneider et al. 1997).

Results and Discussion

Allele Frequencies. The genotype and allele distributions of the four PKLR intragenic polymorphisms in the populations of Central Portugal and Sdo Tome Principe are displayed in Tables 1 and 2. The observed genotype distributions do not showed deviations from Hardy-Weinberg equilibrium.

In the Portuguese population, the biallelic systems showed frequencies of 0.77, 0.77, and 0.78 for the most prevalent alleles 1705C, 1738T, and T10, respectively (Table 1). The (ATT)n microsatellite showed the presence of seven different alleles spanning from 12 to 19 repeats. We did not find the rarer alleles ATT11 and A7T18 previously reported (Baronciani and Beutler 1995; Lenzner et al. 1997). Allele ATT14 was the most common with a frequency of 0.49 followed by alleles ATT15 and ATT12 with a frequency value of 0.19 and 0.13, respectively (Table 2). The four polymorphic sites showed similar allele frequencies to those previously reported in other Caucasian populations (Glenn et al. 1994; Baronciani and Beutler, 1995; van Solinge et al. 1996; Lenzner et al. 1997). No exclusions were detected in 61 mother-child pairs analyzed for the (ATT)n microsatellite.

In Sao Tome e Principe all the biallelic systems showed inverse frequencies relative to those obtained for the Portuguese population. Allele frequencies of 0.61, 0.61, and 0.64 were found for the most common alleles 1705A, 1738C, and T19, respectively (Table 1). Regarding the (ATT)n microsatellite, 10 different alleles were found. Allele 12 was the most frequent (0.38) followed by alleles 14 and 16 with frequencies of 0.16 and 0.14, respectively (Table 2). Two alleles, named 10 and 6 according to the mobility of the corresponding fragments, are reported here for the first time (Figure 1).

Exact test of differentiation between the populations of Portugal and Sao Tome Principe for all polymorphic sites showed highly significant differences (p = 0.000 +/- 0.000), confirming that Sao Tome has not suffered significant Caucasian genetic influence. In accordance, previous studies on mtDNA indicate that Sdo Tomeans share high levels of genetic diversity with the sub-Saharan African populations and can be considered a subset of a mainland African population (Mateu et al. 1997).

Significant statistical differences were found between Sao Tome Principe and a sample of Afro-Americans (Glenn et al. 1994) for the 1705A/C polymorphism (p = 0.0017 +/- 0.0003). Divergence between the two populations was also reported for other polymorphisms (Gusmao et al. 1997; Miranda et al. 1998), reflecting a different genetic background of the two populations, probably caused by distinct patterns of slave traffic during the 16th to 19th centuries.

Haplotype Analysis. Allelic associations between the four polymorphisms were analyzed in the two populations of Sdo Tom6 and Portugal. The tightly linked biallelic polymorphisms 1705A/C and 1738C/T (33 base pairs [bp] distant) and the T10/19 locus, 288 bp distant from the 1705A/C site, showed complete linkage disequilibrium in the Portuguese population. Only two haplotype combinations of these three polymorphic sites were found: a more common (77%) one with the polymorphism 1705C associated to 1738T and T10 (C/T/10 haplotype) and a less common (23%) one with the polymorphism 1705A associated to 1738C and T19 (A/C/19 haplotype). Strong linkage disequilibrium was also observed when analyzing the two haplotypes in conjunction with the (ATT)n microsatellite (chi^sup 2^ = 58.68, p = 0.022, 39 degrees of freedom [df]). The most common C/T/10 haplotype was combined with seven different ATT alleles spanning from 12 to 19 repeats, with the highest frequency (0.53) for the C/T/10/14 association. The rarer A/C/19 haplotype was always combined with the lower repeat-number ATT alleles 12 to 14.

Concerning the population of Sao Tome e Principe, complete linkage disequilibrium was also observed between the 1705A/C and 1738C/T polymorphisms (haplotypes 1705A/1738C and 1705C/1738T). A strong but not total association was present for combinations among these two biallelic systems and the T10/19 site (X2 = 62.06, p = 0.000, 13 df). The presence of four haplotypes, the more common A/C/19 (64%) and C/T/10 (20%) and the two new haplotypes C/T/I9 (13%) and A/C/10 (3%), suggest a crossover event in the region under consideration. These four haplotypes were combined with the (ATT)n microsatellite in 24 different ways. The A/C/19/12 haplotype was the most common with a frequency of 0.41, while the remaining 23 haplotypes showed a frequency ranging from 0.008 to 0.09 each. Regarding the haplotype pairs among the biallelic systems 1705A/C or 1738C/T and the (ATT)n microsatellite, we found 15 different haplotypes and ap value that was not statistically significant (chi^sup 2^ = 47.96, p = 0.738, 55 df). In the same way, linkage disequilibrium was not observed among the T10/19 polymorphism and the (ATT)n microsatellite, which presented 15 different haplotypes (chi^sup 2^ = 52.76, p = 0.817, 63 df).

The higher heterozygosity values obtained for all four single polymorphic sites in Sdo Tome Principe (Tables 1 and 2), together with the presence of more haplotypes and a weaker linkage disequilibrium, were concordant with previous reports on other DNA polymorphisms for Sao Tomeans (Mateu et al. 1997; Seixas et al. 1999) and other sub-Saharan populations (Bowcock et al. 1994; Scozzari et al. 1996; Tishkoff et al. 1996, 1998). The complete linkage disequilibrium at the biallelic intragenic PKLR polymorphisms observed in Portugal and Central Europe (Lenzner et al. 1997) suggests that the chromosomes containing the C/T/10 and A/C/19 combinations were the only ones present in the population of modern humans that left Africa and migrated to Europe.


The present study showed that the analysis of PKLR intragenic polymorphisms may be used not only for haplotype associations with the PKLR mutations on pyruvate kinase-deficient patients, but also for applications in anthropological genetics. The differences observed between both populations of Portugal and Sao Tome Principe on the four PKLR polymorphisms were concordant with the global pattern previously determined on genetic variation between African and non-African populations.

Acknowledgments This work was performed in part with the support of Instituto Ambiente e Vida (IAV) and Centro de Investigacao em Antropologia (CIA), Universidade de Coimbra.

Received 9 March 2000; revision received 18 July 2000.

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1 Departamento de Antropologia, Universidade de Coimbra, Coimbra, Portugal.

2 Unidade de Hematologia Molecular, Centro Hospitalar de Coimbra, Coimbra, Portugal.

3 Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), Porto, Portugal.

4 Faculdade de Ciencias, Universidade do Porto, Porto, Portugal.

Copyright Wayne State University Press Jun 2001
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