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Keywords:

  • SERPINA1 gene;
  • mutation age;
  • population studies

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Abbreviations
  8. Acknowledgments
  9. References
  10. Supporting Information

Alpha 1-antitrypsin (A1AT) deficiency, one of the most common inborn errors of metabolism in Caucasians, is characterized by a low serum concentration of A1AT and a high risk of pulmonary emphysema and liver disease. The allelic frequency for the most common protease inhibitor (PI) Z mutation in the SERPINA1 gene is 2–5% in Caucasians of European descent.

The objective of our study was to estimate the PI Z mutation age using molecular analysis in Latvian and Swedish populations, which have the highest frequency of PI Z mutation.

DNA samples of heterozygous and homozygous PI Z allele carriers from Latvia (n = 21) and Sweden (n = 65) were analysed; 113 unrelated healthy donors from Latvia were used as a control group. MALDI-TOF analysis was performed on all samples. Pairwise Fst was computed to compare the PI Z mutation ages between the two populations and controls. A p value less than 0.05 was considered significant.

Analysis of non-recombinant SNPs revealed that the PI Z mutation age was 2902 years in Latvia (SD 1983) and 2362 years in Sweden (SD 1614) which correlates with previous studies based on microsatellite analysis.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Abbreviations
  8. Acknowledgments
  9. References
  10. Supporting Information

Alpha 1-antitrypsin (A1AT) deficiency, one of the most frequent inborn errors of metabolism in Caucasians, is characterized by a low serum concentration of A1AT and a high risk of pulmonary emphysema and liver disease. The allelic frequency for the most common protease inhibitor (PI) Z mutation is 2–5% in Caucasians of European descent (American Thoracic Society/European Respiratory Society Statement 2003). The previously described gradient of Z mutation frequency shows that the highest rates occur in northern Europe and the lowest rates in southern Europe, suggesting that the mutation originated somewhere in northern Europe, most likely in Sweden (Blanco et al. 2006)

Hutchinson et al. (1998) reported that the frequency of the PI Z mutation is highest on the north-western seaboard of Europe and that the mutation seems to have arisen in the southern part of Sweden, probably about 14,000 years ago (Hutchison 1998).

Beckman et al. (1999) reported that the highest frequency of the PI Z allele identified to date is in the western part of Latvia (Courland) and that the Curonian population has a pronounced west European influence. This explanation was based on archaeological data on the settlement in Courland of people from Sweden and the island of Gotland after the seventh century and evidence of some degree of founder effect there (Beckman et al. 1999).

The most common A1AT deficiency allele PI Z is caused by a single base substitution in exon 5 of the normal M1 allele, which leads to the amino acid change of glutamic acid to lysine at position 342 of the protein (Cox 2001).

Cox et al. (1985) identified several polymorphic restriction sites for the SERPINA1 gene. Linkage disequilibrium was found with the SERPINA1 Z mutation, leading to the conclusion that this PI Z mutation occurs mainly in one haplotype. This indicates a single, relatively recent origin in Caucasians, which was predicted to be an individual who lived in northern Europe about 6000 years or 216 generations ago (Cox 2001, Cox et al. 1985, Cox et al. 1987. Using microsatellite analysis, Byth et al. (1994) recalculated the age of the PI Z allele to be 2000 years or 66 generations, but they did not eliminate the possibility of a high recombination rate within this region.

Others have analysed this unique haplotype of the PI Z allele initially described by Cox et al. (1985), and they have identified a number of common sequence variants in linkage disequilibrium with PI Z. Chappell et al. (2004) identified 16 SNPs throughout the SERPINA1 gene, nine of which had not been reported earlier and which represented significantly different PI Z variants.

The objective of our study was to estimate the PI Z mutation age for the Latvian and Swedish populations using molecular analysis of SNPs and to describe the possible genetic route of this mutation.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Abbreviations
  8. Acknowledgments
  9. References
  10. Supporting Information

DNA was extracted using the standard chloroform–phenol method (John et al. 1991) from unrelated PI Z mutation carriers. DNA samples of heterozygous and homozygous PI Z allele carriers from Latvia (n = 21) and Sweden (n = 65) were analysed. The Swedish individuals were identified and PI typed in the neonatal Swedish screening study 1972–1974 (Sveger et al. 1976). Unrelated healthy PI M (normal) allele carrier donors from Latvia were used as a control group (n = 113).

The data and sample collection was performed in accordance with the regulations issued by the Central Medical Ethics Committee of Latvia.

PI Z typing for the Swedish samples was performed in 1976 using isoelectric focusing within the neonatal screening program (Sveger et al. 1976).

A modified method of directed site mutagenesis PCR (Braun et al. 1996) was used for PI Z genotyping of the Latvian samples.

SNPs and Genotype Analysis

Chappell et al. (2004) described the most frequent haplotype associated with the PI Z allele. Three informative SNPs were chosen from this study: SNPs c.126333C>T localized in exon 1, c.126076C>T localized in intron 1, and c.135575T>C localized in exon 3. Cox et al. (1985) identified the SstI (Isoschizomer Sac1) polymorphism in the intron 1 of SERPINA1 gene using RFLP and Southern blotting. Two sites with SacI recognition sequence were identified in intron 1 from the GenBank sequence with accession Nr. AL132708.3. Primers for these sites were designed with Primer3 software: PF1-GACCTGGGACAGTGAATCGT; PR1-CACCAGCCAAGATACAGCAA, annealing T-60 °C; PF2-CCACCTCTCCTACTGCTTGGGC; and PR2-CAGTGCGTGATTAAGCCTCA, annealing T-60 °C). PCR followed by SacI enzymatic digestion was performed on 50 DNA samples from the control group to identify polymorphism with an allelic frequency of 1%.

After PCR, the MALDI-TOF technique was used to analyse all samples (primers available on request). The SNPs were numbered according to the NCBI sequence Nr.AL132708.3, and nomenclature was used according to that reported by den Dunnen et al. (2001). Genotypes obtained from the analysis were subdivided into two population groups to compare their diversity within Latvian and Swedish PI Z allele carriers. The observed genotype frequencies and diversity were computed by Arlequin software (Excoffier et al. 2005). Pairwise Fst was computed for the population comparisons. The number of permutations was 1000 and the significance level was 0.05.

Most Recent Common Ancestor Identification

Slatkin and Rannala (2000) introduced the coalescence time equation (1) as a method of moment estimator (Slatkin & Rannala, 2000; Rannala & Bertorelle 2001). This equation was applied to samples from Latvian (n = 10) and Swedish (n = 41) PI Z homozygous individuals.

  • image(1)

Where

  • image

Mutation age was analysed in PI ZZ homozygous persons; heterozygous persons were not included in this study.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Abbreviations
  8. Acknowledgments
  9. References
  10. Supporting Information

SNP Variation

The sequencing results of the intron 1 fragment revealed a previously unidentified polymorphic sequence variation 2134 + 45delG (see Supplementary Figure 1), previously named the SacI polymorphic site. The frequency of deletion was 0.093 in the control group and 0.847 in homozygous PI ZZ persons (p<0.001).

To our knowledge, SNP SacI polymorphism has not been reported or described since the report by Cox et al. (1985).

Reference sequence and present Sac1 recognition site sequence are provided as supplementary material.

Genotype Analysis

Four SNPs were analysed in Latvian PI Z mutation carriers (n = 21), hetero- and homozygous Swedish PI Z mutation carriers (n = 65), and the control group (n = 113).

The relative frequencies in these groups are presented in Figure 1.

image

Figure 1. The figure represents relative frequencies of analyzed SNPs. Similar frequencies and patterns are observed between ZZ homozygous persons from Latvia and Sweden, indicating common ancestry. PI Z/M, heterozygous persons; PI Z/Z, homozygous persons; LV, Latvian; SW, Swedish.

Download figure to PowerPoint

PI Z Mutation Age

Mutation Z age was calculated by equation (1) with the assumption of 30 years per generation. A recombination rate of 2.02 cM/Mb for the 14q32 region was obtained from the data of Kong et al. (2004) based on a high-resolution recombination map of the human genome. Marker allele frequencies and calculation results are shown in Table 1.

Table 1.  The PI Z mutation age in samples from Latvian individuals (n = 21)
SNPRecomb. rateFrequency of marker allele in generation tFrequency of marker allele in normal chromosomesGenerationsMutation age
  1. (Average – 97 generations, average mutation age – 2902 years, SD- 1983).

  2. *Recombination was not observed.

2134+45delG0.0020.8890.093651956
g.126076T0.0020.7730.5223229653
g.126333C0.0021*0.578NANA
g.135755C0.0021*0.180NANA

Two SNPs (126333 and 137555) from the analysed homozygous Latvian patient samples were non-recombinant (see Table 1). An average number of generations was 97, which gives mutation age 2902 years (SD, 1983).

The same equation (1) was used to calculate the values in Swedish PI Z mutation carriers. Marker allele frequencies and calculation results are shown in Table 2.

Table 2.  The PI Z mutation age in samples from Swedish individuals (n = 65)
SNPRecomb. rateFrequency of marker allele in generation tFrequency of marker allele in normal chromosomesGenerationsMutation age
  1. (Average – 79 generations, average mutation age – 2362 years, SD- 1614).

2134 + 45delG0.0020.8050.0931213627
g.126076T0.0020.9080.5221063203
g.126333C0.0020.9390.578782340
g.135755C0.0020.9850.1809277

In the Swedish samples, SNP 137555 showed a small number of generations and was localized in close proximity to the Z mutation site (∼2500 bp).

An average number of generations was 79, which gives mutation age 2362 years (SD, 1614).

Using SNPs 126076, 126333 and 2143 + 45delG in the Swedish population, the calculated PI Z allele age was 3057 years (SD, 656).

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Abbreviations
  8. Acknowledgments
  9. References
  10. Supporting Information

The PI Z variant shared a unique genotype that was not present in the control population. This single genotype confirmed the hypothesis of a recent and single origin of the PI Z mutation and is consistent with previous publications (Figure 1).

The SNPs showed high degree of similarity between the PI Z mutation carriers in both Latvian and Swedish populations, indicating a common ancestor.

Microsatellite genotyping of the SERPINA1 gene in four populations with different historical backgrounds (Basque, Portuguese, Canadian of British origin, and Gulf of Guinea inhabitants) showed a common genotype variation. Samples from Portuguese and northern European populations are roughly concordant, and the estimated average PI Z allele age is 4070 years, assuming 30 years per generation (Seixas et al. 2001). The PI Z mutation age for Swedish and Latvian populations had not been calculated previously. Table 3 shows the PI Z mutation age in different populations

Table 3.  The PI Z mutation age in different populations
 Cox et al. 1985[6]Byth et al. 1994[8]Seixas et al. 2001[18]Present study 
PopulationBritish, Ukrainian, German, Dutch and FrenchNorthern EuropeanPortugueseSwedishLatvian
MethodSouthern blot SNP analysisMicrosatellite analysisMicrosatellite analysisSNP analysisSNP analysis
Generations∼216∼66128–1737997
PI Z mutation age600020003200–432523622902

The date of non-recombinant SNPs revealed that the PI Z mutation originated in Latvia 2902 years (SD, 1983) ago and in Sweden 2362 years (SD, 1614) ago. The SERPINA1 gene mutation age of the Swedish and Latvian samples is consistent with the values obtained in previous studies based on microsatellite analysis of a larger region. We speculate that the possible route of this allele was from Sweden to the Baltic countries, where it spread through the waterways into the continent. Lomas (2006) hypothesized that Z allele carriers may have a selective heterozygous advantage. The Z polymers may modulate the immune system so that individuals with the Z and the S allele have an improved inflammatory response to invasive bacterial gastrointestinal and respiratory infections, which in the Neolithic period were major causes of mortality (Lomas, 2006).

The molecular analysis of SNPs to identify the PI Z genotype may be useful in understanding migration patterns and the influence of peoples from north-west Europe on the rest of Europe during the Neolithic period.

Abbreviations

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Abbreviations
  8. Acknowledgments
  9. References
  10. Supporting Information

A1AT – alpha 1- antitrypsin

COPD – Chronic Obstructive Pulmonary Disease

dNTPs – Deoxynucleotide Triphosphates

MALDI-TOF – Matrix-assisted laser desorption/ionization – time of flight mass spectrometer

PCR – Polymerase Chain Reaction

PI Z – protease inhibitor mutation Z

RFLP – restriction fragment length polymorphism

SNP – single nucleotide polymorphism

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Abbreviations
  8. Acknowledgments
  9. References
  10. Supporting Information
  • American Thoracic Society/European Respiratory Society Statement. (2003) Standards for the diagnosis and management of individuals with alpha-1 antitrypsin deficiency. Am J Respir Crit Care Med 168, 818900.
  • Beckman, L., Ambrasiene, D., Krumina, A., Kucinskas, V., Mikelsaar, A. V. & Sikstrom, C. (1999) A1AT (PI) Alleles as markers of West European influence in the Baltic Sea region. Hum Her 49, 5255.
  • Blanco, I., DeSerres, F. J., Bustillo, E. F., Lara, B. & Miravitlles, M. (2006) Estimated numbers and prevalence of PI*S and PI*Z alleles of sum1-antitrypsin deficiency in European countries. Eur Respir J 27, 7784.
  • Braun, A., Meyer, P., Calve, H. & Roscher, A. A. (1996) Rapid and simple diagnosis of the two common protease inhibitor deficiency alleles PI Z and PI S by DNA analyses. Euro J Clin Chem Clin Biochem 34, 761764.
  • Byth, B. C., Billingsley, G. D. & Cox, D. W. (1994) Physical and genetic mapping of the serpin gene cluster at 14q32.1: Allelic association and a unique haplotype associated with alpha 1-antitrypsin deficiency. Am J Hum Genet 55, 126133.
  • Chappell, S., Guetta-Baranes, T., Batowski, K., Yiannakis, E., Morgan, K., O'Connor, C. et al. (2004) Haplotypes of the alpha 1-antitrypsin gene in healthy controls and Z deficiency patients. Human Mutations 765. Online.
  • Cox, D. W. (2001) A1AT deficiency. The metabolic and molecular bases of inherited disease. New York : McGraw – Hill pp.41254158.
  • Cox, D. W., Billingsley, G. D. & Mansfield, T. (1987) DNA restriction site polymorphisms associated with the alpha 1-antitrypsin gene. Am J Hum Genet 41, 891906.
  • Cox, D. W., Woo, S. L. C. & Mansfield, T. (1985) DNA restriction fragments associated with alpha1-antitrypsin indicate a single origin for deficiency allele PIZ. Nature 316, 7981.
  • Den Dunnen, J. T. & Antonarakis, S. E. (2001) Nomenclature for the description of human sequence variations. Hum Genet 109(1), 121124.
  • Excoffier, L., Laval, L. & Schneider, S. (2005) Arlequin 3.0. An integrated software package for population genetics data analysis. Evolutionary Bioinformatics Online 1, 4750.
  • Hutchison, D. C. (1998) Alpha-1- antitrypsin deficiency in Europe: geographical distribution of Pi types S and Z. Resp Med 92, 367377.
  • John, S. W. M., Rozen, R. & Weitzner, G. (1991) A rapid procedure for extracting genomic DNA from leukocytes. Nucleic Acid Research 19(2), 241.
  • Kong, X., Murphy, K., Raj, T., He, C., White, P. S. et al. (2004) A combined linkage-physical map of the human genome. Am J Hum Genet 75(6), 11431148.
  • Lomas, D. A. (2006) A Selective advantage of alpha 1-antitrypsin deficiency. Am J Respir Crit Care Med 173, 10721077.
  • Rannala, B., Bertorelle, G. (2001) Using linked markers to infer the age of a mutation. Human Mutations 18, 87100.
  • Seixas, S., Garcia, O., Trovoada, M. J., Santos, M. T., Amorim, A. & Rosha, J. (2001) Patterns of haplotype diversity within the serpin gene cluster at 14q32.1: insights into the natural history of the alpha 1 antitrypsin polymorphism. Hum Genet 108, 2030.
  • Slatkin, M. & Rannla, B. (2000) Estimating allele age. Annu Rev Genomics Hum Genet 1, 225249.
  • Sveger, T. (1976) Liver disease in alpha1-antitrypsin deficiency detected by screening of 200000 infants. N Eng J Med 294, No. 24, 13161321.

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Abbreviations
  8. Acknowledgments
  9. References
  10. Supporting Information

Figure S1 Sac1 recognition site deletion in the homozygous state.

Legend: SERPINA1 gene sequence analysis. Arrow shows homozygous del site.

Figure S2 Sac1 recognition site deletion in the heterozygous state.

Legend: SERPINA1 gene sequence analysis. Arrow shows heterozygous del site.

This material is available as part of the online article from: http://www.blackwell-synergy.com/doi/abs/10.1111/j.1469-1809.2008.00431.x (This link will take you to the article abstract).

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