The most common, life-threatening autosomal recessive disease of Europeans and Euro-Americans, cystic fibrosis (CF), occurs predominately in patients with the F508del mutation.1 Although F508del is currently detectable as a single allele in 1/30-1/40 Europeans2-4 and Euro-Americans,5 it has not been determined what heterozygote selective advantage(s) might account for its relatively high prevalence. Indirect evidence6 suggests that this mutation was present in Brittany at least 3000 years ago, but no direct analyses of ancient DNA have been reported to identify F508del and clarify its frequency in prehistoric inhabitants of Europe. Here we show that F508del was present in 3 of 32 Iron Age inhabitants of Austria from whom DNA could be recovered from molar teeth using procedures that fulfill authenticity criteria.7 Because these individuals, who were buried in cemeteries along the Danube river, were shown by radiocarbon dating of isolated bone collagen to have lived there during 544-255 BC, this indicates that the F508del mutation is definitely more than 2000 years old and that CF (the disease) was present among them. More generally, the apparent enrichment of this Iron Age population in F508del suggests an evolutionary advantage in their environment that can be investigated by interdisciplinary strategies of paleoepidemiology

Cedric  Le Marechal

Claude Ferec

Malika  Siker

Maria Teschler-Nicola

Philip Farrell

English

Nature Precedings

1 
 
Discovery of the Principal Cystic Fibrosis Mutation (F508del) in Ancient DNA 
from Iron Age Europeans 
 
Philip Farrell
1
, Cedric Le Marechal
2
, Claude Ferec
2
, Malika Siker
1
, and Maria Teschler-Nicola
3
 
 
1 
Departments of Population Health Sciences and Pediatrics 
University of Wisconsin, Madison WI 53726, USA. 
2 
Laboratoire de Génétique Moléculaire et Génétique Epidémiologique, INSERM U613, 
Université de Bretagne Occidentale, 29220 Brest, France. 
and the 
3 
Department of Anthropology 
University of Vienna and the Naturhistorisches Museum, A-1014 Vienna, Austria 
 
To whom correspondence should be addressed. E-mail: pmfarrell@wisc.edu 
 
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The most common, life-threatening autosomal recessive disease of Europeans and Euro-
Americans, cystic fibrosis (CF), occurs predominately in patients with the F508del 
mutation.
1
  Although F508del is currently detectable as a single allele in 1/30-1/40 
Europeans
2-4
 and Euro-Americans,
5
 it has not been determined what heterozygote selective 
advantage(s) might account for its relatively high prevalence.  Indirect evidence
6
 suggests 
that this mutation was present in Brittany at least 3000 years ago, but no direct analyses of 
ancient DNA have been reported to identify F508del and clarify its frequency in prehistoric 
inhabitants of Europe.  Here we show that F508del was present in 3 of 32 Iron Age 
inhabitants of Austria from whom DNA could be recovered from molar teeth using 
procedures that fulfill authenticity criteria.
7
  Because these individuals, who were buried in 
cemeteries along the Danube river, were shown by radiocarbon dating of isolated bone 
collagen to have lived there during 544-255 BC, this indicates that the F508del mutation is 
definitely more than 2000 years old and that CF (the disease) was present among them.  
More generally, the apparent enrichment of this Iron Age population in F508del suggests 
an evolutionary advantage in their environment that can be investigated by 
interdisciplinary strategies of paleoepidemiology. 
 
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 Although more than 1500 mutations have been identified in the cystic fibrosis 
transmembrane conductance regulator (CFTR) gene, the F508del allele
1
 accounts for about 70% 
of cystic fibrosis (CF) chromosomes in known patients and accounts for the relatively high 
incidence of the disease.
2, 3, 8
  To address the gap in knowledge regarding potential selective 
heterozygote advantages proposed
7
 to explain the prevalence of F508del-CF, we have initiated 
an interdisciplinary paleoepidemiology investigation focused on the hypothesis that exposures 
during the post-Neolithic population expansions/migrations
10
 enriched tribal sub-populations in 
this mutant allele of the CFTR gene. Initially, we are seeking direct evidence demonstrating that 
CF was associated with prehistoric Europeans, namely the Celts who emerged as a distinct 
culture after the Bronze Age.  The Celtic tribes as a warrior society with master metal workers 
then flourished during the Iron Age and, after large scale migrations,
10
 occupied the regions of 
Europe where CF is most common.
2, 4, 8
  The availability of relatively large populations of Iron 
Age skeletons provided an opportunity to determine if F508del was present in their DNA.   
 Iron Age cemeteries with Celtic burials in Eastern Austria with access to waterways such 
as the Danube river were the sources of our specimens. After careful excavation, the human 
remains were given to the Department of Anthropology at the Natural History Museum in 
Vienna.  From three collections of Iron Age skeletons buried in cemeteries near Vienna 
[Mannersdorf/Leitha (30 km southeast), Pöttsching (50 km south), and Franzhausen (40km 
west)], we selected molar teeth (replaced with casts) from individuals with at least one intact 
tooth and 8 mm core femur specimens from all skeletons with at least one identifiable femur of 
adequate size. The teeth were individually packaged and transported to Brest, France for the 
molecular studies described below.  The femur specimens were taken to other laboratories for 
analyses such as radiocarbon dating of isolated collagen by accelerator mass spectrometry 
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(AMS) at the Oxford Radiocarbon Accelerator Unit (http://c14.arch.ox.ac.uk/ams.html#prep).  
Each skeleton had been examined and assigned a probable age and sex from its osseous features; 
some were examined again after ancient DNA results became available.  The estimated age of 
these individuals ranged from approximately 3 to 80 years. The AMS-radiocarbon dating of 
isolated collagen
11
 from femurs revealed an average of 386 BC, with a range = 544-255 BC for 
15 specimens analyzed. 
 Processing and analysis of ancient DNA was organized to address recommendations for 
ensuring authenticity.
7, 12
 In planning this investigation, we decided that top priority should be 
given to avoiding contamination with modern DNA.  Thus, it was concluded that molar teeth 
would be preferable to bones because of their protective encasement and the potential for 
multiple specimens.  The key strategy, however, was to perform replicate analyses with 
independent technicians in two separate buildings employing rooms that had never been used for 
any CFTR research. Multiple negative controls were also introduced at each step of the 
procedure, as described by Bramanti et al.
13
  Only two teeth were processed per week, in order to 
avoid any cross contamination. After experiencing failure to recover DNA from partially 
degraded or softened teeth, namely 7 molars from the Mannersdorf/Leitha, collection (Table 1), 
we used only intact teeth from the Pöttsching (N=10) and Franzhausen cemeteries (N=29).  DNA 
analyses were performed by combining PCR amplication of exon 10 of the CFTR gene, which 
contains F508del, with concomitant amplification of short tandem repeat (STR) loci.
14
  This 
permitted detection of any laboratory-related contamination with modern DNA, as well as sex 
determination by assessing Amelogenin polymorphism.
15
  The F508del allele was identified by 
its characteristic 3 base pair (bp) deletion compared to the wild type locus (Figure 1) and 
confirmatory sequencing.   
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 As shown in Table 1, analyzing 46 teeth yielded recoverable DNA in 32 with none from 
7 degraded teeth of Mannersdorf/Leitha collection, 9 of 10 from Pöttsching, and 23 of 29 from 
Franzhausen. Sex concordance was demonstrated in 27 of the 32 individuals, but 3 of 5 
discordant skeletons had uncertain male/female assignments from osseous examinations.  This 
finding adds to fulfillment of authenticity criteria.
7, 12
  DNA fingerprinting of the 32 amplified 
extracts revealed 3 adults with F508del demonstrated by replicated analyses showing the 
characteristic 3bp deletion, as shown in Figure 1.    There were two individuals with the F508del 
allele from Franzhausen and one from Pöttsching. The Franzhausen burials were at widely 
separated graves. As shown in Figure 1C, the presence of F508del was reconfirmed in duplicate 
with another tooth from Franzhausen #203 two-years after the original discovery. The 3bp 
deletion was confirmed by sequencing the PCR product.  This provides unequivocal evidence 
that she (a 25-30 year-old female buried in 351  30 BC) was a CF heterozygote. 
 Demonstrating F508del in these specimens indicates that CF was present in Iron Age 
European populations and that the mutation is at least 2300 years old. The age of the principal 
CF mutation has been matter of debate and uncertainty. Although Morral et al
16
 suggested that 
F508del is 2600 generations (52,000 years) old, this indirect estimation has been criticized by 
Kaplan et al
17
 who proposed 868 generations. In addition, Fichou et al
6
 recently showed that in 
Western Europe (Brittany) the oldest ancestor of the F508del mutation was from ~3000 years 
ago.  
 The presence of the F508del-CF mutation in 3 of 32 ancient DNA extracts from these 
teeth might seem higher than expected, but enrichment of Iron Age European populations in this 
mutation is consistent with a heterozygote selective advantage. Although there has been much 
speculation about factors that might explain the probable evolutionary advantage of F508del,
9
 
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investigation of human populations to test the various hypotheses is sorely needed.  We believe 
that Iron Age skeletons abundant in Europe will permit such studies.  Further paleoepidemiology 
research on other ancient Celtic sub-populations
10
 will also be needed to delineate F508del 
frequency in ancient DNA throughout Europe
8
 where the mutation proliferated over the centuries 
in a geographically specific pattern
4, 8, 18
 aligned with Celtic migrations.
8, 10
  The methods that we 
have established, particularly the use of molar teeth processed to avoid modern DNA 
contamination, will facilitate such an investigation. 
 
Methods 
For ancient DNA analyses in two separated laboratories, we first cleaned selected molar teeth 
with milliQ water and a scrapper, chemical decontamination was accomplished with one hour 
incubation of a cleaning solution (1%SDS, 25mM NaEDTA, 20µg.ml
-1 
proteinase K) and 
periodical wortexing. The solution was then removed, several rinsings with milliQ water were 
performed, and the teeth were air-dried during one night.  The teeth were then crushed in a 
mineralogic mill to obtain a thin powder which was separated in three aliquots. Two of them 
were subjected to immediate analysis and the last one was stored at -20°C. A decalcification 
solution (EDTA 0.5M + 20µg.ml
-1 
proteinase K) was added to the powder and incubated 
overnight at 37°C with agitation before decantation by centrifugation. One ml of the supernatant 
was introduced in the DNA extraction column (Qiamp UltraVirus kit – Qiagen) which was 
processed according to the manufacturer. 
 We quantified the concentration of the extracted DNA (quantifiler kit – 
Appliedbiosytems, Foster City CA) in order to normalize the amount of DNA introduced in the 
PCR (about 1ng). We decided to combine the PCR amplification of the CF gene exon 10 
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(containing F508del) with several STR sequences available in commercial kits in order to be sure 
of the variability between teeth and to identify potential modern, laboratory-related DNA 
contamination.
13
 Thus, the STR sequences of the two technicians were determined and 
monitored with each analysis.  The first 30 teeth were analyzed with the SGM kit 
(Appliedbiosytems, Foster City CA) and the last ones with Powerplex 16 kit (Promega, Madison 
WI) which gave most robust results. Each aliquot was amplified at least twice, with one day 
between each replicate.  Sex was determined by assessing Amelogenin polymorphism with the 
above kits.  Short amplicons were preferentially detected and results interpreted after first 
determining that the no template controls were negative and if at least three STRs were amplified 
in parallel in the two aliquots. Whenever the F508del mutation was detected by its characteristic 
3bp deletion compared to the 90bp of the wild type allele, this result was confirmed by 
sequencing the PCR product (sequence data available upon request--see Supplementary 
Information). 
 
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References  
1. Kerem, B., et al.  Identification of the cystic fibrosis gene:  Genetic analysis.  Science 
245, 1073-1080 (1989). 
2. Southern KW, et al. A survey of newborn screening for cystic fibrosis in Europe.  J Cyst 
Fibros. 6, 57-65 (2007). 
3. Bobadilla, J.L. et al.   Cystic fibrosis: A worldwide analysis of CFTR mutations-
correlation with incidence data and application to screening. Human Mutation 19:575-
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4. Lucotte G, Hazout S, De Braekeleer M. 1995. Complete Map of Cystic Fibrosis Mutation 
DF508 Frequencies in Western Europe and Correlation between Mutation Frequencies an 
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fibrosis: A review.  J Human Biology 64, 167-174 (1992). 
9.  Romeo, G., Devoto, M., Galietta, L.V.J.  Why is the cystic fibrosis gene so frequent?  
Hum. Genet. 84, 1-5 (1989). 
10.  Cunliffe, B.  The Ancient Celts. Penguin Books. (1997). 
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11.  Stafford, T.W., Jr., Hare, P.E., Currie, L.A., Jull, A.J.T. and Donahue, D. Accelerator 
radiocarbon dating at the molecular level.  J Archaeological Sciences 18, 35-72 (1991). 
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(2000). 
13. Bramanti B, Hummel S, Chiarelli B, Herrmann B.  Ancient DNA analysis of the delta 
F508 mutation.  Human Biology 75, 105-115 (2003). 
14. Butler JM.  Genetics and genomics of core short tandem repeat loci used in human 
identity testing.  J Forensic Sci 51, 253-265 (2006). 
15. Sullivan KM, Mannucci A, Kimpton CP, Gill P.  A rapid and quantitative DNA sex test: 
fluorescence-based PCR analysis of X-Y homologous gene amelogenin.  BioTechniques 
15, 636-641 (1993). 
16.  Morral, N., et al.  The origin of the major cystic fibrosis mutation (delta F508) in 
European populations.  Nat Genet 7, 169-175 (1994). 
17. Kaplan NL, Lewis PO, Weir BS.  Age of the ΔF508 cystic fibrosis mutation.  Nature 
Genetics 8, 216-217 (1994). 
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the major CF mutation and its associated haplotypes. Hum Genet 85:436-45 (1990). 
 
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Acknowledgements 
We thank Caroline Benech and Christine Guery for expert ancient DNA analyses, Ronald Mühl 
for invaluable assistance with specimen procurement in Vienna, Thomas Stafford (Colorado) and 
Christopher Ramsey (Oxford) for AMS-radiocarbon dating, and Barry Cunliffe and Peter Ramsl 
(Vienna) for advice on design/interpretation.  We are also grateful to Milan Macek and Kutna 
Hora (Prague) for specimens and encouragement.  NIH grants DK 34108 and M01 RR03186 
supported P. Farrell and M. Siker, while INSERM U613 supports C. Ferec and C. Le Marechal.  
 
Author Information 
The authors declare no competing financial interests.  Correspondence and requests should be 
addressed Philip M. Farrell (pmfarrell@wisc.edu). 
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Table 1.  Ancient DNA Results Using Analysis of Short Tandom Repeat Loci and CFTR 
Gene Exon 10 
 
Cemetery Samples 
available 
Selected DNA 
recovered 
Gender 
concordance 
del3bp 
(F508del) 
Mannersdorf/Leitha 50 7 0   
Pöttsching 17 10 9 8 1 
Franzhausen 52 29 23 19 2
+
 
+
 tooth FRANZ203 was replicated and the 36p deletion confirmed by sequencing (See Figure 1) 
 
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Figure Legend 
Figure 1. This figure provides examples of three "fingerprints" with their corresponding 
electrophoregrams of the blue channel (FAM labeled). The horizontal axis displays the size 
(basepairs) of the PCR products. The vertical axis represents arbitrary fluorescence units. The 
CFTR exon 10 amplicon was added to the nine microsatellites and the gender marker 
Amelogenin of the SGM kit (Appliedbiosystems, Foster City CA) which allowed a simultaneous 
analyzes. The labels below D3S1358 and vWA microsatellites indicate the number of repeats for 
each allele. The 90bp peak corresponds to the wild type allele (solid arrow) of the CFTR exon 
10, whereas the F508del allele was screened based on the deletion of 3bp (ie 87bp) (broken 
arrow). Double peaks with 1bp difference (non template mediated 3' end A adduct) are a 
systematic artifact due to the low DNA quality and additional PCR cycles (35 cycles). Sample 
Franzhausen #74 (panel A from January 2007) is the wild type whereas replicates of the sample 
#203 (panel B from February 2005 and C from January 2007) indicate an heterozygous F508del 
genotype (alleles 87 and 90bp). 
 
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A rapid and quantitative DNA sex test: fluorescence-based PCR analysis of X-Y homologous gene amelogenin.

A survey of newborn screening for cystic fibrosis in Europe.

Accelerator radiocarbon dating at the molecular level.

Age of the ΔF508 cystic fibrosis mutation.

Ancient DNA analysis of the delta F508 mutation.

Ancient DNA: Do it right or not at all. Science;

Ancient DNA.

Cystic fibrosis: A worldwide analysis of CFTR mutationscorrelation with incidence data and application to screening. Human Mutation

Estimating the age of CFTR mutations predominantly found in Brittany (Western France). J Cyst Fibros In press—published online

European Working Group on CF Genetics.

Genetics and genomics of core short tandem repeat loci used in human identity testing.

Identification of the cystic fibrosis gene: Genetic analysis.

The Ancient Celts. Penguin Books.

The application of DNA analysis in a population screening program for neonatal diagnosis of cystic fibrosis: comparison of screening protocols.

The origin of the major cystic fibrosis mutation (delta F508) in European populations.

Why is the cystic fibrosis gene so frequent?

Discovery of the Principal Cystic Fibrosis Mutation (F508del) in Ancient DNA from Iron Age Europeans

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Discovery of the Principal Cystic Fibrosis Mutation (F508del) in Ancient DNA from Iron Age Europeans

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