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The aim of this letter is to make public an unusual skeletal remain with classic features of ankylosing spondylitis (AS). Furthermore, human leucocyte antigen (HLA)-B27 sequences have been studied.
In the National Historical Museum in Stockholm there is a male skeleton (inventory no 14549). It was found during the excavation of St Clemens, a church ruin from the Middle Ages in Visby, Sweden. The preserved parts form a block, with smooth, continuous calcifications of the long ligaments and bony ankyloses of the intervertebral joints. Several ribs are fixed to the thoracic vertebrae through ankyloses of the costovertebral joints (fig 1). Also the sacroiliac joints are fused. The man lived sometime between 900 and 1300 AD. The details of this excavation were published in Swedish in 1911,1 and are therefore not generally known.
DNA was extracted from approximately 20 mg of bone powder for each extraction, from controls as well as the Visby skeleton. The sample from the latter skeleton was extracted in a dedicated ancient DNA facility, separated from areas where modern materials and amplicons are handled. The medieval sample was extracted twice with two negative extraction controls containing ddH2O. The negative ddH2O controls did not yield any pyrosequences originating from the human HLA system. The extraction procedure followed a previously published protocol to minimise the risk of contamination.2
Primers used3 yielded longer (133 base pairs (bp)) and shorter (54 or 55 bp) fragment sizes.
Pyrosequencing was used for allele identification (PSQ 96 MA). The forward primer was also used as the sequencing primer.3 The reaction was carried out according to the instructions supplied by the manufacturer.
During the development of this method, human material with known HLA properties was collected during arthroplastic procedures. All patients gave written consent and the Ethics Committee of Lund University Hospital approved the study (LU 854–03).
Allelic dropout and contamination are known to be major problems in ancient DNA research.4 Attempts to control for contamination have so far proved insufficient.4 5 However, extraction protocols, including heavy bleaching of bone powder,2 6 have been shown to be efficient in eliminating contamination. As ancient DNA is known to be highly fragmented,7 it is possible to use the fragmentation pattern to separate authentic ancient DNA from modern contamination.2 Here we did not see a contradictory result in the typing of the longer and the shorter amplicon. To avoid chimaeric results due to allelic dropout, we typed four amplicons altogether, from two extracts. This is usually enough to control for allelic dropout in highly degraded material.8
In this 700–1100-year-old skeleton, with classic macromorphological changes of AS, HLA-B27 sequences have been found using modern molecular techniques (table 1). The Visby skeleton has many similarities to the often cited skeletal finding with indisputable AS changes reported by Connor9 in 1694. As far as we know HLA-B27 sequences have only once before been reported from examinations of older skeletal material.10
In conclusion, our findings confirm that modern molecular techniques can also be used in investigations of paleopathological skeletons older than that described by Haak et al.10 The use of this technique in such investigations can be expected to give further insight into the prevalence and epidemiology of the HLA-B27 allele and perhaps also about disease susceptibility and severity.
Footnotes
Competing interests: None.
Ethics approval: The Ethics Committee of Lund University Hospital approved the study (LU 854–03).
Patient consent: Obtained.