The pathogenesis of human diseases has been further understood based on findings in the study of ancient populations across thousands of human history . The premise of these work is the diagnosis of ancient diseases . With the progress in biology and medicine, archaeologists and palaeopathologists can obtain a great deal of biological information from ancient human specimens . More information can be obtained from well-preserved specimens, including relatively detailed information at the level of tissue cytology and molecular biology [3-5]. This provides the opportunity for us to make more comprehensive diagnoses and so assist research into ancient human diseases [1,3,6]. However, the diagnosis of ancient human diseases is very difficult if some of the critical information cannot be obtained. Cases of rare diseases reported in the literature for ancient humans are obviously more limited than for modern living humans because the medical history, clinical features, imaging, histopathology and molecular biology can’t be easily obtained [7-11].
CT plays an important role in exploring ancient human diseases [1,12,13]. In HORUS study, clarification proved by CT is the key points in diagnosis of ancient coronary artery diseases . This non-invasive imaging method can find radiographic features in blind spots and reduce any possible damage to the specimen by avoiding other more invasive endoscopic techniques . Various CT reconstruction techniques have had a significant impact on bioarchaeology [1,12,14]. However, reports on CT reconstruction techniques, tissue transition projection (TTP) and CT virtual endoscopy (CTVE), are rare in the archaeological literature. CT reconstruction techniques would be used in our research to manifest the bone lesions of the FD.
Pathology is an important part of modern medicine and is the gold standard for clinical diagnosis. Paraffin section with haematoxylin and eosin stain is the main means of clinicopathologic diagnosis, confirmed with immunohistochemical techniques. Histological staining of bone tissue is of great value in identifying structural changes microscopically, and is therefore routinely used by clinical pathologists, but it is not a method currently widely used in archaeology [2,6,15]. Some researchers have improved the conventional method: a stepwise manual for staining undecalcified dry bone tissue will further expand the palaeopathologist’s diagnostic power . So we attempted to observe the microstructure of the lesions by HE stain in our research. A conventional transmission optical microscope was used to view the sections. At the same time, an optical depth of field microscope, which is generally used in the fields of science and engineering, but seldom be used in clinical practice nor in palaeopathology. This instrument was used in present study to try to directly view the microscopic features of the lesion tissue of the FD.
Usually FD is a progressive bone disorder caused by activate mutation of the GNAS gene that results in inhibition of the differentiation and proliferation of bone-forming stromal cells and leads to the replacement of normal bone and marrow by fibrous tissue and woven bone . In modern medicine, clinical features of FD include cranial asymmetry, facial deformity, hearing loss, proptosis, visual impairment and unilateral blindness when optic foramen was involved . Craniofacial FD is a rare bony disorder with defined epidemiological and clinicopathological features in the modern Chinese population . Only a few case reports of ancient humans with the disease have been found in the literature [7-11] and of ancient Chinese people even fewer.
The abnormal skull specimen was recovered from Jinggouzi (about 2485 B.P.), a Bronze Age cemetery site located in the eastern part of Inner Mongolia. As far as FD is concerned, we obtained as much information as we could by using the methods of modern medicine to improve the credibility of diagnosis. This study developed an applicable combined approach with CT technology and microscopic images to recognize and diagnose the FD. We also tried to use molecular biology to help with diagnosis.
The skull specimen (figure 1) labelled 03LJM55B was discovered at Jinggouzi. The Jinggouzi site is located in Linxi County, Chifeng City, Inner Mongolia Autonomous Region, in northern China. The specimen was preserved in the research center for Chinese frontier archaeology of Jilin University after excavation. The research permit (no. 2013JLU05) was obtained from Chinese frontier archaeology of Jilin University for all aspects of the study.The remains represent one of the earliest skeletal populations in the region and is thought by some to be “DongHu” people, an ethnic group that acted actively in the northeast of Yan country in the history of ancient China from Shang dynasty to Han dynasty with Huimo nationality, Shusen nationality. After excavation, all the finds are preserved at the research centre for Chinese frontier archaeology of Jilin University, (Changchun, Jilin province, China).The skull was identified as being from the late stage of the Spring and Autumn Period to the early stage of warring states (about 2500 years old) from artefacts, the shape of the tombs and radiocarbon dating. The radiocarbon dating of the charcoal indicates that the age is 2485±45BP . The specimen is a male of 22 years old, and the physical age of the specimen is estimated from skeletal indicators, primarily pubic symphyseal morphology and relative dental wear . sex is determined primarily on the basis of pelvic form, supplemented with cranial features .
Imaging and 3D reconstruction
The skull was scanned with a 64-slice CT, GE Discovery High Definition 750 CT scanner (GE Healthcare, Milwaukee, WI, USA). Scans of the whole skull, as well as specific areas of interest, were taken at 120 kV with 300 mAs and the field of view was 25 cm. Cranial data were reconstructed with slice thicknesses of 0.6 mm. All data are archived as Digital Information and Communications in Medicine (DICOM) files on the GE Picture Archiving and Communication System (PACS) server (GE Healthcare). CT scan data were accessed specifically using advanced volumeshare 4.0 (ADW4.0) imaging software on a CT workstation. Images analysis and secondary image reconstruction techniques included multiplanar reconstruction (MPR), shaded surface display (SSD), maximum intensity projection (MIP), TTP and CTVE.
Microscopic and histopathological observation
Some lesion tissue specimens were observed directly with a VHX-2000 series (KEYENCE). Unlike conventional optical microscopes, this microscope has a real-time zoom lens (RZlens) and high dynamic range (HDR) function, synthesizing 3D images by capturing multiple colour images at different brightness levels to observe the specimens. Other lesion tissue specimens were fixed in 10% formalin and embedded in paraffin. The sections of formalin-fixed, paraffin-embedded specimens were stained with HE.
Genomic DNA extraction and amplication
The DNA extraction protocol used followed the reference 22 and 23 [22,23]. Genomic DNA was amplified by arbitrary primer PCR technique, seven arbitrary primers were used as below: (1) 5’-CCGGCTACGG; (2) 5’-CAGGCCCTTC;(3) 5’-AACGGTCACG;(4) 5’-AGCTGCCGGG;(5) 5’-AGGCATTCCC;(6) 5’-GGTCTGAACC;(7) 5’-AAGGCTAACG. Genomic DNA extracted from a healthy tooth from the corresponding author (Dr. Y. Huang) was used as a control.
Clinical features visible by the naked eye
Colour photographs (figure 1) taken with an ordinary camera showed bulging growth in the right frontal and orbital bone. The remains were relatively intact after the fragments had been joined together with glue and parts of the sphenoid and occipital bones are absent. Simultaneously, the optical canal and bone surrounding the nerve could not be seen clearly by the naked eye.
Imaging and 3D reconstruction
Coronary and axial MPR images (figure 2) showed FD lesions in the right of the skull. Localized differential bulging of the disease involved great wings of sphenoid bone, frontal bone and temporal bone. The lesions showed fragmentation of the bone and the central structure of the lesion was absent. The bone around the region of right optic nerve canal was defect in MPR images (figure 2). The wall of the optic canal may have been involved, which maybe cause visual conduction abnormalities, and symptoms of hemianopia would therefore occur. SSD images (figure 1) illustrated that the surface of the bulge lesions was smooth except broken position. MIP images (figure 1) showed low-density lesions visible on the right side. TTP images (figure 3) showed a disturbance of the endometrial structure in the lesions of FD. Virtual endoscopy (figure 4) was used to observe the right orbit and the internal structure of the skull could be observed from any angle. The bones of the lesion area bulged to the internal skull and cortical bone was not complete. Irregular sutures had formed in the lesion.
Microscopic and pathological findings
By 3D deep field microscope, the gross specimen of the lesion showed a loose bone-like tissue. The lesions as seen in low magnified images (figure 5) had an irregular honeycomb-like structure because disordered trabeculae were connected to each other. At higher magnification (figure 5), the trabeculae morphology and the gap between the trabeculae were irregular and varied in size and shape, and part of the margin of the trabeculae showed wormhole-like changes.
By HE staining in Paraffin-embedded specimen, we found that trabeculae with a tortuous irregular arrangement varied in shape and width (figure 6). Irregular trabeculae of woven bone have been described as having a Chinese character-like appearance. The distance between trabeculae varied and the edge of trabeculae was not smooth. The distribution between trabeculae was uneven.
GNAS gene detection
The genomic DNA was extracted from the teeth and amplified by arbitrary primer PCR. Using arbitrary primer 2 and 4 as described in methods, we have successfully got DNA detected. We failed to get DNA enough for subsequent gene analysis using other primers. To detect GNAS gene, we designed a pair of primers to cover the exon 8 and 9 of GNAS gene where the mutation on codon 201 (C→ T) has been indicated in this disease. The GNAS gene exon 8 and 9 coding regions were amplified by PCR using a forward primer, 5’-CCC TCT TTC CAA ACT ACT CC-3’, and a reverse primer, 5’-AAG CCC ACA GCA TCC TAC -3’.
We have successfully got a positive result after amplifying the GNAS gene by PCR from the two teeth specimens of the human skull 03LJM55B, as shown in figure 7. However, when we sequence the amplified PCR fragment, we did not find any gene mutation. Our results suggest that in ancient human, FD might not hold codon 201 mutation, or this specific mutation occurred in the evolution stage.
Modern medical research provided the foundation for our study of ancient human disease [1,3-5] Several CT reconstruction methods, such as MPR and TTP, et al., microscopic pathology techniques and the molecular biology were innovatively used to diagnose the craniofacial FD. The archaeological remains of ancient humans are bones. The skin and soft tissues, including the brain and eyeballs, only rarely survive. Therefore, the clinical characteristics of this patient are speculations based on morphological changes of the bone. Information provided by CT scanning is the basis of a diagnosis of FD. From a radiological point of view, manifestations in CT images include a reduction in bone density and relatively homogeneous lesions of bone [17,24]. The findings from our study and other relevant research show that radiographic features are very similar between ancient and modern cases. The homogeneous density of the lesions is probably because cells and stromal components in the lesion have a stochastic distribution, and after about 2500 years these cells and stromal components have been reduced randomly. Radiographic features and pathologic results confirmed this. There was no damage in the cortical bones surrounding the lesion, except cortical bone thickening as a result of the slow progress of the disease. This change is in accordance with the biological characteristics of FD . Because there were no positive visual findings in other parts of the skeleton, CT scanning of other parts of the skeleton was not undertaken.
Archaeologists and physical anthropologists sometimes apply MPR and SSD when researching ancient human bone specimens . This can help us see obvious radiographic features on the most suitable plane. MPR imaging has direct clinical significance in determining changes in the optic canal. The location and extent of lesions can be displayed clearly from different angles using intuitive and 3D SSD images. Because characteristics of ancient human bone specimens include the presence of a closed or partially closed cavity structure, similar to the airway and intestinal structures of modern living human bodies, we not only used conventional reconstruction techniques, but also applied TTP and CTVE to rebuild the hollow skull and research FD concurrently. TTP can display the delineation of the bowl wall similar to conventional double-contrast examination by enhancing surface transitions while suppressing homogeneous areas . TTP images provide a non-invasive evaluation of the morphology of intracranial membranes. Tissues and organs adjacent to lesions, especially the hollow organs, such as the sinus cavities, display clearly and completely. Virtual endoscopy is an important screening and diagnostic tool in the clinical setting. Some researchers have yet to apply this to ancient human specimens. Virtual endoscopy can be used to observe non-invasively the unknown cavity structure, for example, the complete cranial cavity and the close bone marrow cavity of long bones. Some endoscopes can provide magnified images directly. TTP and virtual endoscopy provides a potential method for future research in physical anthropology, including the identification of race and gender by morphology of intracranial membranes, comparison of femoral morphology (including bone marrow cavity characteristics) and mechanical analyses to determine the aspects of human activity.
Histopathology can be used to differentiate FD from other similar diseases [17,25]. The pathological diagnosis of FD is speculated primarily on changes in the trabeculae. Because the tissue and cellular components that existed between trabeculae have disappeared: the only remains found in ancient specimens are the trabeculae. We placed specimens directly under a 3D deep field microscope for observation. Reports on the application of this method to diagnose ancient disease are rare, particularly regarding FD. In our data, we have reveal some microscopic image features using 3D depth of field observation, including an irregular honeycomb-like structure of lesion and the change of trabeculae morphology(figure 5). Histological staining of bone tissue is of great value in identifying structural changes microscopically, and is therefore routinely used by clinical pathologists, but it is not a method currently widely used in archaeology. By observing paraffin sections with HE staining, we found that the morphology of trabeculae from the archaeological specimen(figure 6) was very similar to modern human sections of FD. This supports the diagnosis of FD. The random distribution of residual trabeculae contributes to the relatively uniform density of CT images.
Usually FD with one or multiple sites of bone involved is the result of GNAS mutation. The disease is called McCune Albright’s syndrome, also known as Albright’s syndrome, if multiple systems are involved [17,27]. Detecting gene mutations contributes to the differential diagnosis and early screening of Albright’s syndrome patients [28-30]. Research on ancient DNA has been a problem because in almost all ancient specimens, DNA is only present in very tiny amount and in various states of degradation. We have succeeded in establishing the DNA ultramicro-analysis techniques from cells using an improved silica method in our laboratory. The genomic DNA was successfully extracted from the teeth although the specimens were from a 2500-year-old ancient human. This is probably because the evacuated area has been dry in China, and the tooth provided a rather close environment in preventing DNA degradation. We have successfully got a positive result after amplifying the GNAS gene by PCR(figure 7). Interestingly, no condon 201 mutation was found by gene sequencing in this case, our results suggest that in ancient human, FD might not hold this specific mutation, although the CT and microscopic images already showed the pathological changes. We recognized that many neoplastic diseases had specific changes at the genetic level, and people generally thought that the occurrence of diseases was due to the accumulation of genetic changes, our study could support that these genetic changes might be the consequence rather than the cause of the disease, at least in FD.
In conclusion, applying modern medicine to research ancient human tissue samples can achieve relatively reliable diagnosis of ancient diseases, including some rare diseases, such as FD. The non-invasive observation of ancient human specimens by CT can provide us with much more detailed morphologic information about the lesions. Clinical pathology and the optical microscopic imaging technology of depth of field enable residual tissue lesions to be more accessible for researchers, and provide important evidence for the diagnosis of FD. The combination of CT and histopathology provides more comprehensive information about ancient human disease and improves the credibility of diagnosis. As well as diagnosing ancient rare diseases by applying techniques from the fields of engineering science and modern medicine, we can also diagnose other ancient human diseases that are common in modern society, such as cancers, cardiovascular disease and trauma. The ultimate aim of our research is to view disease across human history, rather than just focus on the diseases of today, and it will be highly significant to understand the disease progression.
Z Shi participated in designing the research and drafting the manuscript. Qun Zhang contributed to dating analysis of the sample, microscopic imaging using a 3D deep field microscope, photographing of the specimen, and drafting the manuscript. K Cheng conducted CT scanning and analysis of CT reconstruction data. H Shao conducted pathological sections and microscopic analysis. D Zhao conducted extracting and detecting ancient DNA. B Sun and J Yu participated in the CT data analysis and interpretation. L Bi contributed to pathological analysis and manuscript preparation. M Li, Z Sun and L Guo contributed to microscopic imaging using a 3D deep field microscope. H Zhu contributed to the design of the study and archaeological consulting. Quanchao Zhang and Y Huang designed the study and contributed to data analysis, and manuscript preparation.
We thank Dr. Haotong Xu (Postdoctoral Workstation, the General Surgery Center of the Peoples’ Liberation Army, Chengdu Army General Hospital, Chengdu; or Department of Radiology, Second Affiliated Hospital, North Sichuan Medical College, Nanchong, Sichuan, P. R. China), very much for his assistance in the obtaining of parts of three-dimensional structures and his contribution to the writing of the paper.