The paleoepidemiology of Sacral Spina Bifida Occulta in population samples from the Dakhleh Oasis, Egypt

International Journal of Paleopathology 26 (2019) 93–103

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The paleoepidemiology of Sacral Spina Bifida Occulta in population samples from the Dakhleh Oasis, Egypt

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Joseph E. Molto , Casey L. Kirkpatrick, James Keron Department of Anthropology, University of Western University, London, Ontario, Canada

A R T I C LE I N FO

A B S T R A C T

Keywords: Congenital Morphogenetic Morbidity Odds ratio Sacral clefting

Objective: To document sacral spina bifida occulta (SSB0) prevalence in a population sample from the Dakhleh Oasis, Egypt, and address methodological issues in recording and quantifying SSBO variations. Materials: 442 adult sacra from two temporally disjunct samples from the same deme traversing the 3rd intermediate (TIP) and the Roman Periods. Methods: Sacra were scored for SSBO, excluding the sacral hiatus. Risk of SSBO was calculated with the common odds ratio and statistical significance by X2. Data were compared to other archaeological SSBO data. Results: SSBO was present in 15.6% of the sample with a slight, but not significant, temporal increase (TIP to Roman Period) in males, and a significant age-correlated increase in both sexes. Most open sacra occurred in young adults. Conclusions: Data support that SSBO can be considered as a morphogenetic variant. Dakhleh data fall within the prevalence range for most populations, however inter-population comparisons are complicated by methodological inconsistencies. Significance: SSBO can be used in paleogenetic research. Limitations: Methodological differences in scoring SSBO prevent effective comparative study. Suggested future research: Future studies require more rigorous and standardized scoring methods. aDNA may be used to corroborate the morphogenetic value of SSBO and determine its clinical significance.

1. Introduction In contemporary western societies, congenital conditions are relatively more common than in past populations. This statistical anomaly arises because advances in western medicine and public health have considerably reduced childhood mortality from infectious diseases, while most congenital diseases remain difficult to prevent and treat (Barnes, 1994; Roberts and Manchester, 2005). The key reason for the latter is that the etiology of congenital diseases is multifactorial involving the interaction of many environmental and genetic factors, with genes contributing significantly in the majority of them (Albrecht et al., 2007; Au et al., 2010; Barnes, 1994; Bennett, 1972; Boano et al., 2009; Breslin and McCormick, 1979; Holmes et al., 1976; Kibar et al., 2007; Sillence et al., 1979). Defects in the development of the neural tube (NTDs) are among the most common congenital diseases in contemporary western countries (Au et al., 2010; Barnes, 1994; Kibar et al., 2007), and constitute a significant health challenge to western societies (Mohd-Zin et al., 2017). The neural tube is the precursor of the brain and the spinal cord,

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and in the embryo develops via a complicated process called neurulation. It has two phases; primary neurulation (weeks 3–4), which develops into the brain and the vertebral column including the first sacral element, and secondary neurulation (weeks 5–6), which forms the rest of the sacrum and coccygeal region (Kibar et al., 2007). Perturbations of neurulation produce defects which involve the brain and the spine. Anencephaly (no brain) is the most serious form and has absolute mortality (Aufderheide and Rodríguez-Martín, 1998; Kumar and Tubbs, 2011). Other spinal defects are generally grouped into two major forms, spina bifida cystica (SBC) and spina bifida occulta (SBO) (Aufderheide and Rodríguez-Martín, 1998; Kumar and Tubbs, 2011; Mohd-Zin et al., 2017). Most serious is SBC, where the meninges or neural tissue protrude beyond the bony neural arch borders (Kibar et al., 2007; MohdZin et al., 2017). If only the meninges herniate, it is classified as meningocele, whereas if the herniation also includes neural tissue, it is known as myelomeningocele (Kibar et al., 2007; Kumar and Tubbs, 2011; Singh, 2013). While today these can be treated, in antiquity the majority of cases were incompatible with a prolonged survivorship, especially in cases of myelomeningocele.

Corresponding Author. E-mail address: [email protected] (J.E. Molto).

https://doi.org/10.1016/j.ijpp.2019.06.006 Received 1 December 2017; Received in revised form 20 June 2019; Accepted 27 June 2019 1879-9817/ © 2019 Published by Elsevier Inc.

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ongoing (Blom et al., 2006; Mohd-Zin et al., 2017). To fully understand the proximate link in the development of SSBO and its clinical manifestations, it is necessary to briefly review the molecular genesis of the mammalian spine. Spinal development is controlled by a family of genes known as the homeobox or Hox genes, which is very conservative from an evolutionary perspective (Carpenter, 2002; Mallo and Alonso, 2013; Stearns and Medzhitov, 2016). As well, it is colinear in that the sequence of the areas of the spine are matched by the sequence of the HOX genes on chromosomes (Wellick and Capecchi, 2003). The primary development of the vertebral column, including the sacrum, is controlled by the expression of the Hox genes (Mallo and Alonso, 2013). In mice, the deletion of Hox11 results in the agenesis of the sacral vertebrae as they assume a lumbar identity (Wellick and Capecchi, 2003). Sacral agenesis also occurs in humans but is extremely rare, although it has been linked to a homeobox gene HLXB9 (Alison et al., 1998). Kibar et al. (2007) did not find an association of the Hox gene family with spina bifida, but mutations in the folate acid pathway are hypothesized to interfere with the expression of the latter genes, resulting in the various SSBO phenotypes noted. The defect is hypothesized to be a concomitant of the mutations that interfere with tissue union during late primary (affecting S1) and secondary neurulation (Kibar et al., 2007). Potentially, a completely open sacral canal has the most serious clinical implications, as it incorporates mutant genetic changes from all the gene families in the lower spine (Hallock et al., 2012; Kibar et al., 2007; Singh, 2013). These studies, plus genetic studies that link SSBO to a milder expression of the same morphogenetic pathways as more serious forms of spina bifida (Breslin and McCormick, 1979; Fineman et al., 1982; Doudney et al., 2009), suggest that an elevated prevalence of SSBO in a population may increase the risk of severe defects, many of which could lead to spontaneous abortion. In paleoepidemiology, it is not possible to document the prevalence of SSBO in infants and/or children, as the neural arches of S2 and S3 remain unfused until mid-childhood (Kumar and Tubbs, 2011). Particularly problematic is that the S1 neural arch, the element most commonly involved in SSBO (Albrecht et al., 2007; Kumar and Tubbs, 2011), is normally open until mid-adolescence (Merbs and Wilson, 1960; Scheuer and Black, 2002), making it difficult to determine if the hypostosis is normal or variant in a subadult. Also, determining sex in a subadult, even in older adolescents, is generally not possible, although aDNA research of the Alphoid repeats and the Amelogenin genes holds promise in this area (Hildebrandt, 2004). Mummified remains or exceptionally well preserved skeletal remains (e.g., wrapped in linens to preserve soft tissues) may, in some cases, display the presence of severe forms of neural tube defects (Aufderheide and Rodríguez-Martín, 1998; Mathews et al., 2009), but these are rare instances and occur within isolated cases not populations (Boano et al., 2009). In order to minimize potential errors relating to sacral development and age estimation in subadults, we recommend limiting studies to adult sacra. The common genetic link hypothesized between SSBO and the more serious forms of NTDs during embryogenesis (Doudney et al., 2009; Furumato et al., 1999), as well as the fact that SSBO can be studied on skeletal skeletons (Barnes, 1994; Singh, 2013; Kumar and Tubbs, 2011), suggest this research has potential for documenting the natural history and etiology of neural tube defects. We address this herein using paleoepidemiological data on SSBO from large skeletal samples from the Dakhleh Oasis, Egypt (Fig. 1).

The second category is spina bifida occulta (SBO) where the meninges and/or neural tissues remain beneath the skin, and the area of hypostosis is protected by a band of tough fibrous tissue (Barnes, 1994). The defect in the living is sometimes, but rarely, indicated by a skin lesion such as a hairy spot, dermal sinus tract, dimple, hemangioma or lipoma (Albrecht et al., 2007; Gregerson, 1997; Kumar and Tubbs, 2011; Singh, 2013). SBO can be found in any part of the spine but is most common in the sacral region (Aufderheide and Rodríguez-Martín, 1998; Barnes, 1994; Merbs, 2004; Saluja, 1988) and is referred to as sacral spina bifida occulta (SSBO) (Albrecht et al., 2007). Obviously, the spinal cord is not involved in SSBO, although the sacral plexus and its meninges can be. Though ‘occulta’ implies that it is subclinical, this may be misleading as several clinical problems can be associated with its occurrence in the sacrum, such as posterior disc herniation, enuresis and neurological abnormalities of the feet, and functional disorders of the lower urinary tract (Avrahami et al., 1994; Galloway and Tainsh, 1985; Gregerson, 1997; Hallock et al., 2012; Kumar and Tubbs, 2011). Additionally, “some people with sacral spina bifida may experience recurrent and continuous lower back pain that radiates to the hips and legs from impingement of the fifth lumbar spinous process or its unfused apophysis on either the stumps of the laminae of the first sacral vertebra or an intervening fibrous band” (Kumar and Tubbs, 2011: 23). Hallock et al. (2012) and Kibar et al. (2007) note that these clinical manifestations most often occur later in life, and are often associated with other conditions such as a tethered cord and lumbo/sacral canal stenosis (Carpineta et al., 2017; Khoshhal et al., 2012). Fidas et al. (1987) found 23% prevalence of SSBO in a large Scottish population sample, concluding that SSBO was clinically insignificant. Yet, two years later, in a study of 138 patients with urodynamic abnormalities, Fidas et al. (1989) found SSBO in 50% of their sample and noted that the abnormalities were especially stressed in these patients. Other studies argue that there are no clinical outcomes in individuals with SSBO (Nejat et al., 2008; Boone et al., 1985). Although SSBO is discussed in our major paleopathology texts (e.g., Aufderheide and Rodríguez-Martín, 1998; Roberts and Manchester, 2005; Ortner and Putschar, 1985), the clinical implications of the occult type are not discussed, implying that it is clinically insignificant. Treating SSBO as a normal variant and not a pathology is common in bioarchaeological publications (e.g., see Merbs, 2004), although there are exceptions (Papp and Porter, 1994). Overall, the medical and bioarchaeological research is divided on how to classify SSBO: is it an anatomic variant, a pathology, or both? In our opinion, we believe the latter. It is a normal morphogenetic variant that can, in some cases, result in clinical outcomes that can contribute to morbidity in present and past populations. The genetic evidence for the development of spina bifida derives primarily from population (Albrecht et al., 2007; Barnes, 1994; Post, 1966; Roberts and Manchester, 2005) and family studies (Fineman et al., 1982; Yan et al., 2012; Zhang et al., 2012). McKusick (1998) reviewed genetic studies on spina bifida, concluding that the occulta and the more serious cystica phenotypes were variant expressions of the same dominant autosomal gene. Barnes (1994), however, notes that “Studies trying to prove that spina bifida occulta is a minor manifestation of spina bifida cystica have led to some confusion” (Barnes, 1994: 49). Recently, several genes have been associated with the genesis of neural tube defects (Au et al., 2010; Greene et al., 2009; Kibar et al., 2007; Martinez et al., 2009), although it is hypothesized that exogenous environmental factors are required to trigger the development of the defect in genetically susceptible hosts (Barnes, 1994; Boano et al., 2009; Mills et al., 1996). Maternal nutrition, involving folic acid, zinc and/or selenium deficiencies have been identified as primary environmental associations (Barnes, 1994; Armstrong et al., 2013; Kibar et al., 2007; Mills et al., 1996). The candidate genetic loci involving folic acid metabolism are especially noteworthy (Blom et al., 2006; Zhang et al., 2013), as perinatal folic acid supplementation has reduced the risk of NTD by 60–70% (Kibar et al., 2007). Debate on the proximate mechanisms behind the dietary associations of neural tube defects is

2. Materials and methods The examined sacra derive from three cemeteries in the Dakhleh Oasis, Egypt: Ain Tirghi, Kellis 2 (K2) and the Kellis village site (Fig. 2). Ain Tirghi dates to the Third Intermediate Period (circa 1000-800 BCE), and the human remains were interred in tombs carved into the hard rock base of the surrounding hills (Molto, 2001). Most tombs were looted and the remains commingled, but three tombs (AT 31, 34 and 94

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Fig. 1. A. Map of Egypt showing the location of Dakhleh (oval), one of five major oases in the western desert. Also shown are the locations for Bahriyah Oasis (small arrow) and El Giza (large arrow) which have comparative SSBO data that are used in this study. B. Locations of the cemeteries in the Kellis village, Kellis 2 (K2) and Ain Tirghi (AT) which are approximately in the central part of the Oasis.

(Stewart et al., 2004). Village site cemeteries D6 & D7 are small, contained areas (Molto et al., 2003) and, like Ain Tirghi, are hypothesized to represent kinship clusters (Molto, 2001, 2002, Molto et al., 2003; Keron, 2015). Sex determination was based on the scoring of the os pubis (Phenice, 1969). An interobserver reliability test between the lead author and a former member of the bioarchaeology team at DOP (Dr. Scott Fairgrieve) resulted in complete concordance in 65 complete skeletons from these population samples. Age at death determination in the adults for the complete burials from Ain Tirghi and for most of the Kellis burials was based on multiple methods (e.g., morphogenesis

52) had many complete burials. For this study, the commingled remains were reconstructed focusing on the hips and sacra, as this region is useful for both sex and age determinations. The cemeteries within the Kellis village and the large K2 cemetery are represented by single extended burials that date to the Roman Period (Molto, 2001, 2002, Molto et al., 2003). Kellis 2, a large cemetery just northeast of the village, presents a two-dimensional vista which has succinct areas around superstructures that may reflect family groupings (Fig. 3). K2 is estimated to contain 3000–4000 burials, of which more than 700 have been excavated and partially analyzed. K2 has been AMS radiocarbon dated to the Roman Period, c. 50–450 CE

Fig. 2. Views of the three cemeteries in the Dakhleh Oasis. A. Kellis 2 (K2), B. The village site (D6/7), and C. Ain Tirghi. D. Burials in K2 and the town site were well preserved single interments with head to the west. The village site burials were in well delimited areas as were E. the Ain Tirghi burials. Most of the Ain Tirghi burials were commingled from looting, but there were at least three tombs with complete burials that were wrapped in linens or interred in wooden or ceramic coffins (in E). 95

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Fig. 3. The distribution of the burials at the Kellis 2 cemetery. When the people at Kellis converted to Christianity they moved their cemetery and initially constructed tomb superstructures (circles). Burials were placed in and around these structures. As the cemetery was in use for over 400 years, it expanded and areas for the interments encroached (arrows) making it is difficult to assign burials to specific groups. Thus, unlike the Ain Tirghi and village site tombs (D6/7), spatial analysis statistics of genetic traits and SSBO were required to test the familial hypothesis.

S1-S2 centra. S1-S2 fusion normally is complete by the late 20 s to the early 30 s, whereas the neural arch of the first sacral vertebra is usually complete sometime during mid-adolescence (Buikstra and Ubelaker, 1994; Merbs and Wilson, 1960; Scheuer and Black, 2002). These criteria were used in defining the two broad age groups used in this analysis, namely 18–35 and 36+ years. It should be noted that in a

standards of the symphysis pubis (Brooks and Suchey, 1990) and ribs (Iscan et al., 1984 and 1985), dental attrition, and ossification patterns (Buikstra and Ubelaker, 1994)). The commingled Ain Tirghi skeletons were more difficult to determine age at death. The age at death estimates for the commingled Ain Tirghi remains were based on symphysis pubis morphogenesis (Brooks and Suchey, 1990) and the fusion of the 96

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Fig. 4. The two sacra on the left and in the center are from older adult males from Ain Tirghi and Kellis respectively with SSBO of S1S2 and of S1 (arrows). In the Dakhleh sample SSBO is limited to S1 in 69% of the 69 individuals with SSBO, while the S1-S2 phenotype occurred in 10 or 14.5% of the burials with SSBO. The sacrum on the right from a young adult female shows the complete fusion of the neural arches from S1 to the superior border of the S5 centrum. The circles show the various phenotypes of the sacral hiatus. The sacral hiatus to S4 and/or S5 was not scored as SSBO present in this study as it is the normal condition, though in modern clinical contexts it is germane to determine the hiatus level as it is important for caudal epidural procedures.

Following Waldron (2009) and Altman (1991), the OR is calculated as follows:

subsample (n = 103) of complete burials estimated to be at least 40 years of age using multiple criteria, there were 5 skeletons (4.9%) with S1– S2 unfused. Moreover, in 2.2% (2 of 97) of skeletons estimated to be in their early twenties, fusion of S1-S2 had already occurred. As the pubic symphysis can be used to determine age at death between broad cohorts (Loth and Işcan, 1994), these errors appear negligible when assigning individuals to these two broad age categories. SSBO was scored on all of the sacral segments – S1 only, S1 and S2, S1 to S3, and open sacral canal. Figs. 4 and 5 show examples of SSBO in the Dakhleh skeletons. There was no case of S1 being fused but with S2S5 open, which was a subtype previously reported by Barnes (1994) and Singh (2013). As the sacral hiatus (S4 and S5) is normally patent (Albrecht et al., 2007; Barnes, 1994; Hallock et al., 2012; Senoglu et al., 2008; Singh, 2013), it was not scored as SSBO present, but it is necessarily included when the sacral canal was open. The prevalence (p/ n) was computed for males and females in each age cohort for both the Ain Tirghi and Kellis population samples. The K2 and Kellis village samples were combined for this analysis, as both are from the Roman Period at Kellis (Molto et al., 2003). Statistically, an Odds Ratio (OR) and its 95% confidence interval were computed for all pairwise comparisons following Waldron (2009) and Altman (1991), with the significance test (P value) computed according to Seslin (2004). The latter is technically a X2 test and provides a probability estimate, as the 95% CI of an OR is not considered a test of significance (Ahlbom, 1993; Glencross, 2003). The OR can be used to quantify the strength of an association even when not significant.

Odds Ratio =

PG1/(1-PG1) PG2/(1-PG2 )

Where “PG1 “represents the odds of the event (SSBO) of interest for group 1 and “PG2” represents the odds of the event of interest for group 2. This formula can be expressed in tabular form as follows.

Exposure in young adult females Exposure in young adult males

SSBO Present (A) (C)

SSBO Absent (B) (D)

By convention, when the data in contingency table cells were < 5%, a Yates correction factor was applied to compute X2. In this study, we computed the risk of having SSBO in the different population samples (AT and Kellis) and the risks for males and females from both population samples relative to age. Their collective data were also compared by age and sex. Examining the kinship hypothesis was accomplished by computing and interpreting the prevalence data for the Ain Tirghi and Kellis village tombs. In addition, we report a previous result using the binomial theorem, which was used to test the probability of genetic traits clustering within the Kellis village samples. Testing the kinship hypothesis Fig. 5. Completely open sacral canals (arrows) in young adult Kellis males. This SSBO variant was present in 17.4% (12/69) of the Dakhleh sacra with SSBO. Of the 12 cases, 11 occurred in young adults and 10 of these were males. The photo on the left also shows the sacralization of the 5th lumbar vertebra (circle) which can be associated with SSBO.

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Table 1 Age and sex distribution of SSBO in the Ain Tirghi and Kellis samples. Ain Tirghi Total

Age

N

P

%

S1

%

S1-S2

%

S1-S3

%

Open

%

1 st Cohort 2nd cohort Total

116 77 193

23 2 25

19.8 2.6 13

16 2 18

69.6 100 72

1 0 1

4.3 0 4

0 0 0

0 0 0

6 0 6

26.1 0 24

Females

1 st Cohort 2nd cohort Total

52 54 106

10 0 10

19.2 0 9.4

9 0 9

90 0 90

0 0 0

0 0 0

0 0 0

0 0 0

1 0 1

10 0 10

Males

1 st Cohort 2nd cohort Total

64 23 87

13 2 15

20.3 8.7 17.2

7 2 9

53.8 100 60

1 0 1

7.7 0 6.7

0 0 0

0 0 0

5 0 5

38.5 0 33.3

Age

N

1 st Cohort 2nd Cohort Total

119 130 249

Females

1 st Cohort 2nd Cohort Total

Males

1 st Cohort 2nd Cohort Total

Kellis Total

%

S1

%

S1-S2

%

S1-S3

%

0pen

%

29 15 44

24.4 11.5 17.7

20 8 28

69 53.3 63.6

4 5 9

13.8 33.3 20.5

0 1 1

0 6.7 2.3

5 1 6

17.2 6.7 13.6

63 83 146

12 7 19

19 8.4 13

9 5 14

75 71.4 73.7

2 1 3

16.7 14.3 15.8

0 1 1

0 14.3 5.3

1 0 1

8.3 0 5.3

56 47 103

17 8 25

30.4 17 24.3

11 3 14

64.7 37.5 56

2 4 6

11.8 50 24

0 0 0

0 0 0

4 1 5

23.5 12.5 20

of SSBO in males versus females (OR 2.05, 95% CI = 1.22–3.45; P = .007). In AT and K, SSBO is most common in S1 respectively in 72.0% (18/25) and 63.6% (28/44), for a total prevalence of 66.7% (46/ 69). A noteworthy result is that 17.4% (12/69) of individuals with SSBO in the composite sample have completely open canals, and of these 12 individuals (6 each in AT and K), 11 (91.7%) were young adults (18–35 years) and 10 of the 12 were males. Table 3 summarizes SSBO prevalence by sample in each of the Ain Tirghi tombs. As noted, 13.0% of all adults at Ain Tirghi had SSBO. However, the adult sample sizes were variable, ranging from 1 to 34. If we consider only sample sizes > 7, the prevalence varies between 0 and 29% and in 4 of these 10 samples SSBO is absent. In one tomb (#36), the three males with SSBO were all young adults with open sacral canals. In the D6-D7 cemeteries in the Kellis village site, three of seven adults (42.9%), all young adult males, had SSBO; two with S1 and one with S1-S2 (Molto et al., 2003). Also in this group were a number of rare genetic traits (SSBO is not considered rare) that clustered (Molto et al., 2003). One rare morphogenetic trait in particular, the precondylar tubercle, was found in three individuals. Using the binomial distribution, the probability of having three of ten individuals with this trait by chance (when it only occurs in 2% of the combined Kellis and Ain Tirghi samples) is 8 in 10,000 or ˜ 1 per 1000, clearly a highly significant result (Molto et al., 2003). This was interpreted as indicating that the small D6-D7 cemeteries in the Kellis village sample, like those at Ain Tirghi, likely reflect a family group. The clustering of SSBO in this group is similar to the precondylar data, however because SSBO is not a rare trait it precluded using the binominal statistic. We have, however, noted the genetic significance of SSBO. Delineating specific burial areas in the K2 cemetery is difficult, as the cemetery was used for over three hundred years and the spatial separation of burial clusters is not clear due to encroachment of the separate areas over time. Despite this, Fig. 6 clearly shows that SSBO clusters in K2, with the largest number of affected males and females occurring in the western area of the cemetery. The clustering also shows a greater male concentration. The previously noted research using a large battery of morphogenetic nonmetric traits in K2 analysed using spatial statistics (Keron, 2015), rejected the null hypothesis of

for Kellis 2 was more difficult. When the people at Kellis adopted Christianity, they changed and moved their mortuary pattern from a common crypt to single burials in the Christian position (extended with head to the west). Based on spatial clustering of morphogenetic traits, it has been hypothesized that different families established superstructures in the new cemetery (Molto, 2002). Burials in and around the superstructures (see Fig. 3) represent those with the closest affinities and can be readily visualized in terms of the prevalence of genetic traits (i.e., SSBO in this case). Over time, however, the zones demarcating the groups became difficult to delimit, as the zones encroached upon one another (Fig. 3), unlike the well-defined areas in AT and the Kellis village sites. Keron (2015) reports that K2 was organized along kinship lines, as many of the genetic traits clustered around specific superstructures. We report the general results of Keron’s spatial statistical modeling on the SSBO herein, while a full description of the computations are summarized online in Supplement 1. 3. Results Table 1 summarizes SSBO prevalence data by age, sex and sacral element (S1, S1-S2, S1-S3, and open sacral canal) in the Kellis and Ain Tirghi samples. The prevalence of SSBO is slightly higher in Kellis versus Ain Tirghi (17.7% versus 13.0%), but this difference is not statistically significant (OR of 1.44, CI = .84 to 2.45; P = .177). Previous multivariate statistical comparisons using the Mean Measure of Divergence (MMD) of nonmetric cranial morphogenetic data comparing these sample populations did not reject the null hypothesis that these subsamples are from the same evolving deme (Molto, 2001; Rashed, 2010). This similar result for the SSBO data reported herein supports pooling the SSBO data for the AT and K samples (Table 2). The overall prevalence of SSBO (Table 2) is 15.6% (69/442), and there is a clear age regression in prevalence for both sexes (14% difference for females versus 10.7% in males), which collectively shows younger adults have a significantly higher prevalence than older adults (22.1% (52/235) versus 8.2% (17/207), with an OR of 3.17, a 95% CI of 1.77–5.69, and P < .0001). The OR indicates that the risk of SSBO in younger adults is more than three times greater than in the older adults. These data also resulted in a statistically significantly higher prevalence 98

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Table 2 SSBO in the combined Ain Tirghi and Kellis Samples. Total

Age

N

P

%

S1

%

S1-S2

%

S1-S3

%

0pen

%

1 st Cohort 2nd Cohort Total

235 207 442

52 17 69

22.1 8.2 15.6

36 10 46

69.2 58.8 66.7

5 5 10

9.6 29.4 14.5

0 1 1

0 5.9 1.4

11 1 12

21.2 5.9 17.4

Females

1 st Cohort 2nd Cohort Total

115 137 252

22 7 29

19.1 5.1 11.5

18 5 23

81.8 71.4 79.3

2 1 3

9.1 14.3 10.3

0 1 1

0 14.3 3.4

2 0 2

9.1 0 6.9

Males

1 st Cohort 2nd Cohort Total

120 70 190

30 10 40

25 14.3 21.1

18 5 23

60 50 57.5

3 4 7

10 40 17.5

0 0 0

0 0 0

9 1 10

30 10 25

relative to clinical (i.e., radiological) and bioarchaeological studies and have included ours and some additional bioarchaeological population data (e.g., Merbs and Wilson, 1960). From these crude prevalence data it is clear that there is considerable intra- and inter-population variability. The Dakhleh population prevalence of 10.4% for S1 SSBO is virtually identical to the cumulative prevalence of the other bioarchaeological samples reported in this Table (10.5%, 111/1062). In Table 4, the composite clinical data for S1 SSBO (15.2%, 327/2150) were statistically significantly higher than the total bioarchaeological data (OR = 1.54, P = .0002). The prevalence of SSBO in other modern samples varies considerably between 12.4% (Eubanks and Cheruvu, 2009) and 23% (Fidas et al., 1987). Because these and the data in Table 4 are crude and clinical samples and are not real ‘populations’ in the statistical sense, they are difficult to compare and interpret. Moreover, observing SSBO on radiographs can be challenging (Albrecht et al., 2007), since small defects that are readily observed on skeletonized remains may not necessarily be seen on standard radiographs. Clearly, despite the problems of standardization, the Dakhleh results ‘fit’ closely to the published data. The significant age and sex differences in the Dakhleh sample are concordant with modern and bioarchaeological results. The regressive age trend is virtually universal (Fidas et al., 1987; Kumar and Tubbs, 2011; Eubanks and Cheruvu, 2009), whereas the sex differences are more variable (Kumar and Tubbs, 2011; Henneberg and Henneberg, 1999), though in studies with large sample sizes the prevalence is generally significantly higher in males (Fidas et al., 1987; Eubanks and Cheruvu, 2009). The variant results in modern studies with small sample sizes (see Kumar and Tubbs, 2011) are possibly due to sampling error. For example, if parents who have alleles positive for SSBO and the mother has a diet deficient in folic acid, their children, male or female, will have an increased risk of developing a neural tube defect. In such families with a disproportionate number of either sex, the relative frequency of SSBO would be elevated. Though large bioarchaeological and modern samples generally have a higher male prevalence of SSBO, it is noteworthy that more serious open spinal dysraphisms are more common in females than males (Page, 1985; Mohd-Zin et al., 2017), which confounds the interpretation of SSBO. The age regressive trend in SSBO prevalence has been addressed by two hypotheses. One is that the osseous defect is filled in by increased hyperostosis (Fidas et al., 1987; Kumar and Tubbs, 2011; Papp and Porter, 1994). This hypothesis has general support, as hyperostosis into soft tissues tends to increase with age (Ossenberg, 1969; Saunders, 1978). It certainly makes sense when the defects are small as the band of fibrous tissue covering the defect can easily be closed by osteogenic action (Barnes, 1994; Kumar and Tubbs, 2011). The second hypothesis is that SSBO may increase mortality risk, particularly when the defect is large, such as with an open sacral canal. The fact that an open sacral canal was present in 11 of 12 young adults in ancient Dakhleh, and 10 were males, is an interesting association in terms of both age and sex. Unfortunately, there is currently no clinical support for this ‘mortality’ hypothesis, though, as noted, individuals with this phenotype

Table 3 AT SSBO Prevalence by tomb. Tomb

P/N

%

30 31

0/1 4/29

0 13.8

33 34 35 36 37 40 41 42 45 51 52 55 58 Total

0/11 2/34 0/7 4/22 0/13 6/23 2/4 1/4 3/20 2/14 0/8 1/ 2 0/1 25/193

0 5.9 0 18.2 0 26.1 50 25 15 14.3 0 50 0 13

2 males (both with open canals) and 2 females, all young adults both young adult females 1 S1, 3 open sacral canals, all young adult males 4 males (3 young adults, 1 older); 3 young adult females 1 female (open canal) and 1 male; both young adults a young adult female 2 males and a female; young adults 1 young adult female; 1 older adult male a young adult male

randomness in favour of the genetic/familial clustering hypothesis to explain the concentrations. From the application of the same spatial statistics (see attached Supplement) to the SSBO distribution in K2, we can conclude that SSBO is not randomly distributed in K2, which is added support for the genetic/familial clustering hypothesis. 4. Discussion SSBO showed an increasing but statistically insignificant temporal trend between the Ain Tirghi and Kellis samples. These results, in conjunction with MMD coefficients on a large battery of nonmetric morphogenetic traits from previous research (Molto, 2001), support pooling the data, as AT and K are considered part of the same evolving deme. Noteworthy is the fact that, like nonmetric morphogenetic traits, the SSBO clustered (it was not random) in the AT and K tombs, and in the K2 cemetery. These results support the hypothesis that SSBO can be considered a morphogenetic trait with an underlying genetic influence, despite the fact that the precise genetic basis of SSBO is poorly understood since genetic loci studies are based primarily on associations, not strict causality. Overall, SSBO occurred in 15.6% of the adult sacra in Dakhleh. This crude prevalence requires scrutiny, as the data showed statistically significant age regressions for both sexes, with males having a statistically significant higher prevalence. Noteworthy is that the open sacral canal was present in 12 individuals, 10 of whom were males and all but one was in the young adult age category. Comparing and interpreting these data to published skeletal and clinical research is difficult for many reasons. In particular is the lack of standardization in scoring and reporting SSBO (Kumar and Tubbs, 2011; Merbs and Wilson, 1960; Roberts and Manchester, 2005). To illustrate, we use studies of S1 SSBO data, revised from Albrecht et al. (2007), who analyzed SSBO in a contemporary Australian cadaver sample. We have verified and reorganized their S1 SSBO data (Table 4) 99

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Fig. 6. Distribution of SSBO in the Kellis 2 cemetery showing the clustering of males with SSBO (circle) in the north-west area. Note the total absence of SSBO in males associated with the superstructure in the north east area.

P = .0001). Close scrutiny of their data, however, reveals some inherent problems that compromise these comparisons and their interpretations. Sarry El-Din and El Banna’s (2006) Giza sample consisted of 272 adult skeletons from two sample populations: 83 males and 72 females from an upper class group, and 117 lower class workers (64 males and 53 females). The SSBO prevalence of 3.3% (9/272) was based on combined samples. In light of the results herein, it would have been interesting to investigate the prevalence of SSBO in the two subgroups and to compare their age profiles. For example, the elites may have included a greater percentage of older individuals, and given our knowledge of the age regression of SSBO, this may have resulted in a lower prevalence of SSBO. They also report that the prevalence was slightly higher in males (5.18%) than females (3.33%), but again their data were not organized by age. An additional noteworthy result is that

potentially have a higher risk of developing some of the clinical conditions that occasionally cause morbidity in individuals with SSBO. An additional concern with the mortality hypothesis is that that the clinical conditions associated with SSBO, as noted, usually occur later in life. How do our data compare to other SSBO data on ancient Egyptians? Two recent Egyptian bioarchaeological publications deal with this subject. Sarry El-Din and El Banna (2006) report SSBO in 3.33% (9/ 272) of the sacra in ‘Old Kingdom’ Egyptians from Giza, while Hussein et al. (2009) report a prevalence of 62.3% (48/77) in the Greco-Roman inhabitants of the Bahariya Oasis. Hussein et al. (2009) hypothesized that Bahariya data reflect a high degree of endogamy in the GrecoRoman inhabitants of Bahariya Oasis due to their geographical isolation from main Egyptian populations. Obviously, these comparative data show significant differences in the SSBO prevalence (OR = 48, 100

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concentration of SSBO in the Ain Tirghi and Kellis cemeteries has also been interpreted as reflecting familial groups (Molto, 2001, 2002), despite our lack of understanding of genetic mechanisms involved in the development of SSBO. Our interpretation also relies on the fact that dietary and other ubiquitous environmental factors (i.e., diet, water and hyperaridity) influencing our population were similar, even though Ain Tirghi and Kellis are separated by a thousand or more years. Thus, the variance in the prevalence of SSBO between burial areas is interpreted as reflecting underlying genetic mechanisms, as the environment is likely constant. This is a long standing assumption used in the interpretation of morphogenetic traits in bioarchaeological research (Molto, 1983), though it should change once the exact target genes for SSBO have been identified. Of particular interest in spina bifida research is the question of whether or not spina bifida occulta can be classified as a neural tube pathology or a morphogenetic nonmetric hypostotic vertebral variant. The genesis of this query derives from the clinical research of Lawrence et al. (1968) and its bioarchaeological formulation by Barnes (1994), who states: “Developmental (cleft neural arch) delay resulting in hypoplasia or aplasia of the pedicles, laminae, or spinous process can lead to failure of the two halves to coalesce and result in a bifid or cleft neural arch. This is the most commonly known developmental defect of the vertebral column; it is usually referred to as spina bifida. I prefer to not use this term, because it confuses this developmental defect of the neural arch with the defect associated with the neural defect, spina bifida occulta. I prefer the term ‘cleft neural arch” (Barnes, 1994:118-119). Barnes also notes that “Spina bifida occulta resulting from neural tube defect is definitely related to spina bifida cystica, whereas cleft neural arch resulting from neural arch defect is not.” (Barnes, 1994: 49). Barnes also attempts to differentiate the two through photographic evidence of the open sacral canal in one of her figures (Barnes, 1994: 48, Fig. 3.6). She notes, “The spinal canal is widened with neural tube defect, pushing the edges of the bony canal outward. In contrast, the spinal canal remains normal and the edges of the bony cleft are not raised when no neural tube defect is present. Clefting without neural tube defect also occurs in presacral vertebral elements, usually at border areas” (Barnes, 1994: 49). She and Lawrence et al.’s (1968) important research suggests that bioarchaeologists and clinicians should reclassify the vast majority of our observations of spina bifida occulta as ‘neural arch clefting’ in order to be etiologically and anatomically correct. To our knowledge, only Merbs (2004) has followed their recommendation. The fact that both clinicians and bioarchaeologists continue to use spina bifida occulta as proxy for a neural tube defect needs to be addressed in future research. The criteria for scoring ‘spina bifida occulta versus clefting’ must be standardized by rigorous interobserver reliability testing.

Table 4 Crude S1-SSBO prevelence data in bioarchaeological and clinical (imaging) samples revised from Albrecht et al. (2007). Bioarchaeology Sample

P (N) %

Source

Ancient Moroccan Ancient Italian

4 (15) 27 14 (124) 11

Roman British Anglo-Saxon British (6-7 C) Anglo-Saxon British Huguenots in England (10th C) 18-19 C British American Indian Aleuts (combined) Peruvians Ancient Inuit Ancient Egyptians Total

14 (104) 14 10 (27) 37 9 (77) 12 16 (144) 11 17 (112) 15 2 (56) 4 3 (95) 3 2 (175) 1 8 (61) 13 12 (76) 16 111 (1062) 10.5 46 (442) 10.4

Ferembach (1963) Henneberg and Henneberg (1999) Papp and Porter (1994) Papp and Porter (1994) Papp and Porter (1994) Papp and Porter (1994) Saluja (1988) Post (1966) Post (1966) Post (1966) Merbs and Wilson (1960) Post (1966)

Dakhleh, Egyptian Clinical Sample Israeli American British French Australian Total

204 (1200) 17 88 (550) 16 11 (48) 23 24 (299) 8 0 (53) 0 327 (2150) 15.2

present study Avrahami et al. (1994) Southworth and Bersack (1950) Thorpe et al. (1994) Vannier et al. (1981) Albrecht et al. (2007)

S1 SSBO was totally absent (0/272). This sacral element, as noted, has the highest prevalence in all large modern and ancient samples thus far reported, including the present study. As they did not outline their scoring criteria, the low overall prevalence reported suggests they probably correctly scored the sacral hiatus (S4 and S5) as SSBO ‘absent’. The Bahariyah Oasis sample (Hussein et al., 2009) was based on single commingled vertebrae including 77 isolated sacra, the sex of which was determined using a method outlined by Stewart (1979) based on modern samples from the United States. Moreover, age estimation was not possible for the isolated sacra so the authors quite correctly suggested that their prevalence data were ‘crude’. In terms of scoring SSBO, there was no standard listed, although they did show four cases of SSBO, with two being completely open sacral canals. The extremely high prevalence of 62.3% versus the 3.3% for Giza resulted in their endogamy hypothesis. This hypothesis is questionable due to their lack of a scoring standard, which probably included the sacral hiatus as SSBO present. The Dakhleh Oasis was equally, if not more, isolated than the Bahriyah Oasis (Fig. 1), yet Dakhlans in the Roman Period, which is roughly contemporaneous with the Bahriyah sample, had an SSBO prevalence of 17.3% (44 of 249) versus Bahriyah’s 62.3% (48/77). This is a statistically significant difference (P < .0001) and the OR (7.55) suggests that the risk of developing SSBO was 7 times greater in Bahriyah than Dakhleh. We suggest that the comparative SSBO data between Dakhleh and Bahriyah reflects scoring and organizational differences. Their scoring criteria weren’t outlined and their data were not delineated by age and sex, so we have no way of controlling for these variables, making comparisons difficult or impossible. This supports Kumar and Tubbs’ caution that “prevalence means little unless common observational criteria are used, and the groups are truly comparable in terms of age and sex structure, which has seldom been the case” (Kumar and Tubbs, 2011: 28). We conclude that the endogamy hypothesis is a sampling and scoring artifact and should not be seriously entertained by researchers until more refined bioarchaeological data, including demographic information, are utilized. Despite the aforementioned methodological problems, Hussein’s et al. (2009) endogamy hypothesis follows an accepted interpretation of SSBO in bioarchaeology pioneered by Ferembach (1963), who reported 15 cases of SSBO in a small sample (n = 21) in ancient Morocco. The

5. Conclusions SSBO was common in the ancient Egyptian adults (15.6%) from the Dakhleh Oasis, Egypt. The prevalence in our population sample fits into the middle values of the published range for clinical and paleoepidemiological studies that are based on adequate sample sizes. Our results were also concordant with most clinical and bioarchaeological research showing an inverse relationship of SSBO with age in both sexes, and being statistically more common in males than females. The intracemetery analysis shows that SSBO behaves as a morphogenetic variant that can be used in paleogenetic research. However, due to considerable variation in SSBO phenotypes, the significant age regression in prevalence, and the significant sex differences, we recommend using S1SSBO in young adult (18–35) males and females to represent SSBO for inter-population comparisons. The non-union of this sacral element is the most common in ancient samples, including at Dakhleh, and it should have high scoring accuracy and precision. The latter is a fundamental component of the scientific method (Molto, 1983). Our data 101

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could not be reliably compared to recently reported bioarchaeological SSBO results from Egypt due to reporting and standardization issues. Apart from recommending standardization in observation and recording of SSBO, future research should address the potential problem of comparing SSBO prevalence data based on imaging versus dried bone analyses, as each require somewhat different observational standards (Albrecht et al., 2007). The medical community is divided on how to classify SSBO. In our opinion, there are sufficient data showing clinical outcomes of SSBO, particularly the open sacral canal, to conclude that SSBO, in some cases, could contribute to individual and population morbidity. In this regard, it is particularly necessary to address the issue raised by Barnes (1994), namely that SSBO in most cases is not a neural tube defect but a hypostosis, and that these phenotypic differences can be differentiated. Future research may follow Barnes in calling the SSBO variants sacral clefting. A possible resolution to this issue is a meta-analysis of spina bifida, including a major workshop involving paleopathologists and clinicians (i.e., radiologists, surgeons etc.). As part of this, it is worthwhile noting that the complete mtDNA genome from a K2 burial was recently amplified and sequenced using next generation sequencing (Molto et al., 2017). This success implies that in the future it might be possible to determine specific single nucleotide polymorphisms (SNPs) and to track their molecular phylogeny once a candidate gene or genes that cause neural tube defects are found.

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