Possible thalassemia intermedia in a child (16th–18th century) from the westernmost part of Europe: potential association with malaria and past migrations

Abstract

This study describes a non-adult individual with bone features suggestive of β-thalassemia, a disease commonly found in malaria-endemic regions today and in the past. The skeleton of a 5.5- to 6.5-year-old child exhumed from a 16th- to 18th-century CE necropolis in Almeirim (Portugal) was examined macroscopically, with a scanning electron microscope, and by conventional radiology and computed tomography. This individual shows frontoparietal diploic hyperplasia with a slight hair-on-end radiographic appearance and an exuberant serpiginous pattern. The orbital roofs have a plaque-like formation and facial bones display new bone proliferation and porosity. The teeth show caries, calculus deposition, anomalies on the four deciduous canine roots, and linear enamel hypoplasia on the first permanent right upper central incisor. The postcranial skeleton presents osteopenic trabecular appearances, cribra humeralis, and cribra femoralis, the latter associated with malaria. A second non-adult individual (2.5–3.5 years old) had similar lesions in the cranium, raising the question of whether they were siblings. The lesions are consistent with β-thalassemia intermedia, a homozygous or compound heterozygous hemoglobinopathy found in malaria-endemic regions, such as Almeirim, due to the protective advantage conferred by β-thalassemia carriers. Thalassemia presents a wide spectrum of lesions common in other hemolytic anemias, which makes the diagnosis a challenge. Hopefully, biomolecular techniques will assist the diagnosis in skeletonized individuals. To the authors’ knowledge, this is the first possible case of thalassemia in the westernmost part of Europe, in a region currently with high prevalence of hemoglobinopathies, attributed to the past Islamic and sub-Saharan presence, and in an area historically affected by malaria.

Introduction

Almeirim, from the Arabic word meaning wetland, is a municipality located in the district of Santarém, on the south margin of the Tagus River (Figure 1A). Flooded areas are known to be prone to the development of mosquitoes that cause malaria, a disease present in Portugal at least since the Middle Ages (Bruce-Chwatt and Zulueta, 1977). Almeirim expanded socioeconomically and politically by serving as the permanent residence of the Portuguese kings during the 15th–16th centuries CE (Silva, 2007; Custódio, 2008; Simões, 2015). After the 17th century CE, Almeirim became exclusively agricultural (including rice fields) and suffered recurring food and epidemic crises (Couto, 1859). According to Couto (1859), a medical doctor, the region had an unhealthy environment where the winter floods “are slow to be absorbed … and, in the Summer vegetable and animal debris rot, becoming a source of harmful emissions that are blown by the wind and influence the insalubrity of the population” (Couto, 1859). The children were “terribly deformed … affected by intermittent fevers and spleen diseases, which slows down their development.” There are no data for earlier periods, but from December 1855 to December 1858, this doctor treated 887 patients, 25% (220/887) of whom sought cures of their intermittent fevers (a designation for malaria) and 2.7% (24/887) due to splenic hypertrophy and splenitis.

Figure 1.

(A) Geographical location of Almeirim, near the Tagus River. (B) Church of Divino Espírito Santo during the 19th century (Couto 1859: 37). (C) Burial of non-adult individual sk.485.

In regions with endemic malaria, hemoglobinopathies are common because these genetic disorders give protection against this parasitic disease (see Haldane, 1949; Allison, 1954). Genomic studies indicate that the hemoglobinopathies have dispersed during migrations (Almeida, 2015; De Sanctis et al., 2017; Viganó et al., 2017), through intermarriage and becoming established in autochthonous populations due to consanguineous marriage (Denic et al., 2008). The mutations related to sickle cell disease (SCD), another group of hemoglobinopathies, were introduced by migratory flows along the Mediterranean (Lavinha et al., 1992). In Portugal today, there is a relatively higher prevalence of the β-thalassemia trait extending from the south to the Tagus valley (1.06%), a region more influenced by Islamic Berber occupation (8th–12th/13th centuries CE), decreasing to 0.03% north of the Douro River (Martins et al., 1993; Inez et al., 1993). Moreover, after the 15th century enslaved persons from former African colonies arrived on the Portuguese mainland (Lavinha et al., 1992). Despite the existence of a place called Paço dos Negros, 30 km east of Almeirim where “African slaves lived for years and built a family” at least between 1469 and 1521 (https://www.cm-almeirim.pt), so far there is no evidence of individuals of African population affinity in the region of Almeirim.

This study aims to analyze a non-adult individual, sk.485, whose cranial lesions are suggestive of thalassemia, a group of hemoglobinopathies frequent in malaria-endemic regions, not previously found in the paleopathological record of the Iberian Peninsula despite the long presence of Islamic and African populations in what is now Portuguese territory.

Materials and Methods

The individual

During the restoration of the former church of Divino Espírito Santo in Almeirim (Figure 1B), a necropolis dated from the 16th–18th centuries CE was discovered (Macedo, 2015). Twenty-eight burials and nine ossuaries were excavated, with an estimated minimum number of 108 individuals: 70.4% (76/108) adults and 29.6% (32/108) non-adults (Lourenço, 2015). This study focuses on individual sk.485, who was buried supine in a grave without funerary goods. Approximately 80% of the skeleton was recovered (Figure 1C), including 19 deciduous teeth and all the permanent dentition, except for the third molars (not formed yet).

Methodologies

The age at death was estimated through dental development and eruption (AlQahtani et al., 2010), fusion of cranial and postcranial bones (Cunningham et al., 2016), and the length of the long bones (Cardoso et al., 2014). The biological sex estimation was based on mandibular morphology (Schutkowski, 1993) and morphological and metric analyses of the auricular surface (Luna et al., 2017, 2021; Monge Calleja et al., 2020). The ages of enamel hypoplasia formation were obtained following Thomas et al. (2019) and Reid and Dean (2000).

Pathological changes were identified through macroscopic observation. Internal structures were observed with conventional radiology and computerized tomography (Supplement Material 1). To search for possible anemic erythrocyte malformations (see Maat and Baig, 1990; Maat, 1991) fragments of the skull were observed with a TESCAN Vega3SBH scanning electron microscope (SEM) (magnification, 7–10000×; low current intensity, 2–5 kV), without conductive coating to avoid chemical contamination (Monge Calleja, 2015). A differential diagnosis was performed, and the protocol adapted by Appleby et al. (2015) was applied.

Results

This individual died between the age of 5.5 and 6.5 years old according to tooth formation and femoral length. However, tooth eruption and bone development (e.g. unfused pars lateralis of the occipital bone) indicate a slower maturation (c. 3 years). Imaging observation revealed malpositioned lower anterior permanent teeth. The mandible and os coxae morphologies and metrics (correct classification from 83.33% to 86.66%) suggests a male individual.

The cranium exhibits diploic hyperplasia with thinning of the outer table covering the parietal and frontal bosses, with a maximum thickness of 9 mm, and an exuberant serpiginous pattern (Figure 2A). Conventional radiography and computed tomography facilitated visualizing a slight hair-on-end radial striated pattern in the parietals and frontal bones (Figure 2B). The bone proliferation does not involve the sagittal suture or the lower or posterior regions of the parietal or the occipital. No lesions were found on the inner table. Cranial fragments observed by SEM did not reveal erythrocytes. Both orbital roofs are covered by plaque-like layer of highly vascularized new bone that radiates from the lacrimal end (Figure 2C). The zygomatic bones and the lateral and inferior surfaces of the maxilla (Figure 2D) show deposition of new bone and porosity. There are no visible changes in both greater wings of the sphenoid and in the mandible.

Figure 2.

(A) Superior view of sk.485’s skull showing a serpiginous pattern. (B) Diploic hyperplasia visible in computed tomography. (C) Vascularized plaque-like new bone proliferation on the orbital roof. (D) Thickening and porosity of the left maxilla. (E) Radicular constrictions (double arrows) of the four deciduous canines and a pit-like enamel defect (dashed square). (F) Conventional radiology showing osteopenic foci located in the proximal third of the ulna and in the distal end of the right radius (arrows). (G) Anterior projection of humerus and femur showing Harris lines (arrows).

The four deciduous canines show circular constrictions on the roots, reducing the diameter from around 4.37–4.78 mm to 2.78–3.51 mm (Figure 2E). No shortening of root lengths was noticed. The right mandibular canine also has a pit-like enamel defect located on the mid-labial end of the crown (formed around the age of 6 months). In the permanent dentition, the central upper right incisor has four linear enamel hypoplasias, formed between 2 and 5 years of age, and the four first molars present defects in tooth enamel formation. Of the 19 erupted teeth, the anterior dentition shows occlusal wear, and the upper right and the lower left central incisors have interproximal caries. Dental calculus is primarily found on the labial, buccal, and occlusal surfaces of the left maxillary and mandibular dentition (Figure 2D–E).

In the postcranial skeleton, porous areas were recorded on the right humerus (cribra humeralis; left bone absent) and on both femoral necks (cribra femoralis). Four hand phalanges have foramina that appear slightly enlarged. Radiolucent trabecular osteopenic foci are visible in the proximal end of the ulna and in the distal end of the radius (Figure 2F). The distal ends of the right humerus and femur show Harris lines (Figure 2G). A mild proliferation of new bone is visible on the right tibia and fibula. No other alterations were observed macroscopically or radiographically in the skeleton.

Discussion

The most conspicuous lesion found on sk.485 is a severe serpiginous hyperostosis in the neurocranium, with a pronounced thickening of the diploe and slight hair-on-end radiographic appearance. In the skull a plaque-like apposition is visible in the orbital roofs, along with porosity in the facial bones and disturbance in dental development, while the postcranial skeleton shows slow maturation and osteopenia. These lesions led us to consider neoplasia, cyanotic congenital heart disease, acquired and congenital anemias, and leprosy in the differential diagnosis.

Malignant neoplasms can provoke bone growth, but the aspect is different and metastasis “occurs mostly after the fourth decade of life” (Greenspan and Borys, 2015; Marques, 2019). Leukemia is the most common cancer in the young but before modern treatment, children did not survive long enough to develop bone manifestations (Hutter, 2010; Klaus, 2016).

Polycythemia vera and cyanotic congenital heart disease (or critical congenital heart disease) are often considered in the paleopathological differential diagnosis of individuals with hair-on-end. The assumption that polycythemia vera induces diploic hyperplasia is based on a single paper (Dykstra and Halbertsma, 1940). Moreover, this is a myeloproliferative condition, diagnosed only in adults (Spivak, 2018; Peterson, 2000). Cyanotic congenital heart disease refers to any heart defect that reduces the oxygen delivered to the body (Holland et al., 2022) inducing erythroid hyperplasia to overcome chronic hypoxia (Walor et al., 2005). Prior to the development of surgical techniques, these children, so-called ‘blue babies,’ had a very short life expectancy and hair-on-end changes were uncommon (Walor et al., 2005).

Chronic iron deficiency anemia can cause porotic hyperostosis (Stuart-Macadam, 1987; Oxenham and Cavill, 2010). However, there is still controversy among paleopathologists (McIlvaine, 2015). Based on hematological research (Ron, 2002; Arndt et al., 2005), Walker et al. (2009) argued that the lack of iron inhibits, rather than promotes, marrow hypertrophy.

Congenital hemolytic anemias are a heterogeneous group of rare hereditary diseases characterized by erythroid hyperplasia and consequent marrow hypertrophy (Haley, 2017). The most frequent causes are erythrocyte membrane proteins defects such as spherocytosis and elliptocytosis, red cell enzyme deficiencies such as pyruvate kinase deficiency, and defective erythropoiesis as exhibited by hemoglobinopathies. Patients with spherocytosis, elliptocytosis, and pyruvate kinase deficiency usually have mild to moderately well-tolerated hemolytic anemia (Barcellini et al., 2011; Grace et al., 2018). Very rare severe phenotypes have been described causing hydrops fetalis, stillborn babies, or newborns needing hypertransfusion therapy until the spleen is removed (Manco et al., 1999; Ribeiro et al., 2000; Cortesi et al., 2021). Despite its rarity, cases of intermediate severity cannot be excluded. Unfortunately, the erythrocyte shape could not be evaluated in this example.

Severe and intermediate forms of β-thalassemia, SCD, and compounds heterozygotic for both diseases (HbS/β-thalassemia) must be considered in the differential diagnosis of marrow hypertrophy lesions. These hemoglobinopathies can coincide in a variety of skeletal lesions, with only a few characteristics that lead to a specific weighting of one over the other. Hair-on-end changes are more frequent and severe in thalassemia than in other types of anemias (Golding, 1956; Sebes and Diggs, 1979; Lewis, 2010). Also, Hershkovitz et al. (1997), Lagia et al. (2007) and Scianò et al. (2021) demonstrated that in thalassemia there is a more marked predisposition to induce cranial than postcranial lesions, as in the individual sk.485. The parietals and frontal bones shown exuberant lesions with no visible changes in the occipital and mandible bones usually less affected in hemoglobinopathies due to their less active bone marrow and cortical density (Adamopoulos and Petrocheilou, 2020).

Individual sk.485 shows bone growth in both orbital roofs that differs in appearance from the hyperplasia that may occur in thalassemia (Dinan et al., 2013), and from cribra orbitalia. Another disease that leads to porous new bone formation in the orbit is scurvy (Brickley et al., 2020). However, in this hypovitaminosis the porosity covers the cortical bone while in sk.485 it appears as plaque-like apposition. Diagnosis of scurvy also includes porosity in the sphenoid, zygomatics, and maxillae (Ortner and Ericksen, 1997; Brickley et al., 2020). Both zygomatics and maxillae of sk.485 show porosity but there are no visible changes in the greater wing of the sphenoid, considered pathognomonic of scurvy by Ortner and Ericksen (1997). Nonetheless, for Brickley et al. (2020), “[t]here are no pathognomonic features for scurvy.” These authors agree with the criteria that the porosity caused by scurvy must be less than 1 mm in diameter, which is not the case with sk. 485. This may suggest that the porosity do not result from hemorrhagic inflammation but was triggered by a hemolytic condition (Ortner and Ericksen, 1997; Brickley et al., 2020).

In paleopathological studies it is difficult to distinguish between SCD and β-thalassemia (Hershkovitz et al., 1997; Grauer, 2019). The craniofacial and dental involvement (Hattab, 2017; Helmi et al., 2017) in this individual, and the absence of skeletal necrotic signs (Almeida and Roberts, 2005), rule out SCD and potentiate the diagnosis of β-thalassemia, although a delay in maxillary and mandibular tooth eruption may also occur in SCD (Chekroun et al., 2019). Clinical studies emphasize repeated infarctions and ischemic SCD-related changes that may progress to avascular necrosis in the femoral head, shoulder, knee, and talus (Rees et al., 2010; Dinan et al., 2013; Serjeant, 2013), H-shaped or ‘tower’ vertebrae of the spine (Bunn, 1997), and premature closure of the epiphyses (Almeida and Roberts, 2005; Vaishya et al., 2015). As none of these changes were observed in sk.485, the diagnosis of SCD is less likely. The HbS/β-thalassemia comorbidity was also dismissed because, according to Reynolds et al. (1973) and Adekile et al. (2017), it privileges the typical skeletal lesions of SCD over β-Thal.

β-Thalassemia is classified into three phenotypic groups: thalassemia minor, intermedia, and major (Taher et al., 2006). Individuals heterozygous for β-thalassemia (β-thalassemia minor, β⁺/β or β⁰/β, patients with hemoglobin mutation, which can decrease (β⁺) or completely prevent (β⁰) β-globin chain synthesis) are asymptomatic and only have a mild well-tolerated anemia (Forget and Bunn, 2013). β-Thalassemia intermedia and β-thalassemia major have variable manifestations (Taher et al., 2006). In the first group, patients can be asymptomatic (β⁺/β⁺), and blood transfusions are only required in specific situations such as infection, pregnancy, or surgery (Forget and Bunn, 2013; Thein and Rees, 2015). In symptomatic cases (β⁰/β⁺), individuals frequently suffer from osteoporosis, pathological fractures (Asadov et al., 2017), and the iron overload is responsible for cardiovascular complications, the primary cause of morbidity and mortality (Dinan et al., 2013). Without any treatment, symptomatic individuals with β-thalassemia intermedia die before the age of 8 (Taher et al., 2010), while survival in β-Thal major is much lower (Weatherall and Clegg, 2001). Patients with β-thalassemia major are not able to synthesize β-globin chains by both genes (β⁰/β⁰) and consequently do not produce hemoglobin (Hb) A [adult] (α₂β₂) and only have Hb F [fetal] (α₂γ₂), which has high affinity for oxygen (Cao and Galanello, 2011; Forget and Bunn, 2013; Thein and Rees, 2015).

Patients with β-thalassemia intermedia and major can develop postcranial lesions, including subperiosteal marrow proliferation and new bone formation that covers the entire outer costal surface (rib-within-rib), small compression fractures in vertebrae (Adamopoulos and Petrocheilou, 2020), and Erlenmeyer flask deformities affecting particularly the humerus and femurs (Gosein et al., 2019; Adamopoulos and Petrocheilou, 2020). These alterations are not visible in sk.485 and are also scarcely identified in other possible paleopathological cases as can be seen in Table 1 and in the five infants (ages from 3 to 9 months) and an 8-year-old child reported by Tayles (1996) with unspecified genetic anemias, dated from the 2nd century CE, from the Khuk Phanin Di site (Thailand). None of the individuals reported in the bioarcheological record present all the lesions described in the clinical literature; rather, the diagnoses were made mainly due to diploic hyperplasia and hair-on-end radiological appearance. This radiological appearance usually occurs when the clinical symptoms of hemoglobinopathies are present after the 16th month of life, when the fetal hemoglobin (α₂γ₂) production reaches lower levels (Jorge et al., 2016). The study Filon et al. (1995) argued that the diagnosis of β-thalassemia was confirmed genetically but Yang (1997) later questioned the methodology applied.

Table 1.

Synthesis of possible paleopathological cases of β-thalassemia and Sickle Cell Disease (SCD) described in individuals under the age of 12 years old, organized chronologically.

Country	Archaeological site	Chronology	Individual/burial	Age at death	Lesions	Reference
β-Thalassemia cases
Vietnam	Man Bac	3906–3523 cal BP	MB07H1M12	0 y.o.	‘Alteration of the trabecular structure of the ilia’, ‘Enlargement of the scapular spines’ (p. 5)	Vlok et al. (2021)
			MB07H2M26	1.5 y.o.	Marrow hyperplasia, dental displacement, ‘rodent facies deformity’, rib-within-rib, ‘Poor or lack of pneumatization of the paranasal and cranial sinuses sparing the ethmoid sinuses’ (p. 3)
			MB05M12	2 y.o.	Hair-on-end, dental displacement, ‘rodent facies deformity’ (p. 3)
			MB05M3	0.5 y.o.	Hair-on-end, dental displacement, ‘rodent facies deformity’, rib-within-rib, ‘Widening of entire rib’ (p. 3)
Egypt	Luxor	1550–945 BCE	NA	6–8 y.o.	Hair-on-end	Herrerín et al. (2010)
Italy	San Giovenale	3rd cent. BCE	Tomb III	4–5 y.o.	‘[S]keletal changes similar to thalassaemia major and thus to anaemia of genetic origin’	Fornaciari and Malegni (1980) in Facchini et al. (2004)
Italy	Isola Sacra	1st–3rd cents. CE	SCR 8	9–12 m	Diploic thickening, no hair-on-end	Sperduti et al. (1996) in Yang (1997)
			SCR 136	9–12 m	Diploic thickening, slight hair-on-end
			SCR 277	6–12 m	Diploic thickening, no hair-on-end
			SCR 599	3–4 y.o.	Diploic thickening, hair-on-end
			SCR 1353	6–12 m	‘Pronounced rarefaction and disorganization of the trabecular tissue’
UK	Poundbury Camp	1st–5th cents. CE	PC525	1 y.o.	‘Mild hair-on-end formation’, ‘vertebral bodies were thinned and exhibited central depressions’, ‘hypertrophic and apparently fractured ribs’, ‘rib-within-a-rib’	Lewis (2010)
			PC1083	6 m	No evident hair-on-end lesions, ‘femora appear ‘plump’ with flared metaphyseal ends’, ‘areas of necrosis’
			PC920b	9 m	Porotic hyperostosis, hypertrophic sphenoid and zygomatic bone, ‘rodent’ face deformity costal osteomas
UK	Colchester	4th–5th cent. CE	G145	1–2 y.o.	Osteopenia, cortical microporosity, metaphyseal fractures, radiolucent bands, ‘ribs appear flared and thickened at the costochondral ends with increased porosity’	Rohnbogner (2016)
Italy	Piazza dei Miracoli	5th–7th cents. CE	NA	8 y.o.	Hair-on-end, retarded skeletal development.	Baggieri and Mallegni (2001)
Portugal	Almeirim	16th–18th cents. CE	sk.485	5.5–6.5 y.o	Diploic thickening, hair-on-end, delay in maturation of the occipital bone, plaque-like layer covering both orbital roofs, porosity in facial bones, osteopenic foci, postcranial cribra, possible enlarged foramina in four hand phalanges.	Current study
Israel	Akhziv	16th–19th cents. CE	NA	8 y.o.	Thickening of cranial bones, and ‘[h]oneycombed compartments of irregular subperiosteal bone formation. DNA: ‘homozygosity for frameshift in codon 8 of β-globin, causing a β null phenotype’	Filon et al. (1995)
Thailand	Chiengmai	NA	NA	8 y.o.	‘[M]arked thickening of the cranial vault’, hair-on-end, expansion of the facial bones, ‘disorderly eruption of the teeth’, rib expansion	Putschar (1962) in Ortner (2003)
SCD cases
Saint Helena	Rupert’s Valley	19th cent. CE (?)	438	7–12 y.o.	‘[B]ilateral premature fusion of the proximal humeral epiphyses and flattening and elongation of the distal ends of the bone’, necrosis	Witkin (2011) in Lewis (2018)
USA	Hamann–Todd collection	19th–20th cents. CE	HTH 1784	6 y.o.	‘No pathological changes were observed in the maxillary or frontal sinuses’, hair-on-end, ectocranial ‘ballooning’, maxillary thickening, thicker ribs, enlarged nutrient foramina, ‘Posterior calcaneal and specific articular surface disruptive metacarpal lesions’	Hershkovitz et al. (1997)

Cent(s)., century(ies); NA, not available; y.o., years old; m, postnatal months.

Individual sk.485 has constrictions on the roots of the deciduous canines that, to the best of authors’ knowledge, has not been previously reported. The length of the deciduous canine roots seems normal which excludes hypotrophic root anomalies and spiky-shaped roots (Hazza’a and Al-Jamal, 2006; Matos and Santos, 2013), while leprogenic odontodysplasia occur only in permanent teeth (Danielsen, 1968; Matos and Santos, 2013). Moreover, there are no individuals with lesions compatible with a diagnosis of leprosy in this necropolis and skeletal changes hardly happen at such a young age. These root constrictions would have occurred around the sixth postnatal month, coinciding with the age of onset for clinical symptoms of thalassemia and SCD, when the level of fetal hemoglobin is already low (Ashley-Koch et al., 2000; Dinan et al., 2013; Steinberg, 2016). This was also the age of formation of the pit-like enamel defect on the mandibular deciduous teeth. Given the controversy over Harris lines, the age at formation was not estimated. However, by its proximal location on the femur it can be assumed that it was formed in the first years of life (Kulus and Dąbrowski, 2019). These lesions, the delay in maxillary and mandibular tooth eruption, the malpositioned teeth, and the persistence of deciduous dentition, even with high degrees of root resorption, point to a pathochronic condition.

The constellation of non-specific lesions that thalassemia generates in the skeleton (Scianò et al., 2021) include enlarged foramina in the hand phalanges which are possibly present in the individual under study. The non-specificity of the lesions makes the diagnosis challenging. However, the age at death of the individual sk.485, – c. 6 years estimated by dental and femur development – excludes the diagnosis of β-thalassemia major, as this child could not have survived so long without regular blood transfusions. Consequently, the bone manifestation can be considered consistent with β-thalassemia intermedia. This type has been rarely found in the archaeological record from regions where it is prevalent nowadays (Lagia et al., 2007). In this necropolis, only one other individual (sk.436), an infant of c. 2.5–3.5 years, exhibited similar hyperplastic serpiginous lesions in the cranial vault (Figure 3). This skeleton was found in the same stratigraphic layer, 7 m southwest of sk.485. The poor preservation of this individual does not permit a differential diagnosis but the possibility that both non-adults had the same disease and some degree of kinship cannot be excluded.

Figure 3.

Fragment of the cranial vault of individual sk.436 showing a serpiginous pattern and diploic hyperplasia similar to sk.485.

Individual sk.485 also presents cribra humeralis and femoralis. These porous skeletal lesions have been found in individuals from malaria-endemic regions (Buckley and Tayles, 2003; Gowland and Western, 2012; Smith-Guzmán, 2015; Schats 2015, 2021; Gomes et al., 2022) such as Almeirim. However, its presence is not specific of malaria and these cribra can occur with other diseases. According to Couto (1859), this municipality was affected by recurrent floods from the Tagus River and by insect-borne diseases. Carriers of thalassemia are presumably at a selective advantage in malaria-endemic areas (Weatherall and Clegg, 2001; Piel et al., 2010), being frequent in Mediterranean populations (Piel et al., 2013; Angastiniotis et al., 2021).

Although individual sk.485 does not have all the lesions associated with thalassemia, the absence of bone lesions common in SCD patients, and the rarity of the severe forms of other hemolytic anemias, raises the possibility for β-thalassemia intermedia, which is supported by Almeirim being in a malaria-endemic region with a past Islamic Berber occupation, and a community of enslaved sub-Saharan Africans after the 15th century. According to Lewis (2010), the presence of thalassemia in Romano-British culture could be related to migration from the Mediterranean region. Thus, it is surprising that there are no cases of thalassemia in non-adults reported in the territories of France, Spain, and Portugal.

Conclusions

The non-adult individual described in this investigation has grossly and radiologically visible bone alterations suggestive of β-thalassemia intermedia. This individual has relevance because the appearance of this disease is associated with the arrival of populations from the Mediterranean/North Africa and sub-Saharan Africa and may represent the first case identified in the westernmost part of Europe. Moreover, thalassemia confers resistance against malaria, endemic in the region of Almeirim.

The bibliographic research carried out on hemoglobinopathies in non-adult individuals from archaeological contexts, on a global scale, is very scarce, given that these diseases are hereditary. Hopefully, in the near future, it will be possible to carry out genetic analyses to identify the causing mutations, to determine population affinity and possible kinship between the two children in this site with similar cranial lesions and search for Plasmodium aDNA.

Acknowledgments

The authors thank the Councilor of Culture, Eurico M. L. Henriques, and Odete Dias, Municipal Library from the Municipality of Almeirim, ERA-Arqueologia, SA, for allowing this study. The authors acknowledge the editor and the reviewers who helped to improve this work. Acknowledgments are also due to Jane Buikstra, Jorge Correia, Ricardo Gomes, Bruno Magalhães, and Vitor Matos. This research was funded by the Fundação para a Ciência e a Tecnologia (FCT), SFRH/BD/115691/2016 [AMC], and the Research Centre for Anthropology and Health (CIAS, UIDB/00283/2020).

Funding Declaration

This study was supported financially by Fundação para a Ciência e a Tecnologia (FCT), reference SFRH/BD/115691/2016 [AMC] and the Research Centre for Anthropology and Health (CIAS, UIDB/00283/2020).

Conflict of Interest

The authors declare that they have no conflicts of interest.

Authors Contribution

Álvaro M. Monge Calleja conducted the laboratory data collection and analysis, as well as the study conception and design. Marina Lourenço and Marta Macedo were responsible for the bioarchaeological excavation carried out, providing morphometric and photographic field information. Rosa Ramos Gaspar performed the imaging study. Álvaro M. Monge Calleja, M. Letícia Ribeiro and Ana Luísa Santos wrote the manuscript and refined the differential diagnosis, both aided by the suggestions provided by the remaining coauthors. Therefore, all the authors contributed to the elaboration of the manuscript and approved its submission.

References

•  Adamopoulos  S.G. and  Petrocheilou  G.M. (2020) Skeletal radiological findings in thalassemia major. Journal of Research and Practice on the Musculoskeletal System, 4: 76–85. doi: 10.22540/JRPMS-04-076.
•  Adekile  A.D.,  Akbulut  N.,  Azab  A.F.,  Al-Sharida  S., and  Thomas  D. (2017) The sickle β-thalassemia phenotype. Journal of Pediatric Hematology/Oncology, 39: 327–331. doi: 10.1097/MPH.0000000000000747.
•  Allison  A.C. (1954) Notes on sickle-cell polymorphism. Annals of Human Genetics, 19: 39–51. doi: 10.1111/j.1469-1809.1954.tb01261.x.
•  Almeida  L.O. (2015) Human gene mutations and migratory flows—Portugal and the Mediterranean. Advances in Anthropology, 5: 164–169. doi: 10.4236/aa.2015.53015.
•  Almeida  A. and  Roberts  I. (2005) Bone involvement in sickle cell disease. British Journal of Haematology, 129: 482–490. doi: 10.1111/j.1365-2141.2005.05476.x.
•  AlQahtani  S.J.,  Hector  M.P., and  Liversidge  H.M. (2010) The London Atlas of human tooth development and eruption. American Journal of Physical Anthropology, 142: 481–490. doi: 10.1002/ajpa.21258.
•  Angastiniotis  M.,  Cannon  L.,  Antoniou  E.,  Brunetta  A.L.,  Constantinou  G., et al (2021) Hemoglobin disorders in Europe: a systematic effort of identifying and addressing unmet needs and challenges by the Thalassemia International Federation. Thalassemia Reports, 11: 9803. doi: 10.4081/thal.2021.9803.
•  Appleby  J.,  Thomas  R., and  Buikstra  J. (2015) Increasing confidence in paleopathological diagnosis—application of the Istanbul terminological Framework. International Journal of Paleopathology, 8: 19–21. doi: 10.1016/j.ijpp.2014.07.003.
•  Arndt  U.,  Kaltwasser  J.P.,  Gottschalk  R.,  Hoelzer  D., and  Möller  B. (2005) Correction of iron-deficient erythropoiesis in the treatment of anemia of chronic disease with recombinant human erythropoietin. Annals of Hematology, 84: 159–166. doi: 10.1007/s00277-004-0950-z.
•  Asadov  C.,  Gafarova  S., and  Aliyeva  G. (2017) Bone abnormalities in β-thalassemia intermedia. Blood Research & Transfusion Journal, 1: 555551. doi: 10.19080/OABTJ.2017.01.555551.
•  Ashley-Koch  A.,  Yang  Q., and  Olney  R.S. (2000) Sickle hemoglobin (Hb S) allele and sickle cell disease: a HuGE review. American Journal of Epidemiology, 151: 839–845. doi: 10.1093/oxfordjournals.aje.a010288.
•  Baggieri  G. and  Mallegni  F. (2001) Morphopathology of some osseous alterations of thalassic nature. Paleopathology Newsletter, 116: 10–16.
•  Barcellini  W.,  Bianchi  P.,  Fermo  E.,  Imperiali  F.G.,  Marcello  A.P., et al (2011) Hereditary red cell membrane defects: diagnostic and clinical aspects. Blood Transfusion, 9: 274–277. doi: 10.2450/2011.0086-10.
•  Brickley  M.B.,  Ives  R., and  Mays  S. (2020) The Bioarchaeology of Metabolic Bone Disease. Academic Press, London.
•  Bruce-Chwatt  L.J. and  Zulueta  J. (1977) Malaria eradication in Portugal. Transactions of the Royal Society of Tropical Medicine and Hygiene, 71: 232–240. doi: 10.1016/0035-9203(77)90014-1.
•  Buckley  H. and  Tayles  N. (2003) Skeletal pathology in a prehistoric Pacific island sample: issues in lesion recording, quantification, and interpretation. American Journal of Physical Anthropology, 122: 303–324. doi: 10.1002/ajpa.10259.
•  Bunn  H.F. (1997) Pathogenesis and treatment of sickle cell disease. New England Journal of Medicine, 337: 762–769. doi: 10.1056/NEJM199709113371107.
•  Cao  A. and  Galanello  R. (2011) Beta-thalassemia. Genetics in Medicine, 12: 61–76. doi: 10.1097/GIM.0b013e3181cd68ed.
•  Cardoso  H.F.V.,  Abrantes  J., and  Humphrey  L.T. (2014) Age estimation of immature human skeletal remains from the diaphyseal length of the long bones in the postnatal period. International Journal of Legal Medicine, 128: 809–824. doi: 10.1007/s00414-013-0925-5.
•  Chekroun  M.,  Chérifi  H.,  Fournier  B.,  Gaultier  F.,  Sitbon  I.-Y., et al (2019) Oral manifestations of sickle cell disease. British Dental Journal, 226: 27–31. doi: 10.1038/sj.bdj.2019.4
•  Cortesi  V.,  Manzoni  F.,  Raffaeli  G.,  Cavallaro  G.,  Fattizzo  B., et al (2021) Severe presentation of congenital hemolytic anemias in the neonatal age: diagnostic and therapeutic issues. Diagnostics (Basel), 11: 1549. doi: 10.3390/diagnostics11091549.
•  Couto  G.T. (1859) Breve notícia de Almeirim. Associação de Defesa do Património Histórico-Cultural do Concelho, Almeirim (in Portuguese).
•  Cunningham  C.,  Scheuer  L., and  Black  S. (2016) Developmental Juvenile Osteology. Academic Press, London.
•  Custódio  J. (2008) Almeirim, Cronologia. Edições Cosmos (in Portuguese).
•  Danielsen  K. (1968) Leprogenic odontodysplasia (‘dens leprosus’). International Journal of Leprosy, 36: 666–667.
•  De Sanctis  V.,  Kattamis  C.,  Canatan  D.,  Soliman  A.T.,  Elsedfy  H., et al (2017) β-Thalassemia distribution in the Old World: an ancient disease seen from a historical standpoint. Mediterranean Journal of Hematology and Infectious Diseases, 9: e2017018. doi: 10.4084/mjhid.2017.018.
•  Denic  S.,  Nagelkerke  N., and  Agarwal  M.M. (2008) Consanguineous marriages and endemic malaria: can inbreeding increase population fitness? Malaria Journal, 7: 150. doi: 10.1186/1475-2875-7-150.
•  Dinan  D.,  Epelman  M.,  Guimaraes  C.V.,  Donnelly  L.F.,  Nagasubramanian  R., et al (2013) The current state of imaging pediatric hemoglobinopathies. Seminars in Ultrasound, CT and MRI, 34: 493–515. doi: 10.1053/j.sult.2013.05.005.
•  Dykstra  O.H. and  Halbertsma  T. (1940) Polycythaemia vera in childhood. Report of a case with changes in the skull. American Journal of Diseases of Children, 60: 907–916. doi: 10.1001/archpedi.1940.02000040126011.
•  Facchini  F.,  Rastelli  E., and  Brasili  P. (2004) Cribra orbitalia and cribra cranii in Roman skeletal remains from the Ravenna area and Rimini (I–IV century AD). International Journal of Osteoarchaeology, 14: 126–136. doi: 10.1002/oa.717.
•  Filon  D.,  Faerman  M.,  Smith  P., and  Oppenheim  A. (1995) Sequence analysis reveals a β-thalassaemia mutation in the DNA of skeletal remains from the archeological site of Akhziv, Israel. Nature Genetics, 9: 365–368. doi: 10.1038/ng0495-365.
•  Forget  C.G. and  Bunn  H.F. (2013) Classification of the disorders of hemoglobin. Cold Spring Harbor Perspectives in Medicine, 3: a011684. doi: 10.1101/cshperspect.a011684.
•  Golding  J.S. (1956) The bone changes in sickle cell anaemia. Annals of The Royal College of Surgeons of England, 19: 296–315.
•  Gomes  R.A.M.P.,  Petit  J.,  Dutour  O., and  Santos  A.L. (2022) Frequency and co-occurrence of porous skeletal lesions in identified non-adults from Portugal (19th to 20th centuries) and its association with respiratory infections as cause of death. International Journal of Osteoarchaeology, 32: 1061–1072. doi: 10.1002/oa.3132.
•  Gosein  M.,  Maharaj  P.,  Balkaransingh  P.,  Banfield  R.,  Greene  C., et al (2019) Imaging features of thalassaemia. British Journal of Radiology, 92(1095): 20180658. doi: 10.1259/bjr.20180658.
•  Gowland  R. and  Western  A. (2012) Morbidity in the marshes: using spatial epidemiology to investigate skeletal evidence for malaria in Anglo-Saxon England (AD 410–1050). American Journal of Physical Anthropology, 147: 301–311. doi: 10.1002/ajpa.21648.
•  Grace  R.F.,  Bianchi  P.,  van Beers  E.J.,  Eber  S.W.,  Glader  D., et al (2018) Clinical spectrum of pyruvate kinase deficiency: data from the Pyruvate Kinase Deficiency Natural History Study. Blood, 131: 2183–2192. doi: 10.1182/blood-2017-10-810796.
•  Grauer  A.L. (2019) Circulatory, reticuloendothelial, and hematopoietic disorders. In: Buikstra, J.E. (ed.), Ortner’s Identification of Pathological Conditions in Human Skeletal Remains. Academic Press, London, pp. 491–529.
•  Greenspan  A. and  Borys  D. (2015) Radiology and Pathology Correlation of Bone Tumors: A Quick Reference and Review. Lippincott Williams & Wilkins, Philadelphia.
•  Haldane  J.B.S. (1949) The rate of mutation of human genes. Hereditas, 35(S1): 267–273. doi: 10.1111/j.1601-5223.1949.tb03339.x.
•  Haley  K. (2017) Congenital hemolytic anemia. Medical Clinics of North America, 101: 361–374. doi: 10.1016/j.mcna.2016.09.008.
•  Hattab  F.N. (2017) Thalassemia major and related dentomaxillofacial complications: clinical and radiographic overview with reference to dental care. International Journal of Experimental Dental Science, 6: 95–104. doi: 10.5005/jp-journals-10029-1163.
•  Hazza’a  A.M. and  Al-Jamal  G. (2006) Radiographic features of the jaws and teeth in thalasseaemia major. Dentomaxillofacial Radiology, 35: 283–288. doi: 10.1259/dmfr/38094141.
•  Helmi  N.,  Bashir  M.,  Shireen  A., and  Ahmed  I.M. (2017) Thalassemia review: features, dental considerations and management. Electronic Physician, 9(3): 4003–4008. doi: 10.19082/4003.
•  Herrerín  J.,  Baxarias  J.,  Garcia-Guixé  E.,  Núñez  M., and  Dinarés  R. (2010) Betatalasemia em un niño de una necrópolis del Imperio Nuevo (Luxor, Egipto). Estudio macroscópico y radiológico. Imagen Diagnóstica, 1: 61–66. doi: 10.1016/S2171-3669(10)70014-7 (in Spanish).
•  Hershkovitz  I.,  Rothschild  B.M.,  Latimer  B.,  Dutor  O.,  Léonetti  G., et al (1997) Recognition of sickle cell anemia in skeletal remains of children. American Journal of Physical Anthropology, 104: 213–226. doi: 10.1002/(SICI)1096-8644(199710)104:2<213::AID-AJPA8>3.0.CO;2-Z.
•  Holland  J.E.,  Yeager  S.B.,  Glyer  R.H., and  Flyer  J.N. (2022) Neonatal blues: cyanosis and failure to thrive in a newborn. International Journal of Clinical Pediatrics, 11: 51–55. doi: 10.14740/ijcp486.
•  Hutter  J.J. (2010) Childhood leukemia. Pediatrics in Review, 31: 234–241. doi: 10.1542/pir.31-6-234.
•  Inez  F.,  Sequeira  M.,  Santos  P.,  Santos  R., and  Nunes  E. (1993) Contribuição do rastreio de portadores para a prevenção da β-talassémia e da drepanocitose na população portuguesa: estudo multicêntrico. Arquivos INSA, 19: 27–31 (in Portuguese).
•  Jorge  S.E.,  Ribeiro  D.M.,  Santos  M.N.N., and  Sonati  M.F. (2016) Hemoglobin: structure, synthesis and oxygen transport. In: Costa F.F. and Conran N. (eds), Sickle Cell Anemia. Springer, New York, pp. 1–22.
•  Klaus  H. (2016) Probable case of acute childhood leukemia: skeletal involvement, differential diagnosis, and the bioarchaeology of cancer in South America. International Journal of Osteoarchaeology, 26: 348–358. doi: 10.1002/oa.2411.
•  Kulus  M.J. and  Dąbrowski  P. (2019) How to calculate the age at formation of Harris lines? A step-by-step review of current methods and a proposal for modifications to Byers’ formulas. Archaeological and Anthropological Sciences, 11: 1169–1185. doi: 10.1007/s12520-018-00773-5.
•  Lagia  A.,  Eliopoulos  C., and  Manolis  S. (2007) Thalassemia: macroscopic and radiological study of a case. International Journal of Osteoarchaeology, 17: 269–285. doi: 10.1002/oa.881.
•  Lavinha  J.,  Gonçalves  J.,  Faustino  P.,  Romão  L.,  Osório-Almeida  L., et al (1992) Importation route of the sickle cell trait into Portugal: contribution of molecular epidemiology. Human Biology, 64: 891–901.
•  Lewis  M.E. (2010) Thalassemia: its diagnosis and interpretation in past skeletal populations. International Journal of Osteoarchaeology, 22: 685–693. doi: 10.1002/oa.1229.
•  Lewis  M.E. (2018) Paleopathology of Children. Identification of Pathological Conditions in the Human Skeletal Remains of Non-adults. Academic Press, London.
•  Lourenço  A.M.R. (2015) Relatório antropológico de campo. Igreja do Divino Espírito Santo—Almeirim. ERA—Arqueologia, SA (in Portuguese).
•  Luna  L.H.,  Aranda  C.M., and  Santos  A.L. (2017) New method for sex prediction using the human non-adult auricular surface of the ilium in the collection of identified skeletons of the University of Coimbra. International Journal of Osteoarchaeology, 27: 898–911. doi: 10.1002/oa.2604.
•  Luna  L.H.,  Aranda  C.M.,  Monge Calleja  A.M., and  Santos  A.L. (2021) Test of the auricular surface sex estimation method in fetuses and non-adults under 5 years old from the Lisbon and Granada Reference Collections. International Journal of Legal Medicine, 135: 993–1003. doi: 10.1007/s00414-020-02431-9.
•  Maat  G.J.R. (1991) Ultrastructure of normal and pathological fossilized red blood cells compare with pseudopathological biological structures. International Journal of Osteoarchaeology, 1: 209–214. doi: 10.1002/oa.1390010312.
•  Maat  G.J.R. and  Baig  S.D. (1990) Microscopy electron scanning of fossilized sickle-cells. International Journal of Anthropology, 5: 271–275. doi: 10.1007/BF02446253.
•  Macedo  M.L. (2015) Relatório final de trabalhos arqueológicos. Antigo Edifício das Escolas Velhas (Capela do divino Espírito Santo). Projeto No. 1429. Era Arqueología SA (in Portuguese).
•  Manco  L.,  Ribeiro  M.L.,  Almeida  H.,  Freitas  O.,  Abade  A., et al (1999) PK-LR gene mutations in pyruvate kinase deficient Portuguese patients. British Journal of Haematology, 105: 591–595. doi: 10.1046/j.1365-2141.1999.01387.x.
•  Marques  C. (2019) Tumors of bone. In: Buikstra J.E. (ed.), Ortner’s Identification of Pathological Conditions in Human Skeletal Remains. Academic Press, London, pp. 639–717.
•  Martins  M.C.,  Olim  G.,  Melo  J.,  Magalhães  H.Á., and  Rodrigues  M.O. (1993) Hereditary anaemias in Portugal: epidemiology, public health significance, and control. Journal of Medical Genetics, 30: 235–239. doi: 10.1136/jmg.30.3.235.
•  Matos  V.M.J. and  Santos  A.L. (2013) Leprogenic odontodysplasia: new evidence from the St Jørgen’s medieval leprosarium cemetery (Odense, Denmark). Anthropological Science, 121: 43–47. doi: 10.1537/ase.121112.
•  McIlvaine  B.K. (2015) Implications of reappraising the iron-deficiency anemia hypothesis. International Journal of Osteoarchaeology, 25: 997–1000. doi: 10.1002/oa.2383.
•  Monge Calleja  A.M. (2015) Reconstruindo a morte na infância: resultados preliminares da microscopia de varrimento como ferramenta diagnóstica da porosidade em vestígios osteológicos dos séculos XIX–XX. Dissertation, University of Coimbra. http://hdl.handle.net/10316/31298 (in Portuguese).
•  Monge Calleja  A.M.,  Aranda  C.M.,  Santos  A.L., and  Luna  L.H. (2020) Evaluation of the auricular surface method for non-adult sex estimation on the Lisbon documented collection. American Journal of Physical Anthropology, 172: 500–510. doi: 10.1002/ajpa.24012.
•  Ortner  D.J. (2003) Identification of Pathological Conditions in Human Skeletal Remains. Academic Press, London.
•  Ortner  D.J. and  Ericksen  M.F. (1997) Bone changes in the human skull probably resulting from scurvy in infancy and childhood. International Journal of Osteoarchaeology, 7: 212–220. doi: 10.1002/(SICI)1099-1212(199705)7:3<212::AID-OA346>3.0.CO;2-5.
•  Oxenham  M.F. and  Cavill  I. (2010) Porotic hyperostosis and cribra orbitalia: the erythropoietic response to iron-deficiency anaemia. Anthropological Science, 118: 199–200. doi: 10.1537/ase.100302.
•  Peterson  P. (2000) Myeloproliferative disorders. In: Schumacher H.R., Rock W.A., Jr, and Stass S.A. (eds.), Handbook of Hematologic Pathology. Taylor & Francis, New York, pp. 257–274.
•  Piel  F.B.,  Patil  A.P.,  Howes  R.E.,  Nyangiri  O.A.,  Gething  P.W., et al (2010) Global distribution of the sickle cell gene and geographical confirmation of the malaria hypothesis. Nature Communications, 1: 104. doi: 10.1038/ncomms1104.
•  Piel  F.B.,  Patil  A.P.,  Howes  R.E.,  Nyangiri  O.A.,  Gething  P.W., et al (2013) Global epidemiology of sickle haemoglobin in neonates: a contemporary geostatistical model-based map and population estimates. Lancet, 381(9861): 142–151. doi: 10.1016/S0140-6736(12)61229-X.
•  Rees  D.C.,  Williams  T.N., and  Gladwin  M.T. (2010) Sickle-cell disease. Lancet, 376(9757): 2018–2031. doi: 10.1016/S0140-6736(10)61029-X.
•  Reid  D.J. and  Dean  M.C. (2000) The timing of linear hypoplasias on human anterior teeth. American Journal of Physical Anthropology, 113: 135–139. doi: 10.1002/1096-8644(200009)113:1<135::AID-AJPA13>3.0.CO;2-A.
•  Reynolds  J.,  Pritchard  J.A.,  Ludders  D., and  Mason  R.A. (1973) Roentgenographic and clinical appraisal of sickle cell beta-thalassemia disease. American Journal of Roentgenology, 118: 378–400. doi: 10.2214/ajr.118.2.378.
•  Ribeiro  M.L.,  Alloisio  N.,  Almeida  H.,  Gomes  C.,  Texier  P., et al (2000) Severe hereditary spherocytosis and distal renal tubular acidosis associated with the total absence of band 3. Blood, 96: 1602–1604. doi: 10.1182/blood.V96.4.1602.
•  Rohnbogner  A. (2016) Differential diagnosis of a probable case of non-adult thalassaemia from 4th century AD Romano-British Colchester, UK. International Journal of Paleopathology, 15: 39–49. doi: 10.1016/j.ijpp.2016.08.002.
•  Ron  D. (2002) Translational control in the endoplasmic reticulum stress response. Journal of Clinical Investigation, 110: 1383–1388. doi: 10.1172/JCI0216784.
•  Schats  R. (2015) Malaise and mosquitos: osteoarchaeological evidence for malaria in the medieval Netherlands. Excerpta Archaeologica Leidensia, 45: 133–140.
•  Schats  R. (2021) Cribrotic lesions in archaeological human skeletal remains. Prevalence, co-occurrence, and association in medieval and early modern Netherlands. International Journal of Paleopathology, 35: 81–89. doi: 10.1016/j.ijpp.2021.10.003.
•  Schutkowski  H. (1993) Sex determination of infant and juvenile skeletons: I. Morphognostic features. American Journal of Physical Anthropology, 90: 199–205. doi: 10.1002/ajpa.1330900206.
•  Scianò  F.,  Bramanti  B., and  Gualdi-Russo  E. (2021) A new investigative strategy to diagnose β-thalassemia syndrome in past human populations. Archaeological and Anthropological Sciences, 13: 26. doi: 10.1007/s12520-020-01261-5.
•  Sebes  J.I. and  Diggs  L.W. (1979) Radiographic changes of the skull in sickle cell anemia. American Journal of Roentgenology, 132: 373–377. doi: 10.2214/ajr.132.3.373.
•  Serjeant  G.R. (2013) The natural history of sickle cell disease. Cold Spring Harbor Perspectives in Medicine, 3: a011783. doi: 10.1101/cshperspect.a011783.
•  Silva  S.M. (2007) Almeirim: no coração de Lezíria … Héstia Editores, Penamaior (in Portuguese).
•  Simões  C.S. (2015) Almeirim de outros tempos. Centro Paroquial de Bem-Estar Social, Almeirim (in Portuguese).
•  Smith-Guzmán  N.E. (2015) The skeletal manifestation of malaria: an epidemiological approach using documented skeletal collections. American Journal of Physical Anthropology, 158: 624–635. doi: 10.1002/ajpa.22819.
•  Spivak  J.L. (2018) Polycythemia vera. Current Treatment Options in Oncology, 19: 12. doi: 10.1007/s11864-018-0529-x.
•  Steinberg  M.H. (2016) Overview of sickle cell anemia pathophysiology. In: Costa F.F. and Conran N. (eds.), Sickle Cell Anemia. Springer, New York, pp. 49–74.
•  Stuart-Macadam  P. (1987) A radiographic study of porotic hyperostosis. American Journal of Physical Anthropology, 74: 511–520. doi: 10.1002/ajpa.1330740409.
•  Taher  A.,  Isma’eel  H., and  Cappellini  M.D. (2006) Thalassemia intermedia: revisited. Blood Cells, Molecules, and Diseases, 37: 12–20. doi: 10.1016/j.bcmd.2006.04.005.
•  Taher  A.T.,  Musallam  K.M.,  El-Beshlawy  A.,  Kirimi  M.,  Daar  S., et al (2010) Age-related complications in treatment-naïve patients with thalassaemia intermedia. British Journal of Haematology, 150: 480–497. doi: 10.1111/j.1365-2141.2010.08220.x.
•  Tayles  N. (1996) Anemia, genetic diseases, and malaria in prehistoric mainland Southeast Asia. American Journal of Physical Anthropology, 101: 11–27. doi: 10.1002/(SICI)1096-8644(199609)101:1<11::AID-AJPA2>3.0.CO;2-G.
•  Thein  S.L. and  Rees  D. (2015) Haemoglobin and the inherited disorders of globin synthesis. In: Hoffbrand A.V., Higgs D.R., Keeling D.M., and Mehta A.B. (eds.), Postgraduate Haematology. John Wiley, Oxford, pp. 72–97.
•  Thomas  J.A.,  Temple  D.H., and  Klaus  H.D. (2019) Crypt fenestration enamel defects and early life stress: contextual explorations of growth and mortality in colonial Peru. American Journal of Physical Anthropology, 168: 582–594. doi: 10.1002/ajpa.23775.
•  Vaishya  R.,  Agarwal  A.K.,  Edomwonyi  E.O.,  Vijay  V. (2015) Musculoskeletal manifestations of sickle cell disease: a review. Cureus, 7: e358. doi: 10.7759/cureus.358.
•  Viganó  C.,  Haas  C.,  Rühli  F.J., and  Bouwman  A. (2017) 2000 Year old β-thalassemia case in Sardinia suggests malaria was endemic by the Roman period. American Journal of Physical Anthropology, 164: 362–370. doi: 10.1002/ajpa.23278.
•  Vlok  M.,  Buckley  H.R.,  Miszkiewicz  J.J.,  Walker  M.M.,  Domett  K., et al (2021) Forager and farmer evolutionary adaptations to malaria evidenced by 7000 years of thalassemia in Southeast Asia. Scientific Reports, 11: 5677. doi: 10.1038/s41598-021-83978-4.
•  Walker  P.L.,  Bathurst  R.R.,  Richman  R.,  Gjerdrum  T., and  Andrushko  V. (2009) The causes of porotic hyperostosis and cribra orbitalia: a reappraisal of the iron-deficiency anemia hypothesis. American Journal of Physical Anthropology, 139: 109–125. doi: 10.1002/ajpa.21031.
•  Walor  D.M.,  Berdon  W.E., and  Westra  S.J. (2005) ‘Hair-on-end’ skull changes resembling thalassemia caused by marrow expansion in uncorrected complex cyanotic heart disease. Pediatric Radiology, 35: 698–701. doi: 10.1007/s00247-005-1403-0.
•  Weatherall  D.J. and  Clegg  J.B. (2001) The Thalassaemia Syndromes, 4th edn. Blackwell Science, Malden, MA.
•  Yang  D. (1997) DNA Diagnosis of Thalassemia from Ancient Italian Skeletons. Ph.D. dissertation, McMaster University, Hamilton. https://macsphere.mcmaster.ca/bitstream/11375/12939/1/fulltext.pdf