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The molecular Egyptology field started in the mid-eighties with the first publication on the ancient DNA (aDNA) analysis of an Egyptian mummy. Egypt has been a major interest for historians, archeologists, laymen as well as scientists. The aDNA research on Egyptian biological remains has been fueled by their abundance and relatively well-preserved states through artificial mummification and by the advanced analytical techniques. Early doubts of aDNA integrity within the Egyptian mummies and data authenticity were later abated with studies proving successfully authenticated aDNA retrieval. The current review tries to recapitulate the published studies presenting paleogenomic evidence of disease diagnosis and kinship establishment for the Egyptian human remains. Regarding disease diagnosis, the prevailing literature was on paleogenomic evidence of infectious diseases in the human remains. A series of reports presented evidence for the presence of tuberculosis and/or malaria. In addition, there were solitary reports of the presence of leprosy, diphtheria, bacteremia, toxoplasmosis, schistosomiasis and leishmaniasis. On the contrary, paleogenomic evidence of the presence of rare diseases was quite scarce and mentioned only in two articles. On the other hand, kinship analysis of Egyptian human remains, including that of Tutankhamen, was done using both mitochondrial DNA sequences and nuclear DNA markers, to establish family relationships in four studies. It is clear that the field of molecular Egyptology is still a largely unexplored territory. Nevertheless, the paleogenomic investigation of Egyptian remains could make significant contributions to biomedical sciences (e.g. elucidation of coevolution of human host–microbe interrelationship) as well as to evidence-based archeology.

The tenth conference of the African Society of Human Genetics was held in Egypt with the theme "Human Genetics and Genomics in Africa: Challenges for Both Rare and Common Genetic Disorders." Current research was presented, and we discussed visions for the future of genomic research on the African continent. In this report, we summarize the presented scientific research within and relevant to Africa as presented by both African and non-African scientists. We also discuss the current situation concerning genomic medicine and genomic research within the continent, difficulties in implementing genetic services and genomic medicine in Africa, and a road map to overcome those difficulties and meet the needs of the African researchers and patients.

Background Although misdiagnosis of neuromyelitis optica spectrum disorder (NMOSD) with neuropsychiatric systemic lupus erythematosus (NPSLE) or multiple sclerosis (MS) is not infrequent, reliable biomarkers remains an unmet need. Extracellular microRNAs (miRNAs) represent a worthy avenue to identify biomarkers for differential diagnosis. We aimed to explore the potential role of some selected circulating miRNAs as biomarkers for the differential diagnosis in immune-mediated neuroinflammatory diseases. Methods A total of 80 subjects were enrolled in the present study, including 37 patients with MS (relapsing-remitting MS [RRMS; n = 18] and secondary progressive MS [SPMS; n = 19]), 10 patients with NMOSD and 10 patients with NPSLE as well as 23 healthy subjects. Serum expression levels of three selected miRNAs (miR-145, miR-223 and miR-326) were measured using quantitative real-time polymerase chain reaction (qRT-PCR). Whole blood expression levels of cellular immune response-relevant target genes, including signaling mother against decapentaplegic peptide 3 (SMAD3) and specificity protein 1 (SP1), were also measured using qRT-PCR. Results In comparison to healthy subjects, only miR-145 and miR-223 were significantly up-regulated in MS patients, whereas, all the analyzed miRNAs revealed insignificant upregulation in NMOSD patients. All the examined miRNAs were significantly down-regulated in NPSLE patients compared to healthy subjects. miR-145, miR-223 and miR-326 expression profile is a promising diagnostic biomarker for MS and NPSLE, but not for NMOSD. This expression profile is capable of differentiating not only among MS, NMOSD and NPSLE, but also between RRMS and SPMS. Conclusion Specific circulating miRNAs expression signature may have the potential to differentially diagnose immune-mediated neuroinflammatory diseases.

This study aimed at the detection of HSD17B3 gene mutations in Egyptian patients with suspected diagnosis of 46,XY DSD due to 17-β-HSD-3 deficiency and at evaluation of phenotype/genotype relationship of these mutations. The study was conducted on 11 patients of 10 families which were provisionally diagnosed to have 17-β-HSD-3 enzyme deficiency. Karyotyping, hormonal evaluation of testosterone, Δ4-androstenedione, and dihydrotestosterone, and sequencing analysis of the 11 exons of the HSD17B3 gene were done. Mutations in HSD17B3 were detected in exons 2, 7, 8, 10, and 11, and 6 novel mutations were determined in exons 1, 2, 7, and 8. Two patients showed compound heterozygous mutations, while 8 families had probands with homozygous mutations. The current study shows that 17-β-HSD-3 deficiency is not an uncommon disorder among Egyptian DSD cases. It was evidenced that the mutational profile of the disease is rather heterogeneous, relatively different from those reported in other populations, and has a high degree of novel genetic defects.

Background Mutations in the HSD17B3 gene are associated with a 46,XY disorder of sexual development (46,XY DSD) as a result of low testosterone production during embryogenesis. Aim To elucidate the molecular basis of the disorder by chemically analyzing four missense mutations in HSD17B3 (T54A, M164T, L194P, G289S) from Egyptian patients with 46,XY DSD. Methods Expression plasmids for wild-type 17β-hydroxysteroid hydrogenase type 3 (17β-HSD3) and mutant enzymes generated by site-directed mutagenesis were transiently transfected into human HEK-293 cells. Protein expression was verified by western blotting and activity was determined by measuring the conversion of radiolabeled Δ4-androstene-3,17-dione to testosterone. Application of a homology model provided an explanation for the observed effects of the mutations. Outcomes Testosterone formation by wild-type and mutant 17β-HSD3 enzymes was compared. Results Mutations T54A and L194P, despite normal protein expression, completely abolished 17β-HSD3 activity, explaining their severe 46,XY DSD phenotype. Mutant M164T could still produce testosterone, albeit with significantly lower activity compared with wild-type 17β-HSD3, resulting in ambiguous genitalia or a microphallus at birth. The substitution G289S represented a polymorphism exhibiting comparable activity to wild-type 17β-HSD3. Sequencing of the SRD5A2 gene in three siblings bearing the HSD17B3 G289S polymorphism disclosed the homozygous Y91H mutation in the former gene, thus explaining the 46,XY DSD presentations. Molecular modeling analyses supported the biochemical observations and predicted a disruption of cofactor binding by mutations T54A and M164T and of substrate binding by L196P, resulting in the loss of enzyme activity. In contrast, the G289S substitution was predicted to disturb neither the three-dimensional structure nor enzyme activity. Clinical Translation Biochemical analysis of mutant 17β-HSD3 enzymes is necessary to understand genotype-phenotype relationships. Strengths and Limitations Biochemical analysis combined with molecular modeling provides insight into disease mechanism. However, the stability of mutant proteins in vivo cannot be predicted by this approach. Conclusion The 17β-HSD3 G289S substitution, previously reported in other patients with 46,XY DSD, is a polymorphism that does not cause the disorder; thus, further sequence analysis was required and disclosed a mutation in SRD5A2, explaining the cause of 46,XY DSD in these patients.

17-β-Hydroxysteroid dehydrogenase type 3 deficiency is a rare autosomal recessive cause of 46,XY disorder of sex development. Worldwide, about 30 mutations in the hydroxysteroid (17-beta) dehydrogenase 3 (HSD17B3) gene have been reported, involving all exons except exon 1. Herein, we investigated an Egyptian female with 46,XY karyotype and low testosterone/Δ4-androstenedione ratio. Genomic DNA was extracted from blood samples, and then, direct DNA sequencing of HSD17B3 gene was performed. The patient had a homozygous mutation c.198G>A in exon 1 resulting in a stop codon (p.W50X). The study presents the first mutation to be reported in exon 1 of the HSD17B3 gene.

Objectives – This study aimed to examine the genotype–phenotype correlation in Duchenne muscular dystrophy (MD) patients with double deletion (Ddel) mutations in comparison with those having single deletions (Sdel). Materials and methods – The study included 250 Duchenne/Becker MD male patients from whom the 10 Ddel patients were compared with 20 Sdel subjects of same age and disease durations. The patients were subjected to neurological examination including functional disability grading scale (FDGS), molecular analysis of the dystrophin gene and immunohistochemical studies of some muscle biopsies. Results – The mean FDGS value in the Ddel group was lower than that in Sdel patients. The Ddel patients had partial expression of dystrophin in their skeletal muscles, while Sdel cases showed complete absence of the protein. Conclusion – Patients with double deletion mutations within the dystrophin gene have a milder phenotype than patients harboring single deletions at either major or minor hot spots of the gene.

OBJECTIVE Analysis of the 5alpha-reductase type 2 (SRD5A2) gene in Egyptian patients with suspected 5alpha-reductase (5alphaR) deficiency. PATIENTS AND METHODS Eight unrelated patients, originating from different geographical areas of Egypt, were referred to the Department of Pediatrics. Six prepubertal and two postpubertal patients presented with ambiguous genitalia. Four were being reared as females while the others were being reared as males. Six patients were products of consanguineous marriages. All patients had 46,XY karyotype. Basal and post-human chorionic gonadotrophin (hCG) stimulation plasma levels of testosterone and dihydrotestosterone were determined. Sequencing of five exons of the SRD5A2 gene was carried out. RESULTS All patients had normal male testosterone levels, both basal and post-hCG stimulation. The T/DHT ratio was available for six patients and showed values that ranged from normal to high. Three different homozygous mutations were identified. One patient carried a Y235F substitution and two had a N160D substitution. Interestingly, all five of the other patients had the G34R mutation. The parents were heterozygous for the mutations, although the mother of one patient was homozygous for the G34R mutation. CONCLUSION Among eight unrelated Egyptian children with 5alpha-reductase deficiency, the G34R mutation was identified in five patients. The high consanguinity rate in Egypt suggests a common ancestor with a founder gene effect in cases of G34R mutation.

DNA copy number changes were investigated in 69 samples of schistosoma-associated (SA) and nonschistosoma-associated (NSA) squamous cell carcinoma (SCC) and transitional cell carcinoma (TCC) of the bladder by comparative genomic hybridization (CGH). DNA copy number changes were detected in 47 tumors. SA tumors had more changes than NSA tumors (mean, 7 vs. 4), whereas the number of changes in SCC and TCC tumors was similar. SA tumors displayed more gains than losses (1.7:1), whereas NSA tumors showed an equal number of gains and losses. Changes that were observed at similar frequencies in SCC and TCC, irrespective of the schistosomal status, included gains and high-level amplifications at 1q, 8q, and 20q and losses in 9p and 13q. These changes may be involved in a common pathway for bladder tumor development and progression independent of schistosomal status or histological subtype. Losses in 3p and gains at 5p were seen only in SCC (P < 0.01) and losses in 5q were more frequent in SA-SCC than in other tumors (P < 0.05). However, changes that were more frequent in TCC than those in SCC included gains at 17q (P < 0.01) and losses in 4q (P < 0.05) and 6q (P < 0.01). Gains and high-level amplifications at 5p were seen only in SASCC (P < 0.01), whereas gains and high-level amplifications with minimal common overlapping regions at 11q13 were more frequently seen both in SA-SCC and SA-TCC tumors (P < 0.01). In addition to the above mentioned alterations, several other changes were also seen at lower frequencies. The variations in the DNA copy number changes observed in TCC, SCC, SA, and NSA bladder carcinomas suggest that these tumors have different genetic pathways., Facultad de Ciencias Naturales y Museo

Objective To investigate the true character of the harem conspiracy described in the Judicial Papyrus of Turin and determine whether Ramesses III was indeed killed. Design Anthropological, forensic, radiological, and genetic study of the mummies of Ramesses III and unknown man E, found together and taken from the 20th dynasty of ancient Egypt (circa 1190-1070 BC). Results Computed tomography scans revealed a deep cut in Ramesses III's throat, probably made by a sharp knife. During the mummification process, a Horus eye amulet was inserted in the wound for healing purposes, and the neck was covered by a collar of thick linen layers. Forensic examination of unknown man E showed compressed skin folds around his neck and a thoracic inflation. Unknown man E also had an unusual mummification procedure. According to genetic analyses, both mummies had identical haplotypes of the Y chromosome and a common male lineage. Conclusions This study suggests that Ramesses III was murdered during the harem conspiracy by the cutting of his throat. Unknown man E is a possible candidate as Ramesses III's son Pentawere.

We applied, for the first time, next-generation sequencing (NGS) technology on Egyptian mummies. Seven NGS datasets obtained from five randomly selected Third Intermediate to Graeco-Roman Egyptian mummies (806 BC–124AD) and two unearthed pre-contact Bolivian lowland skeletons were generated and characterised. The datasets were contrasted to three recently published NGS datasets obtained from cold-climate regions, i.e. the Saqqaq, the Denisova hominid and the Alpine Iceman. Analysis was done using one million reads of each newly generated or published dataset. Blastn and megablast results were analysed using MEGAN software. Distinct NGS results were replicated by specific and sensitive polymerase chain reaction (PCR) protocols in ancient DNA dedicated laboratories. Here, we provide unambiguous identification of authentic DNA in Egyptian mummies. The NGS datasets showed variable contents of endogenous DNA harboured in tissues. Three of five mummies displayed a human DNA proportion comparable to the human read count of the Saqqaq permafrost-preserved specimen. Furthermore, a metagenomic signature unique to mummies was displayed. By applying a “bacterial fingerprint”, discrimination among mummies and other remains from warm areas outside Egypt was possible. Due to the absence of an adequate environment monitoring, a bacterial bloom was identified when analysing different biopsies from the same mummies taken after a lapse of time of 1.5 years. Plant kingdom representation in all mummy datasets was unique and could be partially associated with their use in embalming materials. Finally, NGS data showed the presence of Plasmodium falciparum and Toxoplasma gondii DNA sequences, indicating malaria and toxoplasmosis in these mummies. We demonstrate that endogenous ancient DNA can be extracted from mummies and serve as a proper template for the NGS technique, thus, opening new pathways of investigation for future genome sequencing of ancient Egyptian individuals.

The 5 alpha-reductase type 2 deficiency is an autosomal recessive disorder of sexual development among 46,XY individuals. In Egypt, there is a prevalence of a G34R disease underlying mutation. This study aimed to devise a rapid diagnostic method based on allele specific PCR (AS-PCR) and a linked polymorphism (V89L). The results showed that one set of primers was capable to differentiate between normal, heterozygous, and affected individuals efficiently. All 34R mutation carrying sequences had 100% linkage to the 89L allele, contrasting normal ones with low 89L frequencies. This linkage infers a founder effect among Egyptians having G34R mutation.

We report a novel mutant of the luteinizing hormone receptor (LHR) in a case of familial Leydig cell hypoplasia and pseudohermaphrotidism. The proband was homozygous for two missense mutations, T1121C and C1175T, causing substitutions I374T and T392I. The molecular effects of the mutations were investigated by heterologous expression of the WT LHR, the double mutant LHR, or receptors with either the I374T or the T392I mutation, and measuring hormone binding and cAMP signaling. All mutant LHRs exhibited severe defects, including loss of ligand binding and cAMP production. Immunoblots showed little difference in protein levels between the WT and mutant receptors.

Analysis of the 5alpha-reductase type 2 (SRD5A2) gene in Egyptian patients with suspected 5alpha-reductase (5alphaR) deficiency.Eight unrelated patients, originating from different geographical areas of Egypt, were referred to the Department of Pediatrics. Six prepubertal and two postpubertal patients presented with ambiguous genitalia. Four were being reared as females while the others were being reared as males. Six patients were products of consanguineous marriages. All patients had 46,XY karyotype. Basal and post-human chorionic gonadotrophin (hCG) stimulation plasma levels of testosterone and dihydrotestosterone were determined. Sequencing of five exons of the SRD5A2 gene was carried out.All patients had normal male testosterone levels, both basal and post-hCG stimulation. The T/DHT ratio was available for six patients and showed values that ranged from normal to high. Three different homozygous mutations were identified. One patient carried a Y235F substitution and two had a N160D substitution. Interestingly, all five of the other patients had the G34R mutation. The parents were heterozygous for the mutations, although the mother of one patient was homozygous for the G34R mutation.Among eight unrelated Egyptian children with 5alpha-reductase deficiency, the G34R mutation was identified in five patients. The high consanguinity rate in Egypt suggests a common ancestor with a founder gene effect in cases of G34R mutation.

Bladder carcinoma accounts for 26% of reported human malignancies in Egypt, and has been strongly associated with urinary schistosomiasis. Nevertheless, the immediate role of schistosomal egg proteins in bladder carcinogenesis is unexplored. We investigated the effects of crude soluble egg antigens (SEA) of Schistosoma hematobium on urothelial cell proliferation. The proliferation of bovine endothelial Endo, human urothelial J82 and smooth muscle SMC cell lines was assessed by low-density growth assays. SEA induced proliferation of both J82 and Endo cells in a dose-dependent fashion, but not SMC. Preboiling or proteinase K treatment of SEA abolished its effect. In addition, SEA enhanced urothelial expression of B-cell translocation protein (BTG1) and human proliferating cell nuclear antigen (PCNA) mRNAs. Given the strong correlation between cell proliferation and carcinogenesis, the findings suggest that crude SEA may play some role in schistosomal bladder carcinogenesis.

The enzyme 5 alpha-reductase (5 alpha R), by virtue of its peripheral 5 alpha-reduction of testosterone (T) to dihydrotestosterone (DHT), is believed to play a major role in the differentiation and the subsequent growth of the penis. However, recent studies have reported 5 alpha R deficiency (5 alpha RD) in patients with isolated micropenis and hypothesized that 5 alpha RD is not invariably associated with genital ambiguity. In Egypt, 5 alpha RD has been reported frequently among intersex patients. The aim of this study was to assess the role of 5 alpha RD in the development of micropenis among Egyptian patients with abnormal sexual development. The study included 29 patients who were categorized into three groups (isolated micropenis, 9 patients; microphallus with genital ambiguity, 11 patients; genital ambiguity with normal-sized phallus, 9 patients). Activity of 5 alpha R was assessed by estimating T/DHT ratios in the basal state in pubertal subjects and following human chorionic gonadotropin (HCG) stimulation test in prepubertals. The results showed that the incidence of 5 alpha RD was much higher in cases of ambiguous genitalia with micropenis (5 families out of 10, 50%) than in those with isolated microphallus (1/9, 11.1%) or those with ambiguous genitalia and normal-sized phallus (1/8, 12.5%). In conclusion, the study showed that isolated micropenis is a heterogeneous disorder and that 5 alpha RD, despite its relative prevalence in Egypt, has a minimal role in the aetiology. On the other hand, 5 alpha RD seems to correlate with penile length in intersex cases.

Context The New Kingdom in ancient Egypt, comprising the 18th, 19th, and 20th dynasties, spanned the mid-16th to the early 11th centuries BC. The late 18th dynasty, which included the reigns of pharaohs Akhenaten and Tutankhamun, was an extraordinary time. The identification of a number of royal mummies from this era, the exact relationships between some members of the royal family, and possible illnesses and causes of death have been matters of debate. Objectives To introduce a new approach to molecular and medical Egyptology, to determine familial relationships among 11 royal mummies of the New Kingdom, and to search for pathological features attributable to possible murder, consanguinity, inherited disorders, and infectious diseases. Design From September 2007 to October 2009, royal mummies underwent detailed anthropological, radiological, and genetic studies as part of the King Tutankhamun Family Project. Mummies distinct from Tutankhamun's immediate lineage served as the genetic and morphological reference. To authenticate DNA results, analytical steps were repeated and independently replicated in a second ancient DNA laboratory staffed by a separate group of personnel. Eleven royal mummies dating from circa 1410-1324 BC and suspected of being kindred of Tutankhamun and 5 royal mummies dating to an earlier period, circa 1550-1479 BC, were examined. Main Outcome Measures Microsatellite-based haplotypes in the mummies, generational segregation of alleles within possible pedigree variants, and correlation of identified diseases with individual age, archeological evidence, and the written historical record. Results Genetic fingerprinting allowed the construction of a 5-generation pedigree of Tutankhamun's immediate lineage. The KV55 mummy and KV35YL were identified as the parents of Tutankhamun. No signs of gynecomastia and craniosynostoses (eg, Antley-Bixler syndrome) or Marfan syndrome were found, but an accumulation of malformations in Tutankhamun's family was evident. Several pathologies including Kohler disease II were diagnosed in Tutankhamun; none alone would have caused death. Genetic testing for STEVOR, AMA1, or MSP1 genes specific for Plasmodium falciparum revealed indications of malaria tropica in 4 mummies, including Tutankhamun’s. These results suggest avascular bone necrosis in conjunction with the malarial infection as the most likely cause of death in Tutankhamun. Walking impairment and malarial disease sustained by Tutankhamun is supported by the discovery of canes and an afterlife pharmacy in his tomb. Conclusion Using a multidisciplinary scientific approach, we showed the feasibility of gathering data on Pharaonic kinship and diseases and speculated about individual causes of death.

A case of familial Leydig cell hypoplasia as a cause of male pseudohermaphroditism is described in two 46, XY female sibs. Biochemical and histologic evidence for such diagnosis is presented.

Prolonged exposure of genital skin fibroblasts (GSF) to dihydrotestosterone (DHT) increases androgen receptor binding of steroid, a process termed ‘up-regulation’. Because the extent of up-regulation appears to be quite variable, we have investigated the optimal conditions and the molecular mechanisms that control this phenomenon in seven strains of normal neonatal GSF. When GSF were incubated for 1–48 h with 3 nM methyltrienolone (R1881), maximal up-regulation was reached by 20 h and remained constant thereafter. With DHT, rapid steroid metabolism required replenishment of DHT for maximum up-regulation. Up-regulation levels following 20 h incubation with DHT (including steroid replenishment) and R1881 were 2.07-fold (range = 1.1–3.3) and 2.35-fold (range = 1.86–3.33), respectively. The greater variability observed with DHT may be related to variable rates of steroid catabolism among cell strains. Half-maximal up-regulation was attained at 0.29 nM R1881, which approximates the Kd. Maximal up-regulation was reached only with continuous exposure to R1881 for 24 h. It was completely inhibited by actinomycin D (0.5 μg/ml) or cycloheximide (10 μg/ml). Following up-regulation, removal of R1881 for 24 h resulted in a highly variable decline of androgen receptors among cell strains. Maximal up-regulation could be reinduced by exposure to R1881 again for an additional 24 h. During up-regulation, androgen receptor levels in nuclei and nuclear matrix rose with increments comparable to those obtained in whole cells. We conclude that the extent of up-regulation and its rate of decline differ greatly among normal cell strains. Hence, its study in cells of patients with androgen insensitivity may have limited value. Our data showed that up-regulation requires DNA transcription and protein synthesis. Receptor stabilization is also involved, as continuous presence of steroid is required for full up-regulation. Finally, our results suggest that up-regulation plays a role in the high affinity binding of androgen receptors hi the nuclear matrix.

Plasma levels of sex-hormone-binding globulin (SHBG) in man are known to be regulated up and down by oestradiol and testosterone, respectively. To determine whether testosterone reduces SHBG level directly or via its conversion into dihydrotestosterone before puberty, the changes of plasma SHBG, testosterone, oestradiol and dihydrotestosterone following human chorionic gonadotrophin stimulation are studied in ten 5 alpha-reductase-deficient and in six normal prepubertal boys. Three main observations provide evidence that dihydrotestosterone plays a major role in SHBG regulation. (1) Basal plasma SHBG in 5 alpha-reductase-deficient is higher than in normal boys (P less than 0.1). (2) Circulating SHBG fails to decrease (P greater than 0.1) after human chorionic gonadotrophin stimulation despite striking elevation of plasma testosterone in 5 alpha-reductase deficiency where negligible dihydrotestosterone response occurs. This is in contrast to normal boys where SHBG is significantly reduced (P less than 0.01) after stimulation. (3). In normal boys the magnitude of plasma dihydrotestosterone response to human chorionic gonadotrophin correlates with that of SHBG (r = 0.72) more than testosterone does versus SHBG (r = 0.36). It is concluded that dihydrotestosterone decreases SHBG concentration in plasma of prepubertal boys. At least part of the observed decrease in SHBG following testosterone administration in earlier reports must have occurred after its conversion to dihydrotestosterone.