Biochemistry & Molecular Biology الكيمياء الحيوية والأحياء الجزيئية
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- ItemScreening of mutations in Polynucleotide Kinase 3' - Phosphatase gene causing Microcephaly, Seizures, and Developmental Delay in Palestine(Al-Quds University, 2025-01-05) Maysa "Mohammad Amer" Kamal Natsheh; ميساء "محمد عامر" كمال نتشةMicrocephaly, Seizures, and Developmental Delay (MCSZ) is an uncommon genetic disorder with an undetermined prevalence. It is inherited in an autosomal recessive pattern associated with either a homozygous or compound heterozygous mutation in the PNKP gene. This gene plays a crucial role in various DNA repair mechanisms, and its mutation leads to the continuous activation of the DNA damage response. This persistent activation is a consequence of the accumulation of double-strand breaks within the affected cells, which may result in the death of sensitive neuronal cells, potentially contributing to the pathogenesis of MCSZ. The disorder is primarily characterized by microcephaly, or an unusually small head size, along with a range of neurological impairments. Purpose: This research aims toscreen PNKP mutations in several Microcephaly, Seizure and Developmental Delay (MCSZ) disorder patients from Palestine. Methods: Three patients from different families were studied, who fulfilled our inclusion criteria: (1) Patients (males and females of different ages) having the general characteristics of MCSZ. (2) Their parents, especially if they are consanguineous in marriage determine if they are carriers for PNKP mutation, and/or family members. Genomic DNA was extracted after obtaining a blood samples which drawn for PNKP gene detection using PCR and Sanger sequencing. PNKP mutations were screenedand targeted the most common published exons (Exon 11, 14 and 15). Results:In this research, we identified a female patient exhibiting microcephaly, significant developmental delays, and early-onset refractory seizures, attributed to a homozygous mutation in the PNKP gene. This mutation, classified as likely pathogenic, is a missense variant denoted as NM_007254.4: c.968 C > T: p. (Thr323Met),located in exon 11 of thePNKP genes. The screening of first-degree relatives revealed that this genetic variant was inherited from both the father and mother who were identified as heterozygous for the MCSZ variant (GA). A pedigree was constructed following the screening of the affected individuals’ first-degree relatives. In contrast, samples from the other two patients did not exhibit any mutations in the PNKP gene. These samples were subsequently subjected to Whole Exome Sequencing, which confirmed the absence of mutations in the PNKP gene for both patients. Conclusion: Microcephaly, Seizures, and Developmental Delay (MCSZ) is underdiagnosed and undertreated in our population. Even simple knowledge among families toward consanguineous marriages, genetic counseling and cascade screening are essentialfor the diagnosis and prevention of MCSZ early in life. The results in this research will be considered as preliminary results on that disease among our population and more cohorts studiesand advanced genetic analysis are still needed.
- Itemmolecular Epidemiology: Prevalence of Human Cutaneous Leishmaniasis in palestine in the period between 2016 and 2024 using Next Generation Sequencing(Al-Quds University, 2025-01-12) Hanan Abdelmajeed Al-Jawabreh; حنان عبد المجيد عبد العزيز المناصرة /الجوابرةBackground: Leishmaniasis is a vector-borne disease caused by protozoan parasite of the genus Leishmania. The infection is transmitted by bite of infected female sandflies of the genus Phlebotomus in the old world or Lutzomyia in the new world. The diagnosis is made by stained smear microscopy, in-vitro culture, and DNA-based detection methods. Materials and Methods: In this study that spanned from 2016 to 2024 included patients from eleven districts in the West Bank of Palestine. The diagnostic methods used in the study included microscopy of Giemsa-stained touch smears, in-vitro culture using NNN medium, and ITS1-PCR. The amplicon-based next-generation sequencing of ITS1-219 was included in the comparison part of the study. The comparison arm of the study compared two groups, CL-confirmed group in w were positive by any of the tests used in the epidemiologic part of the study and a non-CL group which were negative by all diagnostic methods. The NGS1-219-PCR was compared to ITS1-PCR, which is considered as a gold standard owing to its superiority over microscopy and in-vitro culture. Results: The positivity rate during the study period was 17% (213/1262) with a prevalence of 7.0 per 100,000. The annual incidence rate 0.84 per 100,000 (25 cases per year) with approximately equal distribution between males (52%) and females (48%). The age range between 0 to 14 yrs was the most affected by CL. The CL lesions primarily affected the head (45%), followed by the upper extremities (38%) and the lower extremities (17%). The choropleth mapping showed that Jericho is still the district with highest annual incidence rate (21 per 100,000). The study revealed that Leishmania tropica is the predominating species with L. major restricted mainly to Jericho. The year 2017 was the last year to witness a CL peak in Palestine with Jericho contributing to 40% of peak cases. More than half (52%) of the cases appeared in the months of January to March. As for the comparison arm of the study, ITS1-219-NGS was shown to have a higher sensitivity of 94% compared to the imperfect gold standard (ITS1-PCR) of 86%. The agreement between the two tests was fair (Kappa-0.24) only agreed on 56% of the cases only. Furthermore, the standard ITS1-PCR was unable to genotype 29 CL cases but were genotyped by ITS1-219- NGS and confirmed by BLAST search. Unlike the ITS1-PCR, imperfect gold standard, ITS1-219-NGS genotypes all its positive results correctly as confirmed by BLAST search. Moreover, 13 ITS1-PCR weak positive CL cases were found to be negative by ITS1-219- NGS and upon BLAST search shown to be contamination of human and bacterial origin. Conclusion: Jericho area remains the main focus of CL in Palestine with L. tropica as the main species in the country and L. major restricted to Jericho area. Incidence rate has dropped compared to the two decades preceding this study as result of control measures implemented. Amplicon-based NGS is a feasible, highly sensitive, and high throughput diagnostic method with accurate species identification that can be used in clinical practice and epidemiologic survey.
- ItemMolecular Characterization of Borrelia Species as Causative Agents for Tick-Borne Relapsing Fever from Ixodidae and Argasidae in Palestine(Al-Quds University, 2025-01-09) Abbas Khader Hussin Masalma; عباس خضر حسين مسالمةIntroduction: Tick-Borne Relapsing Fever (TBRF) is primarily caused by several Borrelia species that are transmitted through the bites of ticks. It is mainly characterized by multiple recurrences of nonspecific signs and symptoms, including fever, headache, myalgia, arthralgia, and even some neurologic complications. These Borrelia species are mostly vectored by hard ticks like Rhipicephalus genus and soft ticks like Ornithodoros tholozani which is known as the main vector of B. persica in Palestine. However, Molecular based data on Borrelia species and their hosts in Palestine are very scarce as B. persica is the only Borrelia species that had been studied.Objectives: This study aimed to investigate the molecular epidemiology of Borrelia species in ectoparasite hard and soft ticks, collected from different animal hosts throughout Palestine.Methodology: Ticks samples were collected from different animal hosts residing in different districts through Palestine, all ticks were identified at the genus and species levels based on taxonomic keys. DNA extraction was carried out, then screening for the presence of Borrelial DNA was carried out by PCR targeting flagellin (fla B) gene. All positive samples were confirmed by 16sRNA – PCR. PCR products were loaded onto agarose gel for band detection at 250 bp for fla B and 523 bp for 16sRNA, positive samples were sequenced and analyzed for BLAST identification. Statistical analysis was carried out using IBM SPSS v27.0 software. Results: Among all tick samples: 86% (n= 734) were identified as Ixodidae (hard ticks) and 14% (n= 117) were Argasidae (soft ticks). Overall, 76% of the identified hard ticks belonged to the genus Rhipicephalus (92% R. sanguineous and 5.5% R. Turanicus), 12% of Haemaphysalis ticks (80% H. parva and 20% H. adleri), 11% were Hyalomma ticks (84% H. dromedarri, 6% H. impeltatum, and 5% H. eagyptium). All collected soft ticks (n= 117) were identified as Ornithodoros tholozani. Out of 734 hard ticks, 2% (n= 13) were detected to be positive for Borrelia DNA by PCR. Sequence analysis revealed the presence of B. persica in 77% (n=10) of positive samples, while 15% (n=2) were B. turcicae, and 8% (n=1) was B. Lonestari. On the other hand, 9% of soft ticks (n=17) were found to harbor B.persica.Conclusions: Our results highlight the presence of Borrelia persica among hard ticks despite its’ usual presence among soft ticks, this could indicate an incidental acquisition or ecological overlaps. B. persica genotype was also observed in soft ticks. Notably, in Palestine, R. sanguineus is the major hard ticks which vector Borrelia species among hard ticks, while O. tholozani is the major one among soft ticks.
- ItemMolecular and phylogenetic analysis of Enterobius- vermicularis from appendectomy specimens in the West Bank, Palestine(Al-Quds University, 2024-12-22) Issam Bassam Ahmed Jawabra; عصام بسام احمد جوابرهBackground A parasitic nematode known as Enterobius vermicularis (human pinworm) frequently causes gastrointestinal symptoms and, in some cases, acute appendicitis. Despite its widespread prevalence, Palestinian populations have limited molecular data on E. vermicularis. This study aims to investigate the molecular and phylogenetic characteristics of E. vermicularis in appendectomy specimens across Palestine. The study will concentrate on the COX1 gene to comprehend the genetic diversity and transmission patterns. Methods A number of 55 formalin-fixed, paraffin-embedded (FFPE) appendectomy specimens previously collected from 2018-2023, were confirmed for E. vermicularis infection. Samples originated from three government hospitals: Queen Alia Hospital in Hebron, Rafidia Hospital in Nablus, and Beit Jala Hospital in Bethlehem. DNA extraction using FFPE sections was carried out, followed by polymerase chain reaction (PCR) targeting the COX1 gene. Phylogenetic, sequence, and haplotype analysis were performed... Results The male-to-female ratio was 1.2:1, with 54.5% of patients being male and 45.5% female. The average age of patients was 13.3 years. There are no sequence variations among the sequenced samples, suggesting the conservation of this population's genetic structure. The phylogenetic analysis revealed the existence of Type B E. vermicularis in the Palestinian samples, which is exclusively associated with human hosts. Similarly, the haplotype analysis using median-joining network group all Palestinian samples in Group B. Statistical analysis of demographic and clinical variables; including age, gender, WBC, and neutrophil counts, showed no significant correlations with the infection Conclusion The study revealed a strong genetic uniformity among E. vermicularis samples from appendectomy samples in Palestine, suggesting limited transmission dynamics. All samples were identified as Type B, exclusive to human hosts when compared with global patterns. No significant associations were found between demographic or clinical manifestations and infection, indicating stable infection patterns across the population. The findings of the current research provide baseline molecular information about E. vermicularis infection in Palestine and may direct public health efforts by the Palestinian Ministry of Health in future research on the transmission and epidemiology of Enterobius.
- ItemDetection of Genetic Mutations in Inherited Glanzmann Thrombasthenia Patients in Hebron-Palestine.(Al-Quds University, 2025-01-05) Dalal ‘Mohammad Belal’ ‘Mohammad Dawoud’ Al-Hashlamon; دلال "محمد بلال" "محمد داوود" الهشلمونGlanzmann Thrombasthenia (GT) is a rare autosomal recessive bleeding disorder caused by mutations in genes critical for platelet aggregation, particularly the ITGA2B and/or ITGB3 genes, leading to defective platelet function. This disorder affects platelet function through impaired expression or dysfunction of the αIIbβ3 integrin, which plays a key role in blood aggregation. GT can be classified into three types based on αIIbβ3 integrin expression: Type I, where expression is absent or below 5% of normal levels; Type II, where expression is 5–20% of normal levels; and Type III, where integrin is present but functionally abnormal. Diagnosis involves platelet function tests and sequencing of the ITGA2B and ITGB3 genes. Early diagnosis, management, and genetic counselling are vital for improving patient outcomes and preventing complications. The global prevalence of GT is approximately 1 in 1,000,000 individuals. However, in regions with high rates of consanguinity, this prevalence can rise to as high as 1 in 200,000, or even greater. Purpose: This study aims to validate the diagnosis of a clinically suspected GT patient, explore the feasibility of cascade screening for the genetic disorder, and identify pathogenic variants associated with GT within the Palestinian population. Methods: Patients meeting the inclusion criteria were selected based on the following criteria: (1) A lifelong bleeding tendency characterized by a normal platelet count, prolonged bleeding time, normal PT, and normal APTT. (2) Primary symptoms included mucocutaneous bleeding, frequent nosebleeds (epistaxis) and easy bruising. (3) Family history of GT, including being an immediate family member of a diagnosed GT patient. The proband’s whole blood sample was analysed using WES. Based on the WES findings, specific primers were designed, and RT-PCR was performed. Subsequently, first-degree relatives were screened for the identified variant. Results: WES revealed that the proband has a gain variant of uncertain significance (VUS) in the ITGA2B gene: NM_000419.5. This variant involves a duplication spanning exons from 3 to 12, causing an alteration in the reading frame. Notably, this duplication has not been reported in the ClinVar database, suggesting it may be specific to the local population. RT-PCR analysis of the proband's first-degree relatives identified two additional affected family members and six carriers of the variant. However, the results for the proband’s parents remain inconclusive, warranting further investigation. A pedigree was constructed to map the inheritance pattern within the family. Conclusion: GT remains underdiagnosed and undertreated within our population. Our study highlights the potential of WES as a powerful tool for uncovering the molecular basis of GT, suggesting its integration into diagnostic practices could significantly improve both the understanding and management of this condition. Additionally, the duplication mutation identified in our research, spanning multiple exons, may be unique to our population, as it has not been previously reported in genetic databases. However, further research is needed to confirm its pathogenic significance and explore its broader implications for genetic screening.