The genetic landscape of human infertility is complex with diverse etiologies. Identifying the underlying etiology is crucial for guiding reproductive decisions and improving management for infertile couples. Here, we aim to report on the molecular spectrum of monogenic genetic causes of reproductive failure. Over a 3-year period, we recruited all infertile couples considering assisted reproductive technologies (ART) for whom the underlying genetic cause had been identified, in either partner, using exome sequencing (ES). Clinical data of all participants along with their hormonal profiles, sonographic findings and spermograms were recorded. The study included 50 couples with primary infertility. Clinically, male factor infertility was documented in 26 patients, female factor infertility in 10, while reproductive failure was unexplained in the remaining 14 couples. All participating couples had potentially disease-causing variants in infertility genes. ES identified variants related to male infertility in 26 men, while variants in female infertility-related genes were detected in the remaining couples (n = 24). According to ACMG classification criteria, 78% (39/50) of couples harbored pathogenic/likely pathogenic (P/LP) variants, whereas 22% (11/50) carried variants of uncertain significance (VUS). In view of the identified genetic etiologies, the cohort was stratified into two groups based on the predicted reproductive outcome: (1) couples with significantly impaired reproductive potential, and (2) couples who can have biological children using appropriate medical interventions. However, classifications involving VUS were interpreted cautiously and considered exploratory. This study provides further evidence for the molecular heterogeneity of human infertility and highlights the usefulness of genetic testing for infertile couples pursuing ARTs.

PurposeAnophthalmia and microphthalmia (A/M) are among the most severe developmental eye defects. The aim of this study is to describe the genetic landscape of fetal syndromic phenotypes that include A/M.MethodsWe recruited 31 fetuses who underwent prenatal ultrasound examination, postnatal assessment, quantitative fluorescent PCR (QF-PCR) and prenatal exome sequencing (pES).ResultsAll cases displayed A/M associated with at least one extra-ocular malformation and of these, cerebellar hypoplasia was the most common ultrasound finding, detected in 14 (45.2%) cases. Chromosomal aneuploidies were identified in seven cases. Among the other fetuses (n=24), pES identified single nucleotide variants (SNVs) in 21 and copy number variations (CNVs) in three. Recurrent genetic diagnoses within our cohort included muscular dystrophy-dystroglycanopathy type A-3 (MDDGA3; n=3), MDDGA1 (n=2), cerebro-oculo-facio-skeletal syndrome 3 (n=2), syndromic microphthalmia type 9 (n=2), and Fraser syndrome (n=2). We report on seven novel variants.ConclusionsThis study broadens the molecular spectrum of syndromic A/M with 19 distinct variants identified across 16 different genes. Furthermore, some variants were detected in genes that have been rarely, or not previously, linked to human A/M, thereby highlighting atypical clinical findings and suggesting a possible expansion of the phenotypic spectrum associated with these genes.

A 7-day-old female neonate presented with dysmorphic features and multiple congenital anomalies displaying bilateral microphthalmia, congenital heart disease and postaxial polydactyly. Additionally, the brain MRI showed Dandy-Walker malformation, agenesis of corpus callosum, ventriculomegaly and hypoplastic brainstem. G-banded chromosome analysis showed that the child was 47,XX,t(13;18),+mar. Chromosome analysis of the parents showed that the father had a normal karyotype and that the mother had a balance between 13 and 18. MLPA and array CGH showed that the chromosome marker was derived from 18p and 13q. array CGH revealed that the patient had partial trisomy 13q14.3q34 (53,057,362-115,107,733)x3 and partial trisomy 18p11.32p11.21(263,821-14,058,294)x3. This neonate is the first to be liveborn with a condition in which partial trisomy 13q and trisomy 18p coexist. The infant developed hydrocephalus and died at 7 months of age. This report demonstrates that array CGH is a valuable diagnostic tool in determining the origin of additional small genetic materials. Proper genetic and prenatal counseling is very important for future family planning.

Celiac disease (CD) is one of the most common autoimmune disorders. It is triggered by exposure to dietary gluten proteins resulting in small intestine mucosal injury. Previous studies showed that CD is highly associated with human leukocyte antigens (HLA) class II DQ heterodimers, mainly HLA-DQ2.5 and HLA-DQ8. The aim of the work was to evaluate the distribution of the CD associated risk HLA-DQ haplotypes in CD patients, CD patients with Type 1 diabetes mellitus (T1DM) comorbidity, in at-risk and healthy individuals in an Egyptian cohort. The study included 124 individuals, divided into 4 groups. They were 28 CD patients, 21 CD and T1DM patients diagnosed with T1DM comorbidity, 50 at-risk group including relatives of CD patients and T1DM patients and finally 25 normal individuals as controls. The multiplex ligation-dependent probe amplification (MLPA) assay was performed using peripheral blood DNA. HLA-DQ2.5 was the most frequent haplotype among CD patients (69.3%) and among the combined groups in the cohort population (58.1%), either homozygous or heterozygous (together with HLA-DQ8 or -DQ2.2).HLA-DQ8 was the second most frequent haplotype followed by HLA-DQ2.2 and HLA-DQ7.5. In the control group, two individuals carried HLA-DQ2.3 and one carried a single DQB1*02:01 allele. In the at-risk group, 7 individuals were negative for all the haplotypes investigated. In conclusion, CD is a multifactorial disease where HLA-DQ haplotypes are a major genetic predisposing factor for both development and progress of the CD disease.

To evaluate the diagnostic yield of prenatal exome sequencing (pES) in fetuses with structural anomalies detected by prenatal ultrasound in a consanguineous population. This was a prospective study of 244 anomalous fetuses from unrelated consanguineous Egyptian families. Detailed phenotyping was performed throughout pregnancy and postnatally, and pES data analysis was conducted. Genetic variants were prioritized based on the correlation of their corresponding human phenotype ontology terms with the ultrasound findings. Analyses were carried out to determine the diagnostic efficiency of pES and its correlation to the organ systems involved. The largest clinical category of fetuses referred for pES was those manifesting multisystem anomalies (104/244, 42.6%). pES provided a definitive diagnosis explaining the fetal anomalies in 47.1% (115/244) of the cases, with the identification of 122 pathogenic or likely pathogenic variants completely fitting with the phenotype. Variants of uncertain significance associated with the fetal phenotypes were detected in 84 fetuses (34%), while 18.44% (45/244) had negative results. Positive consanguinity is associated with a high diagnostic yield of ES. The novel variants and new fetal manifestations, described in our cohort, further expand the mutational and phenotypic spectrum of a wide variety of genetic disorders presenting with congenital malformations.

Introduction: The fluorescence in situ hybridization (FISH) is a very important technique, as it can diagnose many genetic disorders and cancers. Molecular cytogenetic analysis (FISH) can diagnose numerical chromosome aberrations, sex chromosomes anomalies, and many genetic disorders. Aim: With the limited number of commercially available probes that do not cover all research needs and the high prices of the commercial probes, our goal is to apply recent technologies to produce FISH probes that can accurately and sensitively diagnose genetic diseases and cancer in Egypt and establishing the inhouse production of different FISH probes. We intend to adhere to the published guidelines and validation procedures to ensure the production of accurate FISH probes for clinical diagnosis. Methods: We used specific DNA segments extracted from BAC clones, and we performed nick translation to label the segment with fluorescence labeled dye. The second method involved the use of specific primers for the centromere of certain chromosomes and using PCR technique for amplification and labeling. The probes were tested on metaphase and interphase cells derived from cultured human peripheral blood samples. We followed standard guidelines to test the adequacy of probe slide hybridization, proper probe localization, probe sensitivity and specificity, probe reproducibility, cut-off values, and overall probe validation. Results: In this research, we presented the generation of three dual-color probes, each probe has a control locus. We offered three dual-color probes targeted 9p21, Xp21 and 17p13.1 loci. chromosome 9p21probe for diagnosis of structural abnormalities in chromosome 9, the Xp21 to test for structural abnormalities of chromosome X, and the 17p13.1 for TP53 gene to detect the loss of p53. We also produced probes for Down syndrome specific region, Rb gene and centromeres for chromosomes X, 17, and 18. Conclusion: The produced probes are specific and sensitive and can be produced at the commercial level in the laboratory. The production of FISH probes in Egypt can be used as a powerful diagnostic marker for genetic disorders and cancers and our work can be consider as a base to start national project to produce our needs of FISH probes.

Trisomy 4p is a rare constitutional chromosomal rearrangement that leads to severe intellectual disability, characteristic facial features including a characteristic nose with a flat bridge and a bulbous tip (boxer nose), and extremities abnormalities. Pure trisomy 4p syndrome is due to duplication of the entire p arm of chromosome 4. Genotype-phenotype correlations in pure trisomy 4p cases are not well understood. Only five cases of pure trisomy 4p was previously reported in form of iso 4p, derivative formation and marker formation. Here, we present the clinical and laboratory findings of fifth case of pure trisomy 4p with interesting MRI manifestations. Our patient's karyotype was defined as 47,XY,+(4)(pter→q11) mat that was inherited from his mother who had balanced translocation defined as 46, XX, t(4;12)(q12;q24.33). Our case is the sixth case of pure trisomy 4p and the 2nd report of pure trisomy 4p due to the presence of marker. Our findings emphasize and strengthen the clinical features of patients with pure trisomy 4p. More accurate clinical reports of patients with pure trisomy 4p is needed for more elucidation of the pathogenesis of pure trisomy 4p syndrome.

Background Fanconi anemia is a genetically heterogeneous recessive disorder distinguished by cytogenetic instability, hypersensitivity to DNA crosslinking agents, increased chromosomal breakage, and disturbed DNA repair. To date, Fanconi anemia complementation group (FANC) includes 23 FANC genes identified of which, FANCA gene is the most commonly mutated. The mutation spectrum of the FANCA gene is highly heterogeneous with large intragenic deletions due to Alu elements-mediated recombination.The study aimed to identify different deletion mutations on FANCA gene in Egyptian Fanconi anemia patients by multiplex ligation-dependent probe amplification (MLPA) technique to define the spectrum of FA molecular pathology as a step for disease control. The study included 80 FA patients (36 females and 44 males) whose ages ranged from 4 months to 17 years descending from unrelated consanguineous families referred to the Hereditary Blood Disorders Clinic, National Research Centre (NRC), Egypt. Patients were diagnosed with classical clinical presentation of FA and were confirmed by chromosomal breakage using Diepoxybutane (DEB). Results The common clinical presentation in our FA patients were the presence of café au lait spots with hyperpigmentation in 65/80 (81%) followed by skeletal defects in 40/80 (50%). MLPA revealed a total of five different intragenic homozygous deletions of FANCA gene in 16 /80 (20%) patients, among them two deletion patterns were novel. Conclusion Molecular analysis using MLPA could detect pathogenic mutations in 20% of FA patients, our study generated considerable data on causative mutations that was used for genetic counseling and prenatal diagnosis.

Objective: The purpose of this study was to improve our understanding of severe serine biosynthesis defects through a comprehensive description of prenatal, and postnatal manifestations and the mutational spectrum in a new cohort of 12 unrelated Egyptian Families. Methods: Detailed fetal ultrasound examination, postnatal assessment, and whole exome sequencing (WES) were performed in a cohort of 12 fetuses with suspected Neu-Laxova syndrome (NLS), the most severe expression of serine biosynthesis defects. Additionally, a comprehensive review of the literature was conducted by merging the data from all the molecularly-confirmed cases with ours to gain a better understanding of the clinical variability of NLS. Results: Novel clinical manifestations including intrauterine convulsions, hemivertebrae, natal teeth, holoprosencephaly, and rhombencephalosynapsis were observed. Molecular analysis identified 7 and 2 likely disease-causing variants in the PSAT1 and PHGDH genes, respectively. Four of them were novel, including the c.734G>A missense variant in PSAT1, which has been proposed to be a founder variant among Egyptians. Conclusion: The present cohort expands the spectrum of serine biosynthesis disorders. Moreover, it illuminates the role of prenatal exome sequencing in lethal conditions constituting the most severe end of already-known human diseases.

Background: Telomere length quantification had evolutionary been used in assessment and linking the phenotype and severity of many diseases, among these progeroid syndromes. Aim: This study aims to measure the individual telomere length in premature aging syndromes through establishment of the new technique of Quantitative Fluorescence In Situ Hybridization (Q-FISH) and the use of telomere length as an early diagnostic tool, prognostic factor and follow up tool for the premature aging syndromes patients. Patients and methods: This study was conducted on 27 patients and 10 normal controls matching in age and sex, patients were grouped into Fanconi anemia (FA) and non-Fanconi anemia groups. All patients and normal controls were subjected to thorough clinical assessment and blood sample were taken for Q-FISH. Results: Non-FA group had shown shorter telomere length than that of FA group. Patients having high number of clinical abnormalities had shown shorter telomere length, while patients with better hemoglobin and higher platelet levels had shown longer telomeres. Conclusion: Q-FISH technique was now well-established in our cytogenetics department as a diagnostic method for the measurement of telomere length, and it was used in this study to assess the telomere length in all patients’ and control groups. Q-FISH as a powerful tool for measurement and assessment of telomere length is recommended to be done in all patients suffering from premature aging syndromes and progeroid features, and could be considered as a prognostic marker for these syndromes.

Introduction: Pontocerebellar hypoplasia (PCH) represents a group of rare disorders with prenatal onset and time-dependent loss of brain parenchyma, predominantly affecting the cerebellum and pons with variable involvement of supratentorial structures. Radiologically and pathologically, they are characterized by small cerebellum and pons. Our study aimed to screen for the TSEN54 gene variants in Egyptian patients with PCH for proper counseling and to describe the brain MRI and the clinical phenotype and compare, them to those described in the literature. Methods: Thirty patients from thirty Egyptian families with a diagnosis of PCH based on neuroimaging findings were selected. Clinical evaluation, radiological findings, and genetic investigations were done for all patients. Results: The common missense variant c.919G>T (p.A307S) was identified in only 6 patients from six unrelated families (6/30; 20%) who showed different degrees of pontocerebellar malformations on brain imaging. Conclusion: The presence of a dragonfly/butterfly-like pattern in the coronal section of the cerebellum recommends genetic testing of TSEN54 as a first step. For negative cases, whole-exome sequencing is essential to reach a definite diagnosis and determine the etiology.

MAD2L1BP-encoded p31comet mediates Trip13-dependent disassembly of Mad2-and Rev7-containing complexes and, through this antagonism, promotes timely spindle assembly checkpoint (SAC) silencing, faithful chromosome segregation, insulin signaling, and homology-directed repair (HDR) of DNA double-strand breaks. We identified a homozygous MAD2L1BP nonsense variant, R253*, in 2 siblings with microcephaly, epileptic encephalopathy, and juvenile granulosa cell tumors of ovary and testis. Patient-derived cells exhibited high-grade mosaic variegated aneuploidy, slowed-down proliferation, and instability of truncated p31comet mRNA and protein. Corresponding recombinant p31comet was defective in Trip13, Mad2, and Rev7 binding and unable to support SAC silencing or HDR. Furthermore, C-terminal truncation abrogated an identified interaction of p31comet with tp53. Another homozygous truncation, R227*, detected in an early-deceased patient with lowlevel aneuploidy, severe epileptic encephalopathy, and frequent blood glucose elevations, likely corresponds to complete loss of function, as in Mad2l1bp-/- mice. Thus, human mutations of p31comet are linked to aneuploidy and tumor predisposition.

Objective: To describe the prenatal findings, associated anomalies, chromosomal abnormalities of fetuses with ventral wall defects (VWDs).Methods: Detailed fetal anomaly scan, postnatal assessment, and chromosomal analysis were performed in 200 fetuses with VWDs.Results: The omphalocele subtype was the most frequently encountered with 121 cases (60%), among them Pentalogy of Cantrell (POC) and OEIS complex (omphalocele, exstrophy of bladder, imperforate anus, spinal defects) were presented in 6 cases and 5 cases, respectively.The second most common variety was gastroschisis in 63 cases (31.5%).Additionally, 12 fetuses were found to have body stalk anomaly, while ectopia cordis and bladder exstrophy each were detected in 2 fetuses.Abnormal karyotype was found in 93 cases (49.4 %); the most frequently was trisomy 18(64.5%)followed by trisomy 13 (22.5%),trisomy 21(5.37%) and 45,X (4.3%).One case of POC had ring chromosome 13 karyotyping, one case with ectopia cordis had 45,X and a case of body stalk anomaly with sacrocooygeal teratoma was associated with monosmy 21.Major structural anomalies were detected in 104 cases (86%) with omphalocele and 22 cases (34.9%) with gastroschisis. Conclusion:Our study highlights the clinical and genetic heterogeneity of VWD especially the severe forms.

Background: Currently identified genetic and environmental factors account only for 50% of celiac disease (CD) pathogenesis. Recently, scientists directed special focus on the role of epigenetic factors particularly DNA methylation in predisposition of CD to open new windows for better disease pathogenesis elucidation, leading to identifying novel therapeutic targets. We aimed for the first time to investigate the DNA methylation level of the promoter area of MTHFR, MALT1 and MAP3K7 genes in peripheral blood of CD patients. Materials and methods: This pilot study included 50 Egyptian newly diagnosed pediatric patients with CD and 30 healthy subjects. DNA methylation status in peripheral blood was evaluated using methyl specific polymerase chain reaction. Results: The level of MTHFR methylation in patients was significantly higher in comparison with controls (p = 0.001), while that of MAP3K7 and MALT methylation showed no statistically significant difference between the two groups (p = 0.2, 0.4 respectively). Conclusions: Our data may suggest the possible role of MTHFR promotor hypermethylation in the pathogenesis of CD in Egyptian pediatric patients. Further studies should be constructed to evaluate the effect of gluten free diet on methylation level.

Background Aplastic anemia (AA) is a bone marrow disorder characterized by peripheral pancytopenia and marrow hypoplasia which can lead to life-threatening complications. Our objective was to study the telomerase genes ( TERT and TERC ) variants, explore their relationship to telomere shortening and TERT gene expression, and to identify variants in the MPL gene within Egyptian AA patients. Methods Forty AA patients and 40 sex- and age-matched healthy individuals as the control group were studied through sequencing of TERT , TERC , and MPL genes. Quantitative real-time PCR (qRT-PCR) was used for measuring TERT gene expression. Telomere length (TL) was measured using the Quantitative Fluorescence In Situ Hybridization (Q-FISH) technique. In silico analysis was performed for the prediction of the pathogenicity of resultant variants. Results Sequencing of MPL , TERT , and TERC genes identified 26 variants. Eleven variants were identified in the MPL gene. Three of them are pathogenic: two missense [c.305 G>A, c.1589 C>T] and one splice site [g.9130T>G]. TERT gene sequencing showed thirteen variants, among them, four novel [c.484G>A, c.499G>A, c.512G>A, c.3164C>G] and two previously reported [c.835G>A, c.2031C>T] were predicted to be pathogenic. Two variants were characterized within the TERC gene; n.514A>G and n.463 C>T. TERT gene expression was downregulated in 70% of studied patients and the Q-FISH technique detected telomere shortening in 82.5% of patients. Conclusions Twenty-six pathogenic and benign variants within the TERC , TERT , and MPL genes were identified among the studied AA patients that were in several cases associated with shortened telomeres and/or lower TERT gene expression. Genotype/phenotype correlation in AA patients is of great importance in explaining the disease severity and guiding therapeutic decisions.

Background The deletions of azoospermic factor regions (AZF) are considered risk factor of spermatogenic failure. AZF duplications or complex copy number variants (CNVs) were rarely studied because STS-PCR could not always detect these changes. The application of multiplex ligation-dependent probe amplification (MLPA) as a valuable test for detection of the deletion and or duplication was introduced to investigate the AZF sub-region CNVs. The MLPA technique is still not applied on a large scale, and the publications in this area of research are limited. The aim of this work was to evaluate the efficacy of MLPA assay to detect AZF-linked CNVs in idiopathic spermatogenic failure patients and to evaluate its importance as a prognostic marker in the reproduction outcome. Results Forty infertile men (37 with azoospermia and 3 with severe oligozoospermia) and 20 normal fertile men were subjected to thorough clinical, pathological, and laboratory assessment, chromosomal study, MLPA, STS-PCR assays, histopathology study, and testicular sperm retrieval (TESE). Out of the 40 patients, 7 patients have shown CNV in the AZFc region, 6 patients have partial deletion, and one patient has partial duplication. Only one of the normal control has AZFc duplication. STS-PCR was able to detect the deletion in only 4 out of the 7 positive patients and none of the control. Conclusion We concluded that MLPA should be applied on a larger scale for the detection of Y chromosome microdeletion as a rapid, efficient, and cheap test.

BACKGROUND:The prenatal diagnosis of syndromes caused by chromosomal abnormality is a long-established part of obstetric care. Several DNA-based molecular approaches have provided rapid prenatal diagnosis of of cytogenomic abnormalities. MLPA has become available for rapid aneuploidy detection of the most common chromosome abnormalities.OBJECTIVES:The aim of this study is to introduce the MLPA technique as a method for the prenatal detection of aneuploidy in Egypt by its validation compared to the FISH technique.METHODS:Fifty AF samples were collected for this study and were subjected to MLPA and FISH assays to detect the most common prenatal chromosomal abnormality.RESULTS AND CONCLUSIONS:Our study confirmed previous reports that MLPA is analogous to FISH for detecting common aneuploidies and could be a quick and dependable tool for prenatal diagnosis. Therefore, initial prompt testing of AF samples for the copy number of the most common occurring aneuploidies is recommended.