EMC10 homozygous variant identified in a family with global developmental delay, mild intellectual disability, and speech delay.

In recent years, several genes have been implicated in the variable disease presentation of global developmental delay (GDD) and intellectual disability (ID). The endoplasmic reticulum membrane protein complex (EMC) family is known to be involved in GDD and ID. Homozygous variants of EMC1 are associated with GDD, scoliosis, and cerebellar atrophy, indicating the relevance of this pathway for neurogenetic disorders. EMC10 is a bone marrow-derived angiogenic growth factor that plays an important role in infarct vascularization and promoting tissue repair. However, this gene has not been previously associated with human disease. Herein, we describe a Saudi family with two individuals segregating a recessive neurodevelopmental disorder. Both of the affected individuals showed mild ID, speech delay, and GDD. Whole-exome sequencing (WES) and Sanger sequencing were performed to identify candidate genes. Further, to elucidate the functional effects of the variant, quantitative real-time PCR (RT-qPCR)-based expression analysis was performed. WES revealed a homozygous splice acceptor site variant (c.679-1G>A) in EMC10 (chromosome 19q13.33) that segregated perfectly within the family. RT-qPCR showed a substantial decrease in the relative EMC10 gene expression in the patients, indicating the pathogenicity of the identified variant. For the first time in the literature, the EMC10 gene variant was associated with mild ID, speech delay, and GDD. Thus, this gene plays a key role in developmental milestones, with the potential to cause neurodevelopmental disorders in humans.

Congenital disorder of glycosylation with defective fucosylation 2 (FCSK gene defect): The third report in the literature with a mild phenotype.

BACKGROUND: Congenital disorders of glycosylation (CDG) are a group of heterogeneous disorders caused by abnormal lipid or protein glycosylation. Variants in the FCSK gene have been reported to cause CDG. Defective FCSK-induced CDG (FCSK-CDG) has only been reported previously in three unrelated children. METHODS: In this study, we genetically and clinically examined a 3-year-old proband with resolved infantile spasms and normal development. Standard whole-exome sequencing (WES) and Sanger sequencing were performed to identify the functional impact of the variant. RESULTS: WES revealed a rare biallelic missense variant (c.3013G>C; p.Val1005Leu) in FCSK. RT-qPCR showed a significant depletion in FCSK gene expression in the affected individual. Western blotting revealed reduced FCSK expression at the protein level compared to that in the control. Furthermore, 3D protein modeling suggested changes in the secondary structure, which might affect the overall FCSK protein function. CONCLUSION: This study broadens the mutation and phenotypic spectrum of FCSK-associated developmental disorders.

Novel homozygous pathogenic mitochondrial DNAJC19 variant in a patient with dilated cardiomyopathy and global developmental delay.

BACKGROUND: Dilated cardiomyopathy with ataxia syndrome (DCMA) or 3-methylglutaconic aciduria type V is a rare global autosomal recessive mitochondrial syndrome that is clinically and genetically heterogeneous. It is characterized by early-onset dilated cardiomyopathy and increased urinary excretion of 3-methylglutaconic acid. As a result, some patients die due to cardiac failure, while others manifest with growth retardation, microcytic anemia, mild ataxia, and mild muscle weakness. DCMA is caused by variants in the DnaJ heat shock protein family (Hsp40) member C19 gene (DNAJC19), which plays an important role in mitochondrial protein import machinery in the inner mitochondrial membrane. METHODS: We describe a single affected family member who presented with cardiomyopathy, global developmental delay, chest infection, seizures, elevated excretion of 3-methylglutaconic acid, and 3-methylglutaric acid in the urine. RESULTS: Whole-exome sequencing followed by Sanger sequencing revealed a homozygous frameshift variant in the reading frame starting at codon 54 in exon 4 in the DNAJC19 gene (c.159del [Phe54Leufs*5]), which results in a stop codon four positions downstream. Quantitative gene expression analysis revealed that DNAJC19 mRNA expression in this patient was substantially reduced compared to the control. CONCLUSIONS: We present a novel variant in the DNAJC19 gene that causes rare autosomal recessive mitochondrial 3-methylglutaconic aciduria type V. By comparing the current case with previously reported ones, we conclude that the disease is extremely heterogeneous for reasons that are still unknown.

Non-invasive prenatal testing for autosomal recessive disorders: A new promising approach.

Background: In pregnant women at risk of autosomal recessive (AR) disorders, prenatal diagnosis of AR disorders primarily involves invasive procedures, such as chorionic villus sampling and amniocentesis. Methods: We collected blood samples from four pregnant women in their first trimester who presented a risk of having a child with an AR disorder. Cell-free DNA (cfDNA) was extracted, amplified, and double-purified to reduce maternal DNA interference. Additionally, whole-genome amplification was performed for traces of residual purified cfDNA for utilization in subsequent applications. Results: Based on our findings, we detected the fetal status with the family corresponding different genes, i.e., LZTR1, DVL2, HBB, RNASEH2B, and MYO7A, as homozygous affected, wild-type, and heterozygous carriers, respectively. Results were subsequently confirmed by prenatal amniocentesis. The results of AmpFLSTR™ Identifiler™ presented a distinct profile from the corresponding mother profile, thereby corroborating the result reflecting the genetic material of the fetus. Conclusion: Herein, we detected AR disease mutations in the first trimester of pregnancy while surmounting limitations associated with maternal genetic material interference. Importantly, such detection strategies would allow the screening of pregnant women for common AR diseases, especially in highly consanguineous marriage populations. This technique would open avenues for the early detection and prevention of recessive diseases among the population.

Identification of the TTC26 Splice Variant in a Novel Complex Ciliopathy Syndrome with Biliary, Renal, Neurological, and Skeletal Manifestations.

Ciliopathies constitute heterogeneous disorders that result from mutations in ciliary proteins. These proteins play an important role in the development of organs, physiology, and signaling pathways, and sequence variations in the genes encoding these proteins are associated with multisystem disorders. In this study, we describe a severe ciliopathy disorder that segregates in an autosomal recessive manner in a nonconsanguineous Saudi family. The proband exhibited features such as cholestasis, cystic dilatation of intrahepatic biliary ducts, diabetes insipidus, dysmorphic facial features, optic atrophy, pituitary hypoplasia, hydrocephalus, aqueductal stenosis, hyperextensible knee joints, bilateral knee dislocation, polydactyly, and syndactyly. Whole-genome sequencing and Sanger sequencing revealed a homozygous splice site variant (c.4-1G>C; NM_024926.3) in the tetratricopeptide repeat domain 26 (TTC26) gene located in chromosome 7q34, which cosegregated perfectly with the disease phenotype. qRT-PCR revealed a substantial decrease in the expression of the TTC26 gene as compared to the normal control, suggesting the pathogenicity of the identified variant. This report further strengthens the evidence that homozygous variants in the TTC26 gene cause severe ciliopathies with diverse phenotypes. We named this newly characterized condition as BRENS syndrome, which stands for biliary, renal, neurological, and skeletal features.

Mutated VWA8 Is Associated With Developmental Delay, Microcephaly, and Scoliosis and Plays a Novel Role in Early Development and Skeletal Morphogenesis in Zebrafish.

Von Willebrand A domain-containing protein 8 (VWA8), also named KIAA0564, is a poorly characterized, mitochondrial matrix-targeted protein having a putative ATPase activity. VWA8 is comprising of ATPase-associated domains and a VWFA domain associated with ATPase activity inside the cell. In the present study, we describe a large consanguineous family of Saudi origin segregating a complex developmental syndrome in an autosomal recessive fashion. All the affected individuals exhibited severe developmental disorders. DNA from three patients was subjected to whole-exome sequencing followed by Sanger sequencing. VWA8 knock-down zebrafish morpholinos were used to study the phenotypic effect of this gene on zebrafish development. A homozygous missense variant [c.947A > G; p.(Asp316Gly)] was identified in exon 8 of the VWA8 gene, which perfectly segregated with the disease phenotype. Using zebrafish morpholino, we observed delayed development at an early stage, lack of movement, light sensitivity, severe skeletal deformity such as scoliosis, and facial dysmorphism. This is the first homozygous variant identified in the VWA8 gene underlying global developmental delay, microcephaly, scoliosis, limbs, and cardiovascular malformations in humans. We provide genetic and molecular evidence using zebrafish morpholino for a homozygous variant in the VWA8 gene, associated with such a complex developmental syndrome in humans.

Next Generation Sequencing Based Non-invasive Prenatal Testing (NIPT): First Report From Saudi Arabia.

Background: Non-invasive prenatal testing (NIPT) for aneuploidy in pregnant women screening has been recently established in Saudi Arabia. We aim from this study to report our experience in the implementation of this new technology in clinical practice and to assess factors influencing cell-free fetal (cffDNA) fraction and successful NIPT reporting. Methods: In total, 200 pregnant women were subjected to the NIPT test using standard methods. Next-generation sequencing (NGS) was used to analyze cffDNA in maternal plasma. Results: Out of the 200 NIPT cases, the average age of pregnant women was 35 ± 6 years (range: 21-48 years). The average cffDNA fraction of reported cases was 13.72% (range: 3-31%). Out of these 200 cases, 187 (93.5%) were at low risk, while 13 (6.5%) cases revealed high risk for aneuploidy. Among these chromosomal abnormalities, 7 (3.5%) cases of Down's syndrome, 5 (2.5%) Edwards' Syndrome, and only 1 case of (0.5%) Patau's syndrome was observed. Out of the 13 high-risk cases, 2 (15.3%) were found in women below the age of 30. Conclusion: This is the first study reporting the successful implementation of an in-house NIPT screening service in Saudi Arabia. Our data showed high accuracy and sensitivity to detect high-risk cases indicating the usefulness of such a technique as an alternative to invasive testing and (hopefully) will change the common screening practice for pregnant women in Saudi Arabia.

Next-Generation Sequencing-Based Pre-Implantation Genetic Testing for Aneuploidy (PGT-A): First Report from Saudi Arabia.

Recently, high-throughput next-generation sequencing (NGS)-based preimplantation genetic testing for aneuploidies techniques came into use. This technique is essential for successful embryo transfer and accomplishing pregnancy, thus reducing the time and cost of additional cycles. In this study, we describe our first experience in introducing an NGS-based preimplantation genetic testing for aneuploidy (PGT-A) service using next-generation sequencing in King Abdulaziz Medical City located in Riyadh, Saudi Arabia. Our main goal was to report the successful implementation of this new technology in clinical practice and highlight the factors that may affect the results. In total, 200 blastomere biopsies were obtained from 36 in vitro fertilization (IVF) cycles from Saudi couples suffering from prolonged infertility or recurrent embryo transfer failure. NGS-based PGT-A was performed in all embryos. The results were analyzed in five age groups, showing that aneuploidy rates increased with maternal age. Moreover, the results also showed that complex abnormal embryos with (2-5) aneuploidy are the most common type of embryos. Additionally, our data showed that chromosome 16-related abnormality was the most frequent abnormality detected among all reported abnormalities. In conclusion, our study suggests that NGS-based PGT-A is an applicable and reliable technique for routine-based embryo screening, especially for couples suffering from recurrent miscarriages or multiple embryo transfer failures.

Pancytopenia, Recurrent Infection, Poor Wound Healing, Heterotopia of the Brain Probably Associated with A Candidate Novel de Novo CDC42 Gene Defect: Expanding the Molecular and Phenotypic Spectrum.

CDC42 (cell division cycle protein 42) belongs to the Rho GTPase family that is known to control the signaling axis that regulates several cellular functions, including cell cycle progression, migration, and proliferation. However, the functional characterization of the CDC42 gene in mammalian physiology remains largely unclear. Here, we report the genetic and functional characterization of a non-consanguineous Saudi family with a single affected individual. Clinical examinations revealed poor wound healing, heterotopia of the brain, pancytopenia, and recurrent infections. Whole exome sequencing revealed a de novo missense variant (c.101C > A, p.Pro34Gln) in the CDC42 gene. The functional assays revealed a substantial reduction in the growth and motility of the patient cells as compared to the normal cells control. Homology three-dimensional (3-D) modeling of CDC42 revealed that the Pro34 is important for the proper protein secondary structure. In conclusion, we report a candidate disease-causing variant, which requires further confirmation for the etiology of CDC42 pathogenesis. This represents the first case from the Saudi population. The current study adds to the spectrum of mutations in the CDC42 gene that might help in genetic counseling and contributes to the CDC42-related genetic and functional characterization. However, further studies into the molecular mechanisms that are involved are needed in order to determine the role of the CDC42 gene associated with aberrant cell migration and immune response.

A Missense Mutation in the UGDH Gene Is Associated With Developmental Delay and Axial Hypotonia.

UDP-glucose dehydrogenase (UGDH) encodes an oxidoreductase that converts two successive oxidations of UDP-glucose to produce UDP-glucuronic acid, a key component in the synthesis of several polysaccharides such as glycosaminoglycan and the disaccharide hyaluronic acid. UGDH is critical to the production of extracellular matrix components which are essential to the migration and connectivity of neurons early in human brain development. In this report, we describe one child of a consanguineous family who presented with distinct clinical features including global developmental delay, axial hypotonia, bilateral undescended testis, and subtle dysmorphic features. Whole genome sequencing and a segregation was performed to identify the genetic cause of the disease within the family. Though mutations in the UGDH protein have been described as causing developmental delay in various model organisms, to our knowledge, this is the first identification of the novel homozygous missense variant in exon8 of UGDH NM_003359.3: c.950 G>A (p.Arg317Gln) and most likely the cause of the patient's phenotype. This variant falls in an active region and replaces the highly conserved Arginine 317 residues across mammals.

Mutated RAP1GDS1 causes a new syndrome of dysmorphic feature, intellectual disability & speech delay.

BACKGROUND: RAP1GDS1 (RAP1, GTP-GDP dissociation stimulator 1), also known as SmgGDS, is a guanine nucleotide exchange factor (GEF) that regulates small GTPases, including, RHOA, RAC1, and KRAS. RAP1GDS1 was shown to be highly expressed in different tissue types including the brain. However, mutations in the RAP1GDS1 gene associated with human diseases have not previously been reported. METHODS: We report on four affected individuals, presenting intellectual disability, global developmental delay (GDD), and hypotonia. The probands' DNA was subjected to whole-genome sequencing, revealing a homozygous splice acceptor site mutation in the RAP1GDS1 gene (1444-1G > A). Sanger sequencing was performed to confirm the segregation of the variant in two Saudi families. The possible aberrant splicing in the patients' RNA was investigated using RT-PCR and changes in mRNA expression of the patients were confirmed using qRT-PCR. RESULTS: The identified splice variant was found to segregate within the two families. RT-PCR showed that the mutation affected RAP1GDS1 gene splicing, resulting in the production of aberrant transcripts in the affected individuals. Quantitative gene expression analysis demonstrated that the RAP1GDS1 mRNA expression in all the probands was significantly decreased compared to that of the control, and Sanger sequencing of the probands' cDNA revealed skipping of exon 13, further strengthening the pathogenicity of this variant. CONCLUSION: We are the first to report the mutation of the RAP1GDS1 gene as a potential cause of GDD and hypotonia. However, further investigations into the molecular mechanisms involved are required to confirm the role of RAP1GDS1 gene in causing GDD and hypotonia.

Preparation of anastrozole loaded PEG-PLA nanoparticles: evaluation of apoptotic response of breast cancer cell lines.

PURPOSE: Anastrozole (ANS) is an aromatase inhibitor that is widely used as a treatment for breast cancer in postmenopausal women. Despite the wide use of ANS, it is associated with serious side effects due to uncontrolled delivery. In addition, ANS exhibits low solubility and short plasma half-life. Nanotechnology-based drug delivery has the potential to enhance the efficacy of drugs and overcome undesirable side effects. In this study, we aimed to prepare novel ANS-loaded PLA-PEG-PLA nanoparticles (ANS-NPs) and to compare the apoptotic response of MCF-7 cell line to both ANS and ANS-loaded NPs. METHOD: ANS-NPs were synthesized using double emulsion method and characterized using different methods. The apoptotic response was evaluated by assessing cell viability, morphology, and studying changes in the expression of MAPK3, MCL1, and c-MYC apoptotic genes in MCF-7 cell lines. RESULTS: ANS was successfully encapsulated within PLA-PEG-PLA, forming monodisperse therapeutic NPs with an encapsulation efficiency of 67%, particle size of 186±27.13, and a polydispersity index of 0.26±0.11 with a sustained release profile extended over 144 hours. In addition, results for cell viability and for gene expression represent a similar apoptotic response between the free ANS and ANS-NPs. CONCLUSION: The synthesized ANS-NPs showed a similar therapeutic effect as the free ANS, which provides a rationale to pursue pre-clinical evaluation of ANS-NPs on animal models.