Biallelic variants in HECT E3 paralogs, HECTD4 and UBE3C, encoding ubiquitin ligases cause neurodevelopmental disorders that overlap with Angelman syndrome.

PURPOSE: Pathogenic variants in genes encoding ubiquitin E3 ligases are known to cause neurodevelopmental syndromes. Additional neurodevelopmental disorders associated with the other genes encoding E3 ligases are yet to be identified. METHODS: Chromosomal analysis and exome sequencing were used to identify the genetic causes in 10 patients from 7 unrelated families with syndromic neurodevelopmental, seizure, and movement disorders and neurobehavioral phenotypes. RESULTS: In total, 4 patients were found to have 3 different homozygous loss-of-function (LoF) variants, and 3 patients had 4 compound heterozygous missense variants in the candidate E3 ligase gene, HECTD4, that were rare, absent from controls as homozygous, and predicted to be deleterious in silico. In 3 patients from 2 families with Angelman-like syndrome, paralog-directed candidate gene approach detected 2 LoF variants in the other candidate E3 ligase gene, UBE3C, a paralog of the Angelman syndrome E3 ligase gene, UBE3A. The RNA studies in 4 patients with LoF variants in HECTD4 and UBE3C provided evidence for the LoF effect. CONCLUSION: HECTD4 and UBE3C are novel biallelic rare disease genes, expand the association of the other HECT E3 ligase group with neurodevelopmental syndromes, and could explain some of the missing heritability in patients with a suggestive clinical diagnosis of Angelman syndrome.

Biallelic loss of function variant in the unfolded protein response gene PDIA6 is associated with asphyxiating thoracic dystrophy and neonatal-onset diabetes.

Protein disulfide isomerase A6 (PDIA6) is an unfolded protein response (UPR)-regulating protein. PDIA6 regulates the UPR sensing proteins, Inositol requiring enzyme 1, and EIF2AK3. Biallelic inactivation of the two genes in mice and humans resulted in embryonic lethality, diabetes, skeletal defects, and renal insufficiency. We recently showed that PDIA6 inactivation in mice caused embryonic and early lethality, diabetes and immunodeficiency. Here, we present a case with asphyxiating thoracic dystrophy (ATD) syndrome and infantile-onset diabetes. Whole exome sequencing revealed a homozygous frameshift variant in the PDIA6 gene. RNA expression was reduced in a gene dosage-dependent manner, supporting a loss-of-function effect of this variant. Phenotypic correlation with the mouse model recapitulated the growth defect and delay, early lethality, coagulation, diabetes, immunological, and polycystic kidney disease phenotypes. In general, the phenotype of the current patient is consistent with phenotypes associated with the disruption of PDIA6 and the sensors of UPR in mice and humans. This is the first study to associate ATD to the UPR gene, PDIA6. We recommend screening ATD cases with or without insulin-dependent diabetes for variants in PDIA6.

NR2F1 mediated down-regulation of osteoblast differentiation was rescued by bone morphogenetic protein-2 (BMP-2) in human MSC.

Endochondral ossification is the process by which long bones are formed; the process of long bone formation is regulated by numerous factors such as transcription factors, cytokines, and extracellular matrix molecules. Human hormone Nuclear receptors (hHNR) are a family of ligand-regulated transcription factors that are activated by steroid hormones, such as estrogen and progesterone, and various lipid-soluble signals, including retinoic acid, oxysterols, and thyroid hormone. Whole genome microarray data from our previous study revealed that most hHNR's are up-regulated during osteoblast differentiation in hMSCS. NR2F1 was among the highest expressed hHNR during osteogenesis, NR2F1 belongs to the steroid/thyroid hormone nuclear receptor superfamily. NR2F1 is designated as an orphan nuclear receptor because its ligands are unknown. NR2F1 plays a wide range of roles, including cell differentiation, cancer progression, and central and peripheral neurogenesis. Identifying signaling networks involved in osteoblast differentiation is important in orchestrating new therapeutic and clinical applications in bone biology. This study aimed to identify alterations in signaling networks mediated by NR2F1 in osteoblast differentiation. siRNA-mediated down-regulation of NR2F1 leads to impairment in the differentiation of hBMSC-TERT to osteoblast; gene-expression results confirmed the down-regulation of osteoblast markers such as RUNX2, ALPL, OSC, and BSP. Global whole gene expression analysis revealed that most down-regulated genes were associated with osteoblast differentiation (DDIT3, BMP2). Pathway analysis revealed prominent signaling pathways that were down-regulated, including the TGFß pathway and MAPK pathway. Functional studies on NR2F1 transfected cells, during osteoblast differentiation in combination with TGFß1 and BMP-2, showed that TGFß1 does not recover osteoblast differentiation, whereas BMP-2 rescues osteoblast differentiation in NR2F1 siRNA transfected cells. Thus, our results showed that BMP-2 could intervene in NR2F1 down-regulated signaling pathways to recover osteoblast differentiation.

Identification of Novel and Recurrent Variants in BTD, GBE1, AGL and ASL Genes in Families with Metabolic Disorders in Saudi Arabia.

Background and Objectives: Inherited metabolic disorders (IMDs) are a group of genetic disorders characterized by defects in enzymes or transport proteins involved in metabolic processes. These defects result in an abnormal accumulation of metabolites and thus interfere with the body's metabolism. A variety of IMDs exist and differential diagnosis is often challenging. Our objective was to gain insight into the genetic basis of IMDs and the correlations between specific genetic mutations and clinical presentations in patients admitted at various hospitals in the Madinah region of the Kingdom of Saudi Arabia. Material and Methods: Whole exome sequencing (WES) has emerged as a powerful tool for diagnosing IMDs and allows for the identification of disease-causing genetic mutations in individuals suspected of IMDs. This ensures accurate diagnosis and appropriate management. WES was performed in four families with multiple individuals showing clinical presentation of IMDs. Validation of the variants identified through WES was conducted using Sanger sequencing. Furthermore, various computational analyses were employed to uncover the disease gene co-expression and metabolic pathways. Results: Exome variant data analysis revealed missense variants in the BTD (c.1270G > C), ASL (c.1300G > T), GBE1 (c.985T > G) and AGL (c.113C > G) genes. Mutations in these genes are known to cause IMDs. Conclusions: Thus, our data showed that exome sequencing, in conjunction with clinical and biochemical characteristics and pathological hallmarks, could deliver an accurate and high-throughput outcome for the diagnosis and sub-typing of IMDs. Overall, our findings emphasize that the integration of WES with clinical and pathological information has the potential to improve the diagnosis and understanding of IMDs and related disorders, ultimately benefiting patients and the medical community.

Exome sequence analysis identifies a homozygous, pathogenic, frameshift variant in the MAN2B1 gene underlying clinical variant of α-mannosidosis.

Background: α-mannosidosis (MAN) is a rare genetic condition that segregates in an autosomal recessive manner. Lack of lysosomal alpha-mannosidase is the underlying cause of the disease. Symptoms of the disease gradually worsen with the age. Newborns are usually asymptomatic, however, some cases are reported with either congenital ankle equinus or hydrocephalus during the first year. Primary symptoms are characterized by immune deficiency, hearing loss, skeletal abnormalities, progressive mental, motor and speech functions' impairment followed by facial asymmetry. Methods: We studied two Saudi families (A and B) with bilateral moderate hearing loss (family A) and clubfoot with glaucoma (family B). Clinical diagnosis was not reached based on phenotype of patients. Therefore, hypothesis-free whole exome sequencing (WES) was performed on DNA samples from affected individuals of both the families, followed by Sanger sequencing and segregation analysis to validate the segregation of the identified variant. Furthermore, 3D protein modelling was performed to determine the in silico effects of the identified variant on the protein structure and function. Results: Re-examination of clinical features revealed that the patients in family A have speech delay and hearing impairment along with craniostenosis, whereas the patients from family B have only clubfoot and glaucoma. WES identified a well known pathogenic homozygous frameshift variant (NM_000528.4: c.2402dupG; p.S802fs*129) in MAN2B1 in both the families. Sanger sequencing confirmed the segregation of the variant with the disease phenotype in both the families. 3D structural modeling of the MAN2B1 protein revealed significant changes in the tertiary structure of the mutant protein, which would affect enzyme function. This report presents a new case where partial and novel α-mannosidosis phenotypes are associated with a MAN2B1 gene pathogenic variant. Conclusion: Patients in both the families have manifested peculiar set of clinical symptoms associated with α-mannosidosis. Family A manifested partial clinical symptoms missing several characteristic features like intellectual disability, dysmorphic features, neurological and abdominal manifestations, whereas family B has no reported clinical symptoms related to α-mannosidosis except the novel symptoms including club foot and glaucoma which has never been reported earlier The current findings support the evidence that biallelic variants of MAN2B1 are associated with new clinical variants of α-mannosidosis.

Centromere protein I (CENPI) is a candidate gene for X-linked steroid sensitive nephrotic syndrome.

BACKGROUND: Individuals with proteinuria in association with hypoalbuminemia, edema, and hyperlipidemia are considered as having nephrotic syndrome (NS). NS is the most common kidney disease seen in the paediatric age group. NS is usually classified into steroid resistant nephrotic syndrome (SRNS) and steroid sensitive nephrotic syndrome (SSNS). More than 58 genes have been identified as a monogenic cause of SRNS, however, the genetic architecture of childhood SSNS remains poorly understood. METHODS: Here in this study, we performed sequencing of 66 NS candidate genes followed by whole genome SNP genotyping and whole exome sequencing in SSNS families with multiple affected individuals. RESULTS: NS candidate genes sequencing did not identify any pathogenic variant in the known genes. Homozygosity mapping based on an autosomal recessive model failed to detect any shared loss of heterozygosity region in the genome. An unbiased and hypothesis-free exome data analysis identified a missense variant (c.383G>A; p.Arg128Gln) in the CENPI gene. Sanger sequencing of both parents, unaffected and affected individuals confirmed an X-linked inheritance pattern of the variant (c.383G>A) with SSNS phenotype. The variant (c.383G>A) is very rare and is potentially damaging. CONCLUSION: Collectively, these observations suggest that a specific pathogenic link between SSNS development and alteration in CENPI exists. However, human mutations in CENPI causing SSNS have not been reported hitherto. Identification of genetic defects underlying SSNS will help in understanding the precise aetiology of SSNS and improved management of children with NS.

An 18 bps in-frame deletion mutation in RUNX2 gene is a population polymorphism rather than a pathogenic variant.

We recruited a family with an affected child exhibiting features of cleidocranial dysplasia with some phenotypic variations from reported cases. Whole exome sequencing data analysis identified an 18-bps heterozygous in-frame deletion variant (c.243-260delGGCGGCTGCGGCGGCGGC) in the RUNX2 gene. Sanger sequencing validated the presence of deletion in affected individual. Initially, we considered this variant as a causal mutation for the patient's phenotype based on previous report(s). However, further analysis of variant revealed that it is present in high frequency in variety of genome variation databases. Moreover, segregation analysis discovered the presence of variant in mother as well. Furthermore, screening of population matched control individuals revealed that the variant is present in apparently healthy individuals as well. Three-dimensional structures of the wild-type and mutant RUNX2 protein (p.Ala82_Ala87del) were analysed and it was found that both wild type and mutant protein show similar secondary structure pattern. Presence of RUNX2 deletion variant (c.243-260delGGCGGCTGCGGCGGCGGC) in control individuals, its high population frequency, benign effect on the overall protein structure lead to the argument that this variant is a population polymorphism and not a pathogenic mutation.

Novel homozygous loss-of-function mutations in RP1 and RP1L1 genes in retinitis pigmentosa patients.

Background: Retinitis pigmentosa (RP) is a heterogeneous group of ocular dystrophy. It is challenging to identify the underlying genetic defect in individuals with RP due to huge genetic heterogeneity. This study was designed to delineate the genetic defect(s) underlying RP in extended Saudi families and to describe the possible disease mechanism.Materials and Methods: Fundus photography and a high definition optical coherence tomography (HD-OCT) were performed in order to detect the earlier stages of macular degeneration. Genomic DNA was extracted followed by genome-wide SNP genotyping and whole exome sequencing (WES). Exome data was filtered to identify the genetic variant(s) of interest.Results: Clinical examination showed that affected individuals manifest key features of RP. The fundus exam shows pale optic disc and bone spicules at the periphery. OCT shows macular degeneration as early as at the age of 4 years. Whole genome scan by SNPs identified multiple homozygous regions. WES identified a 10 bps novel insertion mutation (c.3544_3545insAGAAAAGCTG; p.Ala1182fs) in the RP1 gene in both affected individuals of family A. Affected individual from family B showed a large insertion of 48 nucleotides in the coding part of the RP1L1 gene (c.3955_3956insGGACTAAAGTAATAGAAGGGCTGCAAGAAGAGAGGGTGCAGTTAGAGG; p.Ala1319fs). Sanger sequencing validates the autosomal recessive inheritance of the mutations.Conclusion: The results strongly suggest that the insertion mutations in the RP1 and RP1L1 genes are responsible for the retinal phenotype in affected individuals from two families. Heterozygous individuals are asymptomatic carriers. We propose that the protective allele in other homozygous regions in heterozygous carriers contribute to the phenotypic variability in asymptomatic individuals.

Silicon carbide whisker-mediated embryogenic callus transformation of cotton (Gossypium hirsutum L.) and regeneration of salt tolerant plants.

A silicon carbide whisker-mediated gene transfer system with recovery of fertile and stable transformants was developed for cotton (Gossypium hirsutum L.) cv. Coker-312. Two-month-old hypocotyl-derived embryogenic/non-embryogenic calli at different days after subculture were treated with silicon carbide whiskers for 2 min in order to deliver pGreen0029 encoding GUS gene and pRG229 AVP1 gene, encoding Arabidopsis vacuolar pyrophosphatase, having neomycin phosphotransferaseII (nptII) genes as plant-selectable markers. Three crucial transformation parameters, i.e., callus type, days after subculture and selection marker concentration for transformation of cotton calli were evaluated for optimum efficiency of cotton embryogenic callus transformation giving upto 94% transformation efficiency. Within six weeks, emergence of kanamycin-resistant (kmr) callus colonies was noted on selection medium. GUS and Southern blot analysis showed expression of intact and multiple transgene copies in the transformed tissues. Kanamycin wiping of leaves from T1, T2, and T3 progeny plants revealed that transgenes were inherited in a Mendelian fashion. Salt treatment of T1 AVP1 transgenic cotton plants showed significant enhancement in salt tolerance as compared to control plants. Thus far, this is first viable physical procedure after particle bombardment available for cotton that successfully can be used to generate fertile cotton transformants.

Whole exome sequencing identification of a novel insertion mutation in the phospholipase C ε­1 gene in a family with steroid resistant inherited nephrotic syndrome.

Nephrotic syndrome (NS) represents a heterogeneous group of kidney disorders characterized by excessive proteinuria, hypoalbuminemia and edema. Defects in the filtration barrier of the glomeruli results in the development of NS. The genetic cause of NS remains to be fully elucidated. However, previous studies based on positional cloning of genes mutated in NS have provided limited insight into the pathogenesis of this disease. Mutations in phospholipase C ε­1 (PLCE1) have been reported as a cause of early onset NS characterized by histology of diffuse mesangial sclerosis. In the present study, the underlying cause of NS in a consanguineous family was identified. Clinical and molecular aspects of a consanguineous Saudi family comprised of five individuals with steroid resistant NS were examined. Seven healthy individuals from the same family were also studied. Whole exome sequencing (WES) was performed to detect the genetic defect underlying NS. WES identified a homozygous novel insertion mutation (c.6272_6273insT) in the PLCE1 gene. Pedigree and segregation analysis confirmed an autosomal recessive inheritance pattern. This mutation may result in a bi­allelic loss of the C­terminal Ras­associating domain in PLCE1 that results in NS. The present study expanded the mutational spectrum of PLCE1 in NS. In addition, the present study provided further evidence that supports the important involvement of PLCE1 in the physiological function of the glomerular filtration barrier.

Whole exome sequencing identified a novel single base pair insertion mutation in the EYS gene in a six generation family with retinitis pigmentosa.

Retinitis pigmentosa (RP) is a group of inherited progressive retinal dystrophies (RD) and is characterized by photoreceptor degeneration. RP is clinically and genetically heterogeneous disorder. More than 70 genes are known and, thus, identification of causative genes and mutations in known genes is challenging. This study was designed to identify the underlying genetic defect in a large extended Saudi family with multiple RP affected members. Fundus photography, Optical Coherence Tomography (OCT) and visual field perimetry were performed for affected individuals. Whole exome sequencing was used to detect the underlying genetic defect in a large family with 12 affected individuals showing autosomal recessive isolated RP. WES data analysis identified a novel insertion mutation in the EYS (eyes shut homolog) gene (c.910_911insT; p.Trp304LeufsTer8). Sanger sequencing validates the variant discovered through exome in all 12 affected individuals and showed that this mutation is segregating with RP phenotype in an autosomal recessive manner in 51 individuals of the family tested here. Our study expands the mutation spectrum of EYS gene in RP patients and extends the body of evidence that supports the importance of EYS gene in eye development.

TGFß1-Induced Differentiation of Human Bone Marrow-Derived MSCs Is Mediated by Changes to the Actin Cytoskeleton.

TGFß is a potent regulator of several biological functions in many cell types, but its role in the differentiation of human bone marrow-derived skeletal stem cells (hMSCs) is currently poorly understood. In the present study, we demonstrate that a single dose of TGFß1 prior to induction of osteogenic or adipogenic differentiation results in increased mineralized matrix or increased numbers of lipid-filled mature adipocytes, respectively. To identify the mechanisms underlying this TGFß-mediated enhancement of lineage commitment, we compared the gene expression profiles of TGFß1-treated hMSC cultures using DNA microarrays. In total, 1932 genes were upregulated, and 1298 genes were downregulated. Bioinformatics analysis revealed that TGFßl treatment was associated with an enrichment of genes in the skeletal and extracellular matrix categories and the regulation of the actin cytoskeleton. To investigate further, we examined the actin cytoskeleton following treatment with TGFß1 and/or cytochalasin D. Interestingly, cytochalasin D treatment of hMSCs enhanced adipogenic differentiation but inhibited osteogenic differentiation. Global gene expression profiling revealed a significant enrichment of pathways related to osteogenesis and adipogenesis and of genes regulated by both TGFß1 and cytochalasin D. Our study demonstrates that TGFß1 enhances hMSC commitment to either the osteogenic or adipogenic lineages by reorganizing the actin cytoskeleton.

Characterization of Cellular and Molecular Heterogeneity of Bone Marrow Stromal Cells.

Human bone marrow-derived stromal stem cells (hBMSC) exhibit multiple functions, including differentiation into skeletal cells (progenitor function), hematopoiesis support, and immune regulation (nonprogenitor function). We have previously demonstrated the presence of morphological and functional heterogeneity of hBMSC cultures. In the present study, we characterized in detail two hTERT-BMSC clonal cell populations termed here CL1 and CL2 that represent an opposing phenotype with respect to morphology, markers expression: alkaline phosphatase (ALP) and CD146, and ex vivo differentiation potential. CL1 differentiated readily to osteoblasts, adipocytes, and chondrocytes as shown by expression of lineage specific genes and proteins. Whole genome transcriptome profiling of CL1 versus CL2 revealed enrichment in CL1 of bone-, mineralization-, and skeletal muscle-related genes, for example, ALP, POSTN, IGFBP5 BMP4, and CXCL12. On the other hand, CL2 transcriptome was enriched in immune modulatory genes, for example, CD14, CD99, NOTCH3, CXCL6, CFB, and CFI. Furthermore, gene expression microarray analysis of osteoblast differentiated CL1 versus CL2 showed significant upregulation in CL1 of bone development and osteoblast differentiation genes which included several homeobox genes: TBX15, HOXA2 and HOXA10, and IGF1, FGFR3, BMP6, MCAM, ITGA10, IGFBP5, and ALP. siRNA-based downregulation of the ALP gene in CL1 impaired osteoblastic and adipocytic differentiation. Our studies demonstrate the existence of molecular and functional heterogeneity in cultured hBMSC. ALP can be employed to identify osteoblastic and adipocytic progenitor cells in the heterogeneous hBMSC cultures.

High frequency and poor prognosis of late childhood BCR-ABL-positive and MLL-AF4-positive ALL define the need for advanced molecular diagnostics and improved therapeutic strategies in pediatric B-ALL in Pakistan.

BACKGROUND: Fusion oncogenes (FOs) resulting from chromosomal abnormalities have an important role in leukemogenesis in pediatric B cell acute lymphoblastic leukemia (ALL). The most common FOs are BCR-ABL, MLL-AF4, ETV6-RUNX1, and TCF3-PBX1, all of which have important prognostic and drug selection implications. Moreover, frequencies of FOs have ethnic variations. We studied Pakistani frequencies of FOs, clinical pattern, and outcome in pediatric B-ALL. METHODS: FOs were studied in 188 patients at diagnosis using reverse transcriptase-polymerase chain reaction (RT-PCR) and interphase fluorescent in situ hybridization (FISH). Data were analyzed using SPSS version 17 (SPSS Inc., Chicago, IL, USA). RESULTS: FOs were detected in 87.2 % of patients. Mean overall survival was 70.9 weeks, 3-year survival was 31.9 %, and 3-year relapse-free survival was 18.1 %. Four patients died of drug toxicities. ETV6-RUNX1 (19.14 %) had better survival (110.9 weeks; p = 0.03); TCF3-PBX1 (2.1 %) was associated with inferior outcome and higher central nervous system (CNS) relapse risk; MLL-AF4 (18.1 %) was more common in the 8- to 15-year age group (24/34; p = 0.001) and was associated with organomegaly, low platelet count, and poor survival; and BCR-ABL (47.9 %) was associated with older age (7-15 years, 52/90), lower remission rates, shorter survival (43.73 ± 4.24 weeks) and higher white blood cell count. Overall, MLL-AF4 and BCR-ABL were detected in 66 % of B-ALL, presented in later childhood, and were associated with poor prognosis and inferior survival. CONCLUSIONS: This study reports the highest ethnic frequency of BCR-ABL FO in pediatric ALL, and is consistent with previous reports from our region. Poor prognosis BCR-ABL and MLL-AF4 was detected in two-thirds of pediatric B-ALL and is likely to be the reason for the already reported poor survival of childhood ALL in South-East Asia. Furthermore, MLL-AF4, usually most common in infants, presented in later childhood in most of the ALL patients, which was one of the unique findings in our study. The results presented here highlight the need for mandatory inclusion of molecular testing for pediatric ALL patients in clinical decision making, together with the incorporation of tyrosine kinase inhibitors, as well as hematopoietic stem cell transplantation facilities, to improve treatment outcome for patients in developing countries.