Derivation of two iPSC lines (KAIMRCi004-A, KAIMRCi004-B) from a Saudi patient with Biotin-Thiamine-responsive Basal Ganglia Disease (BTBGD) carrying homozygous pathogenic missense variant in the SCL19A3 gene.

The neurometabolic disorder known as biotin-thiamine-responsive basal ganglia disease (BTBGD) is a rare autosomal recessive condition linked to bi-allelic pathogenic mutations in the SLC19A3 gene. BTBGD is characterized by progressive encephalopathy, confusion, seizures, dysarthria, dystonia, and severe disabilities. Diagnosis is difficult due to the disease's rare nature and diverse clinical characteristics. The primary treatment for BTBGD at this time is thiamine and biotin supplementation, while its long-term effectiveness is still being investigated. In this study, we have generated two clones of induced pluripotent stem cells (iPSCs) from a 10-year-old female BTBGD patient carrying a homozygous mutation for the pathogenic variant in exon 5 of the SLC19A3 gene, c.1264A > G (p.Thr422Ala). We have confirmed the pluripotency of the generated iPS lines and successfully differentiated them to neural progenitors. Because our understanding of genotype-phenotype correlations in BTBGD is limited, the establishment of BTBGD-iPSC lines with a homozygous SLC19A3 mutation provides a valuable cellular model to explore the molecular mechanisms underlying SLC19A3-associated cellular dysfunction. This model holds potential for advancing the development of novel therapeutic strategies.

Generation of iPSC lines (KAIMRCi003A, KAIMRCi003B) from a Saudi patient with Dravet syndrome carrying homozygous mutation in the CPLX1 gene and heterozygous mutation in SCN9A.

The most prevalent form of epileptic encephalopathy is Dravet syndrome (DRVT), which is triggered by the pathogenic variant SCN1A in 80% of cases. iPSCs with different SCN1A mutations have been constructed by several groups to model DRVT syndrome. However, no studies involving DRVT-iPSCs with rare genetic variants have been conducted. Here, we established two DRVT-iPSC lines harboring a homozygous mutation in the CPLX1 gene and heterozygous mutation in SCN9A gene. Therefore, the derivation of these iPSC lines provides a unique cellular platform to dissect the molecular mechanisms underlying the cellular dysfunctions consequent to CPLX1 and SCN9A mutations.

HLA-based banking of induced pluripotent stem cells in Saudi Arabia.

BACKGROUND: Human iPSCs' derivation and use in clinical studies are transforming medicine. Yet, there is a high cost and long waiting time associated with autologous iPS-based cellular therapy, and the genetic engineering of hypo-immunogenic iPS cell lines is hampered with numerous hurdles. Therefore, it is increasingly interesting to create cell stocks based on HLA haplotype distribution in a given population. This study aimed to assess the potential of HLA-based iPS banking for the Saudi population. METHODS: In this study, we interrogated the HLA database of the Saudi Stem Cell Donor Registry (SSCDR), containing high-resolution HLA genotype data from 64,315 registered Saudi donors at the time of analysis. This database was considered to be a representative sample of the Saudi population. The most frequent HLA haplotypes in the Saudi population were determined, and an in-house developed iterative algorithm was used to identify their HLA matching percentages in the SSCDR database and cumulative coverage. Subsequently, to develop a clinically relevant protocol for iPSCs generation, and to illustrate the applicability of the concept of HLA-based banking for cell therapy purposes, the first HLA-based iPS cell line in Saudi Arabia was generated. Clinically relevant methods were employed to generate the two iPS clones from a homozygous donor for the most prevalent HLA haplotype in the Saudi population. The generated lines were then assessed for pluripotency markers, and their ability to differentiate into all three germ layers, beating cardiomyocytes, and neural progenitors was examined. Additionally, the genetic stability of the HLA-iPS cell lines was verified by comparing the mutational burden in the clones and the original blood sample, using whole-genome sequencing. The standards set by the American College of Medical Genetics and Genomics (ACMG) were used to determine the clinical significance of identified variants. RESULTS: The analysis revealed that the establishment of only 13 iPSC lines would match 30% of the Saudi population, 39 lines would attain 50% coverage, and 596 lines would be necessary for over 90% coverage. The proof-of-concept HLA-iPSCs, which cover 6.1% of the Saudi population, successfully demonstrated pluripotency and the ability to differentiate into various cell types including beating cardiomyocytes and neuronal progenitors. The comprehensive genetic analysis corroborated that all identified variants in the derived iPSCs were inherently present in the original donor sample and were classified as benign according to the standards set by the ACMG. CONCLUSIONS: Our study sets a road map for introducing iPS-based cell therapy in the Kingdom of Saudi Arabia. It underscores the pragmatic approach of HLA-based iPSC banking which circumvents the limitations of autologous iPS-based cellular therapies. The successful generation and validation of iPSC lines based on the most prevalent HLA haplotype in the Saudi population signify a promising step toward broadening the accessibility and applicability of stem cell therapies and regenerative medicine in Saudi Arabia.

Generation of myoglobin (MB)-knockout human embryonic stem cell (hESC) line (KAIMRCe002-A-1S) using CRISPR/Cas9 technology.

Myoglobin (MB) is a cytoplasmic hemoprotein that is predominantly expressed in the heart and oxidative myofibers of skeletal muscle. It has been demonstrated that MB binds to oxygen and promotes its diffusion for energy production in the mitochondria. Recently, MB was found to be expressed in different forms of malignant tumors and cancer cell lines. Further studies using gene disruption technology will enhance the understanding of MB's role in human cardiovascular biology and cancers. Here, we describe the generation of a homozygous MB knockout in human embryonic stem cells (hESC-MB-/-) via CRISPR/Cas9 to study MB function in human biology and diseases.

Clinically significant findings of high-risk mutations in human SLC29A4 gene associated with diabetes mellitus type 2 in Pakistani population.

This study conducted an in-depth analysis combining computational and experimental verifications of the deleterious missense mutations associated with the SLC29A4 protein. The functional annotation of the non-synonymous single nucleotide polymorphism (nsSNPs), followed by structure-function analysis, revealed 13 single nucleotide polymorphisms (SNP) as the most damaging. Among these, six mutants P429T/S, L144S, M108V, N86H, and V79E, were predicted as structurally and functionally damaging by protein stability analysis. Also, these variants are located at evolutionary conserved regions, either buried, contributing to the structural damage, or exposed, causing functional changes in the protein. These mutants were further taken for molecular docking studies. When verified via experimental analysis, the SNPs M108V (rs149798710), N86H (rs151039853), and V79E (rs17854505) showed an association with type 2 diabetes mellitus (T2DM). Minor allele frequency for rs149798710 (A > G) was 0.23 in controls, 0.29 in metformin responders, 0.37 in metformin non-responder, for rs151039853 (A > C) was 0.21 in controls, 0.28 in metformin responders, 0.36 in metformin non-responder and for rs17854505 (T > A) was 0.20 in controls, 0.25 in metformin responders, 0.37 in metformin non-responder. Hence, this study concludes that SLC29A4 M108V (rs149798710), N86H (rs151039853), and V79E (rs17854505) polymorphisms were associated with the increased risk of T2DM as well as with the increased risk towards the failure of metformin therapeutic response in T2DM patients of Pakistan. Communicated by Ramaswamy H. Sarma.

T cell receptor repertoire as a potential diagnostic marker for celiac disease.

An individual's T cell repertoire is skewed towards some specificities as a result of past antigen exposure and subsequent clonal expansion. Identifying T cell receptor signatures associated with a disease is challenging due to the overall complexity of antigens and polymorphic HLA allotypes. In celiac disease, the antigen epitopes are well characterised and the specific HLA-DQ2-restricted T-cell repertoire associated with the disease has been explored in depth. By investigating T cell receptor repertoires of unsorted lamina propria T cells from 15 individuals, we provide the first proof-of-concept study showing that it could be possible to infer disease state by matching against a priori known disease-associated T cell receptor sequences.

Exploiting antigen receptor information to quantify index switching in single-cell transcriptome sequencing experiments.

By offering high sequencing speed and ultra-high-throughput at a low price, Illumina next-generation sequencing platforms have been widely adopted in recent years. However, an experiment with multiplexed library could be at risk of molecular recombination, known as "index switching", which causes a proportion of the reads to be assigned to an incorrect sample. It is reported that a new advance, exclusion amplification (ExAmp) in conjunction with the patterned flow cell technology introduced on HiSeq 3000/HiSeq 4000/HiSeq X sequencing systems, potentially suffers from a higher rate of index switching than conventional bridge amplification. We took advantage of the diverse but highly cell-specific expression of antigen receptors on immune cells to quantify index switching on single cell RNA-seq data that were sequenced on HiSeq 3000 and HiSeq 4000. By utilizing the unique antigen receptor expression, we could quantify the spread-of-signal from many different wells (n = 55 from total of three batches) due to index switching. Based on full-length T cell receptor (TCR) sequences from all samples reconstructed by TraCeR and TCR gene expression quantified by Kallisto, we found index switching in all three batches of experiments investigated. The median percentage of incorrectly detected markers was estimated to be 3.9% (interquartile range (IQR): 1.7%-7.3%). We did not detect any consistent patterns of certain indices to be more prone to switching than others, suggesting that index switching is a stochastic process. Our results confirm that index switching is a problem that affects samples run in multiplexed libraries on Illumina HiSeq 3000 and HiSeq 4000 platforms.

Genetic link of type 1 diabetes susceptibility loci with rheumatoid arthritis in Pakistani patients.

Rheumatoid arthritis (RA) and type 1 diabetes (T1D) are two autoimmune disorders that have been reported to co-occur in the same subjects or in different subjects from the same family. This suggests the sharing of disease susceptibility loci between RA and T1D. This study was aimed to find out such susceptibility loci that are common in both T1D and RA in Pakistani population. A total of 366 Pakistanis comprising related and unrelated RA cases and controls were recruited. Blood samples were collected from all patients followed by DNA isolation. Thirty-one single-nucleotide polymorphisms (SNPs) previously reported to be associated with T1D were genotyped in RA cases and controls using TaqMan SNP genotyping assays. Data was analyzed using FamCC software. We have identified seven SNP associations that survived multiple testing corrections using false discovery rate: SKAP2/rs7804356 (p = 2.47E-04), GLIS3/rs7020673 (p = 2.86E-04), GSDMB/rs2290400 (p = 23.48E-04), BACH2/rs11755527 (p = 9.16E-04), C6orf173/ rs9388489 (p = 3.11E-03), PRKCQ/DKFZp667F0711/ rs947474 (p = 4.53E-03), and DLK1/ rs941576 (p = 9.51E-03). Our results support the presence of overlapping loci between RA and T1D in Pakistani patients.

Association of 32 type 1 diabetes risk loci in Pakistani patients.

AIM: To identify risk alleles contributing towards type 1 diabetes in Pakistani patients. INTRODUCTION: Type 1 diabetes (T1D) is an autoimmune disease which is caused by destruction of insulin producing ß cells by immune system. Genetic predisposition as well as environmental factors contribute to its etiology. To date more than 40 risk loci have been identified for T1D. METHODOLOGY: A total of 191 family-based and unrelated T1D cases and controls were recruited. DNA was extracted and 32 genome-wide significant single nucleotide polymorphisms (SNPs) previously reported in Europeans were genotyped. Genotyping was performed using TaqMan SNP genotyping assays and the data was analyzed using FamCC software. RESULTS: Our results showed significant association of 10 single nucleotide polymorphisms (SNPs) with T1D at p<0.01, including HLA-DQA1/rs9272346, ERBB3/rs2292239, SIRPG/rs2281808, IL2-KIAA1109/rs4505848, GLIS3/rs7020673, CD226/rs763361, PTPN2/rs478582, IKZF1/rs10272724, BACH2/rs11755527, C6orf173/rs9388489, whereas 5 more SNPs showed their association at 0.01

Data interpretation: deciphering the biological function of Type 2 diabetes associated risk loci.

AIMS: Type 2 diabetes (T2D) is a complex multifactorial disorder with more than 40 loci associated with disease susceptibility. Most of these genome-wide significant loci reside in noncoding regions, it is important to decipher the potential regulatory function of these variants and to differentiate between true and tag signals. Nowadays, databases are being developed to study and predict the function of these associated variants, and RegulomeDB is one such database. METHODS: We used RegulomeDB to analyze the potential function of the associated variants reported in five genome-wide association studies (GWAS) of T2D. RESULTS: We investigated the 1,567 single nucleotide polymorphisms (SNPs) with 989 SNPs with a score of 1-6. Of those 989 SNPs, only 64 returned with RegulomeDB score <3 (evidence of regulatory function), and only four of these were GWAS significant SNPs (THADA/rs10203174, score = 1b; UBE2E2/rs7612463, score = 2a; ARAP1/rs1552224 and TP53INP1/rs8996852, score = 2b). But only 63 % of the annotated SNPs showed regulatory function that is an important limitation of the RegulomeDB as this database only provides information of few regulatory elements. CONCLUSION: This study further supports that some of the noncoding GWAS variants are the true associations and not the tag ones. This study also proves the utility and importance of the RegulomeDB and other such databases. Although it is an extensive database of regulatory elements but has certain limitation due to utilization of only few types of regulatory elements and pathways.