MOLECULAR ANALYSIS OF THE CAMP- RESPONSE ELEMENT [CRE] ELEMENTS IN THE PROMOTER REGION AND EXON 1 OF THE SURVIVAL OF MOTOR NEURON 2 [SMN2] GENE IN MALAYSIAN SPINAL MUSCULAR ATROPHY PATIENTS; TO ELUCIDATE THEIR ROLE IN CIRCUMSCRIBING THE CLINICAL SEVERITY

Objective: In the Spinal muscular atrophy [SMA] genes [SMN1 and SMN2 genes]; the CRE-II elements at -400 bp in the promoter region of the SMN genes and CRE-I element at +108 bp in the exon 1 of the SMN genes, are reported to have a role in c-AMP induce expression of the SMN genes through its binding affinity to CREB-1. This study was designed to determine the role of CRE sites in the circumscribing the clinical severity of SMA. Methods: Direct sequencing was performed for the PCR products of the promoter regions of the SMA patients with homozygous deletion of SMN1, different copy number of SMN2 and NAIP non deletion. Results: No variation among the CRE-I and CRE-II sites was found in all the clinical types as compare to normal healthy control showing no role of CRE sites in circumscribing the clinical severity of SMA. Conclusion: There was no sequence variation found in the CRE binding sites in the three different clinical types of SMA reflecting no role of CRE binding sites in circumscribing the clinical severity of SMA

Genetically Confirmed Spinal Muscular Atrophy Type 3 With Epilepsy In A Malay Patient, A Case Report

Spinal Muscular Atrophy (SMA) is an autosomal recessive disease affecting the anterior horn cells of the spinal cord. The diagnosis is usually based on the clinical presentation with or without muscle biopsy and the molecular detection of mutation in the SMNI gene. There have been a few reported cases of SMA with central nervous system involvement, but these were without genetic diagnoses. We report a Malay girl with genetically confirmed SMA complicated by epilepsy. She first presented with motor weakness at the age of 17 months and recurrent seizures a month later. The molecular genetic analysis of her SMN gene showed homozygous deletion of exon 7 and 8 of the SMN1 gene. The seizure responded well to carbamazepine. To the best of our knowledge, this is the first case of genetically comfirmed Malay SMA patient with an association with epilepsy

De novo Ring Chromosome 6 in a Child with Multiple Congenital Anomalies

Ring chromosome 6, especially if it is de novo, is a rare occurrence. The phenotype of patients with ring chromosome 6 can be highly variable ranging from almost normal to severe malformations and mental retardation. The size and structure of the ring chromosome as well as the level of mosaicism are important factors in determining the clinical phenotype. Here we report an eight month-old child, a product of a non consanguineous marriage, who presented with developmental retardation, hypertelorism, microcephaly, flat occiput, broad nasal bridge, large ears, micrognathia, wide spaced nipples, protruding umbilicus, short stubby fingers, clinodactyly, single palmar crease, short neck with no obvious webbing, and congenital heart defect. Conventional karyotyping and Whole Chromosome Paint of the peripheral leukocytes showed 46,XY,r(6)(p25q27) karyotype with plausible breakpoints at p25 and q27 end. Conventional karyotyping of both parents showed normal karyotype. To the best of our knowledge, this is the first report of a Malay individual with ring chromosome 6, and this report adds to the collective knowledge of this rare chromosome abnormality

Molecular analysis of promoter region of the SMN2 gene in the patients of spinal musculatr atrophy.

Spinal muscular atrophy (SMA) is a neurodegenerative disorder caused by the absence of the full length SMN protein (FL-SMN) as a result of mutation or deletion of SMN1 gene. The isoform to this gene, SMN2 gene, with mutation in 1 base pair, encodes for 10% of FL-SMN protein and is reported to decrease the severity of the disease when there is an increase gene dosage. There are 3 clinical types of SMA; type I, type II and type III. Type I SMA is the most severe type and only a small amount of FL-SMN protein is present in these individuals. We postulated that the difference in the promoter region of SMN2 gene produces the different level of FL-SMN protein. To verify this hypothesis, the DNA samples of 69 SMA patients who were referred to the Human Genome Center, USM were extracted from their blood. The SMN1 deletion analysis was performed, followed by the SMN2 copy no. analysis and NAIP deletion analysis to remove any clinical bias as NAIP gene deletion and SMN2 copy number have been reported to be associated with SMA disease severity. Only 10 SMA patients from different clinical types (type I=2, type II=3, type III=5) with homozygous deletion of the SMN1 and 2 copies of the SMN2 and deletion in NAIP were finally recruited. Primers were designed for the amplification of the SMN2 promoter region. Bioinformatics analysis was performed to identify the crucial transcription factor binding sites within the reported ~4.6 kb promoter region. As the core promoter region is still unknown (unreported), we analyzed 15 ORFs and 24 nested ORFs with 15 TATA boxes reflecting the diverse functional integrity of this region. The promoter prediction and core promoter prediction was also performed. Based on the bioinformatics analysis and the designed primers, PCR amplification was done for different regions in the promoter and the PCR products were subjected to direct DNA sequencing. The results were analyzed by Vector NTI suite 9, ClustalX and Gene Doc softwares. The molecular analysis confirmed the absence of any mutation in the promoter region of the SMN2 gene between normal healthy individuals (total 2) and SMA patients. In 4 patients and 1 normal healthy individual the CA repeats were found to be increased which we think cause no effect in disease progression and severity. In conclusion, there was no mutation found in the promoter region of the SMN2 gene among the SMA patients of different clinical types and normal controls. Further analysis involving the cloning of the promoter regions with highest probability of involvement in expression of the SMN2 gene using luciferase assay is ongoing. The results will be useful for the subsequent phase of the study involving the transcription initiation of the SMN2 gene

Development of comparative genomic hybridization(CGH) technique for the study of nasopharyngeal carcinoma(NPC)

NPC is a disease in which malignant cells are formed in the tissue of nasopharynx. It is a highly prevalent disease in Southern China and Southeast Asia including Malaysia. CGH is a molecular cytogenetic technique which is used to identify imbalanced genetic alterations in this malignancy. Twenty eight samples were obtained. Out of this; twelve tumors were extracted from twelve NPC biopsies while twelve references DNA was extracted from twelve normal controls peripheral blood. Tumor DNA and normal DNA was labeled by nick translation method with green and red fluorescent dyes. These were hybridized at metaphase chromosomes DNA and counterstained with DAPL Finally, the image was captured and analyzed. Chromosomal gains that were found in this study were 4q26, llql3-ql4, 9pl3, 8ql3-q22 and 10q22- q26 while chromosomal losses were found at region 20p12 and 13q21-q31. We believe this study has provided the platform for further investigations to locate possible tumorsuppressor genes and oncogenes in our NPC patients

Global Globin Network and adopting genomic variant database requirements for thalassemia

\ua9 The Author(s) 2024. Published by Oxford University Press.Thalassemia is one of the most prevalent monogenic disorders in low- and middle-income countries (LMICs). There are an estimated 270 million carriers of hemoglobinopathies (abnormal hemoglobins and/or thalassemia) worldwide, necessitating global methods and solutions for effective and optimal therapy. LMICs are disproportionately impacted by thalassemia, and due to disparities in genomics awareness and diagnostic resources, certain LMICs lag behind high-income countries (HICs). This spurred the establishment of the Global Globin Network (GGN) in 2015 at UNESCO, Paris, as a project-wide endeavor within the Human Variome Project (HVP). Primarily aimed at enhancing thalassemia clinical services, research, and genomic diagnostic capabilities with a focus on LMIC needs, GGN aims to foster data collection in a shared database by all affected nations, thus improving data sharing and thalassemia management. In this paper, we propose a minimum requirement for establishing a genomic database in thalassemia based on the HVP database guidelines. We suggest using an existing platform recommended by HVP, the Leiden Open Variation Database (LOVD) (https://www.lovd.nl/). Adoption of our proposed criteria will assist in improving or supplementing the existing databases, allowing for better-quality services for individuals with thalassemia. Database URL: https://www.lovd.nl/

Adapting the ACMG/AMP variant classification framework: a perspective from the ClinGen Hemoglobinopathy Variant Curation Expert Panel

Accurate and consistent interpretation of sequence variants is integral to the delivery of safe and reliable diagnostic genetic services. To standardize the interpretation process, in 2015, the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) published a joint guideline based on a set of shared standards for the classification of variants in Mendelian diseases. The generality of these standards and their subjective interpretation between laboratories has prompted efforts to reduce discordance of variant classifications, with a focus on the expert specification of the ACMG/AMP guidelines for individual genes or diseases. Herein, we describe our experience as a ClinGen Variant Curation Expert Panel to adapt the ACMG/AMP criteria for the classification of variants in three globin genes (HBB, HBA2, and HBA1) related to recessively inherited hemoglobinopathies, including five evidence categories, as use cases demonstrating the process of specification and the underlying rationale.Genetics of disease, diagnosis and treatmen

A whole genome analyses of genetic variants in two Kelantan Malay individuals

The sequencing of two members of the Royal Kelantan Malay family genomes will provide insights on the Kelantan Malay whole genome sequences. The two Kelantan Malay genomes were analyzed for the SNP markers associated with thalassemia and Helicobacter pylori infection. Helicobacter pylori infection was reported to be low prevalence in the north-east as compared to the west coast of the Peninsular Malaysia and beta-thalassemia was known to be one of the most common inherited and genetic disorder in Malaysia.By combining SNP information from literatures, GWAS study and NCBI ClinVar, 18 unique SNPs were selected for further analysis. From these 18 SNPs, 10 SNPs came from previous study of Helicobacter pylori infection among Malay patients, 6 SNPs were from NCBI ClinVar and 2 SNPs from GWAS studies. The analysis reveals that both Royal Kelantan Malay genomes shared all the 10 SNPs identified by Maran (Single Nucleotide Polymorphims (SNPs) genotypic profiling of Malay patients with and without Helicobacter pylori infection in Kelantan, 2011) and one SNP from GWAS study. In addition, the analysis also reveals that both Royal Kelantan Malay genomes shared 3 SNP markers; HBG1 (rs1061234), HBB (rs1609812) and BCL11A (rs766432) where all three markers were associated with beta-thalassemia.Our findings suggest that the Royal Kelantan Malays carry the SNPs which are associated with protection to Helicobacter pylori infection. In addition they also carry SNPs which are associated with beta-thalassemia. These findings are in line with the findings by other researchers who conducted studies on thalassemia and Helicobacter pylori infection in the non-royal Malay population.Wan Khairunnisa Wan Juhari, Nur Aida Md Tamrin, Mohd Hanif Ridzuan Mat Daud, Hatin Wan Isa, Nurfazreen Mohd Nasir, Sathiya Maran, Nur Shafawati Abdul Rajab, Khairul Bariah Ahmad Amin Noordin, Nik Norliza Nik Hassan, Rick Tearle, Rozaimi Razali, Amir Feisal Merican and Bin Alwi Zilfali

Analysis of sequence variations in low-density lipoprotein receptor gene among Malaysian patients with familial hypercholesterolemia

<p>Abstract</p> <p>Background</p> <p>Familial hypercholesterolemia is a genetic disorder mainly caused by defects in the low-density lipoprotein receptor gene. Few and limited analyses of familial hypercholesterolemia have been performed in Malaysia, and the underlying mutations therefore remain largely unknown.</p> <p>We studied a group of 154 unrelated FH patients from a northern area of Malaysia (Kelantan). The promoter region and exons 2-15 of the LDLR gene were screened by denaturing high-performance liquid chromatography to detect short deletions and nucleotide substitutions, and by multiplex ligation-dependent probe amplification to detect large rearrangements.</p> <p>Results</p> <p>A total of 29 gene sequence variants were reported in 117(76.0%) of the studied subjects. Eight different mutations (1 large rearrangement, 1 short deletion, 5 missense mutations, and 1 splice site mutation), and 21 variants. Eight gene sequence variants were reported for the first time and they were noticed in familial hypercholesterolemic patients, but not in controls (p.Asp100Asp, p.Asp139His, p.Arg471Gly, c.1705+117 T>G, c.1186+41T>A, 1705+112C>G, Dup exon 12 and p.Trp666ProfsX45). The incidence of the p.Arg471Gly variant was 11%. Patients with pathogenic mutations were younger, had significantly higher incidences of cardiovascular disease, xanthomas, and family history of hyperlipidemia, together with significantly higher total cholesterol and low density lipoprotein levels than patients with non-pathogenic variants.</p> <p>Conclusions</p> <p>Twenty-nine gene sequence variants occurred among FH patients; those with predicted pathogenicity were associated with higher incidences of cardiovascular diseases, tendon xanthomas, and higher total and low density lipoprotein levels compared to the rest. These results provide preliminary information on the mutation spectrum of this gene among patients with FH in Malaysia.</p