UNASSIGNED: TP53 is a highly conserved tumor suppressor gene present on chromosome 17 and comprised 11 exons and 12 introns. The TP53 protein maintained the genomic integrity of the cell by regulating different pathways. The association of TP53 with leukemia and the increasing prevalence of leukemia in Pakistan instigated us to initiate the current study.
UNASSIGNED: The TP53 gene of acute myeloid leukemia patients (n = 23) and normal individuals (n = 30) was amplified through polymerase chain reaction (PCR). The PCR amplified products of 3 samples 1 normal (NC-30) and 2 cancerous (LK-6 and LK-19) were subjected to deoxyribonucleic acid (DNA) sequence analysis. Bioinformatics analysis of the obtained DNA sequences helped to identify nature, type, and functional impact of mutations, if any.
UNASSIGNED: Results revealed 2 novel mutations in Case No. 1 (c. G >A10987 and c. InsA13298_13299) and Case No. 2 (c. InsC13284_13285, c. T >A13365) which generate a premature codon (ocher) at position 239 and lead to truncated TP53 protein. In Case No. 3, 16 novel mutations were identified and c. delC11093 mutation created a premature codon (opal) at 59th position. Hence, the resultant protein will lack its tetramerization and N-terminal domain required for its normal functioning. Moreover, some intronic mutations were noticed and found to have a negative impact on splicing related regulatory sequences.
UNASSIGNED: Results suggest the role of TP53 inactivating mutations in pathogenesis of leukemia.

Prediction of the effect of a single-nucleotide variant (SNV) in an intronic region on aberrant pre-mRNA splicing is challenging except for an SNV affecting the canonical GU/AG splice sites (ss). To predict pathogenicity of SNVs at intronic positions -50 (Int-50) to -3 (Int-3) close to the 3\' ss, we developed light gradient boosting machine (LightGBM)-based IntSplice2 models using pathogenic SNVs in the human gene mutation database (HGMD) and ClinVar and common SNVs in dbSNP with 0.01 ≤ minor allelic frequency (MAF) < 0.50. The LightGBM models were generated using features representing splicing cis-elements. The average recall/sensitivity and specificity of IntSplice2 by fivefold cross-validation (CV) of the training dataset were 0.764 and 0.884, respectively. The recall/sensitivity of IntSplice2 was lower than the average recall/sensitivity of 0.800 of IntSplice that we previously made with support vector machine (SVM) modeling for the same intronic positions. In contrast, the specificity of IntSplice2 was higher than the average specificity of 0.849 of IntSplice. For benchmarking (BM) of IntSplice2 with IntSplice, we made a test dataset that was not used to train IntSplice. After excluding the test dataset from the training dataset, we generated IntSplice2-BM and compared it with IntSplice using the test dataset. IntSplice2-BM was superior to IntSplice in all of the seven statistical measures of accuracy, precision, recall/sensitivity, specificity, F1 score, negative predictive value (NPV), and matthews correlation coefficient (MCC). We made the IntSplice2 web service at https://www.med.nagoya-u.ac.jp/neurogenetics/IntSplice2.

Stargardt disease is a progressive retinal disorder caused by bi-allelic mutations in the ABCA4 gene that encodes the ATP-binding cassette, subfamily A, member 4 transporter protein. Over the past few years, we and others have identified several pathogenic variants that reside within the introns of ABCA4, including a recurrent variant in intron 36 (c.5196+1137G>A) of which the pathogenicity so far remained controversial. Detailed clinical characterization of this variant confirmed its pathogenic nature, and classified it as an allele of intermediate severity. Moreover, we discovered several additional ABCA4 variants clustering in intron 36. Several of these variants resulted in aberrant splicing of ABCA4, i.e., the inclusion of pseudoexons, while the splicing defects caused by the recurrent c.5196+1137G>A variant strongly increased upon differentiation of patient-derived induced pluripotent stem cells into retina-like cells. Finally, all splicing defects could be rescued by the administration of antisense oligonucleotides that were designed to specifically block the pseudoexon insertion, including rescue in 3D retinal organoids harboring the c.5196+1137G>A variant. Our data illustrate the importance of intronic variants in ABCA4 and expand the therapeutic possibilities for overcoming splicing defects in Stargardt disease.

BACKGROUND: In about 5% of patients with hereditary angioedema due to C1-inhibitor deficiency (C1-INH-HAE) no mutation in the SERPING1 gene is detected.
METHODS: C1-INH-HAE cases with no mutation in the coding region of SERPING1 after conventional genotyping were examined for defects in the intronic or untranslated regions of the gene. Using a next-generation sequencing (NGS) platform targeting the entire SERPING1, 14 unrelated C1-INH-HAE patients with no detectable mutations in the coding region of the gene were sequenced. Detected variants with a global minor allele frequency lower than the frequency of C1-INH-HAE (0.002%), were submitted to in silico analysis using ten different bioinformatics tools. Pedigree analysis and examination of their pathogenic effect on the RNA level were performed for filtered in variants.
RESULTS: In two unrelated patients, the novel mutation c.-22-155G > T was detected in intron 1 of the SERPING1 gene by the use NGS and confirmed by Sanger sequencing. All bioinformatics tools predicted that the variant causes a deleterious effect on the gene and pedigree analysis showed its co-segregation with the disease. Degradation of the mutated allele was demonstrated by the loss of heterozygosity on the cDNA level. According to the American College of Medical Genetics and Genomics 2015 guidelines the c.-22-155G > T was curated as pathogenic.
CONCLUSIONS: For the first time, a deep intronic mutation that was detected by NGS in the SERPING1 gene, was proven pathogenic for C1-INH-HAE. Therefore, advanced DNA sequencing methods should be performed in cases of C1-INH-HAE where standard approaches fail to uncover the genetic alteration.

BACKGROUND: The Fat mass and obesity-associated gene (FTO) and its involvement in weight gain and obesity is well-known. However, no reports have been published on the Indian population regarding the relationship between single nucleotide polymorphisms (SNPs) in its intronic region and obesity. The aim of this pilot study was to evaluate the frequency and association of SNPs in intron-1 of the FTO gene in obese and overweight Indian adults.
METHODS: This study group consisted of 80 adults, aged 23.5 ± 8.9 yr, with a mean BMI of 28.8 ± 6.2 kg/m2. Genomic DNA was isolated, exons1-3 & intron1 of FTO were amplified using polymerase chain reaction and sequenced by ABI sequencing detection system. The reported SNPs rs1420185, rs8050136, rs1121980 and rs55872725 were checked for their presence or absence in this group of the adult Indian population.
RESULTS: No mutations were found in the exonic sequence of FTO, however, the association of rs1420185, rs8050136, rs1121980 and rs55872725 SNPs was identified in this population. The genotypic frequency at FTO rs8050136 was 32.2% for C>A, at rs55872725 it was 45.7% for C>T, at rs1420185 it was 27.1% for T>C and at rs1121980 it was 30.5% for G>A. All four SNPs in combination were observed in 6 participants (10.2%), all of whom were found to be either obese or overweight.
CONCLUSIONS: These findings indicate that Indians with these SNPs are most likely to be at increased risk of obesity.

Tangier disease is a rare disorder of lipoprotein metabolism that presents with extremely low levels of HDL cholesterol and apoprotein A-I. It is caused by mutations in the ATP-binding cassette transporter A1 (ABCA1) gene. Clinical heterogeneity and mutational pattern of Tangier disease are poorly characterized. Moreover, also familial HDL deficiency may be caused by mutations in ABCA1 gene. ATP-binding cassette transporter A1 (ABCA1) gene mutations in a patient with Tangier disease, who presented an uncommon clinical history, and in his family were found and characterized. He was found to be compound heterozygous for two intronic mutations of ABCA1 gene, causing abnormal pre-mRNAs splicing. The novel c.1510-1G > A mutation was located in intron 12 and caused the activation of a cryptic splice site in exon 13, which determined the loss of 22 amino acids of exon 13 with the introduction of a premature stop codon. Five heterozygous carriers of this mutation were also found in proband\'s family, all presenting reduced HDL cholesterol and ApoAI (0.86 ± 0.16 mmol/L and 92.2 ± 10.9 mg/dL respectively), but not the typical features of Tangier disease, a phenotype compatible with the diagnosis of familial HDL deficiency. The other known mutation c.1195-27G > A was confirmed to cause aberrant retention of 25 nucleotides of intron 10 leading to the insertion of a stop codon after 20 amino acids of exon 11. Heterozygous carriers of this mutation also showed the clinical phenotype of familial HDL deficiency. Our study extends the catalog of pathogenic intronic mutations affecting ABCA1 pre-mRNA splicing. In a large family, a clear demonstration that the same mutations may cause Tangier disease (if in compound heterozygosis) or familial HDL deficiency (if in heterozygosis) is provided.

Charcot-Marie-Tooth disease (CMT) refers to a group of clinically and genetically heterogeneous inherited neuropathies. Ganglioside-induced differentiation-associated protein 1 GDAP1-related CMT has been reported in an autosomal dominant or recessive form in patients presenting either axonal or demyelinating neuropathy. We report two Sri Lankan sisters born to consanguineous parents and presenting with a severe axonal sensorimotor neuropathy. The early onset of the disease, the distal and proximal weakness and atrophy leading to major disability, along with areflexia, and, most notably, vocal cord and diaphragm paralysis were highly evocative of a GDAP1-related CMT. However, sequencing of the coding regions of the gene was normal. Whole-exome sequencing (WES) was performed and revealed that the largest region of homozygosity was around GDAP1 with several variants, mostly in non-coding regions. In view of the high clinical suspicion of GDAP1 gene involvement, we examined the variants in this gene and this, along with functional studies, allowed us to identify an alternative splicing site revealing a cryptic in-frame stop codon in intron 4 responsible for a severe loss of wild-type GDAP1. This work is the first to describe a deleterious mutation in GDAP1 gene outside of coding sequences or intronic junctions and emphasizes the importance of interpreting molecular analysis, and in particular WES results, in light of the clinical and electrophysiological phenotype.

BACKGROUND: Blood group genotyping is used to predict RhD phenotype in transfusion and obstetric medicine. Prediction of antigen D is based on molecular techniques which targets most common RHD-specific polymorphism. However, inactive RHD variants can suggest false-positive RhD phenotype. Their types and frequencies vary among ethnicities. Our study aimed to identify RHD variants among Moroccan blood donors who are serologically D negative.
METHODS: DNA from 53 blood donors who are serologically D negative RhC and/or RhE positive were screened for RHD exon 10 by PCR-SSP. RHD-positive samples were further tested by multiplex PCR covering exons 3, 4, 5, 6, 7 and 9 and then sequenced by targeted next-generation sequencing method. Mutations\' impact on mRNA splicing was predicted using alamut software version 2·0.
RESULTS: PCR-SSP revealed 9 of 53 (16·9%) RHD-positive samples. Five of nine samples were positive for all tested exons, two of nine were positive for exon 9, and two of nine were undetermined. Sequencing revealed four novel RHD variants based on six mutations in introns 1, 3, 5 and 6. In silico analysis revealed aberrant splicing of three mutations (RHD c.487-1024delG, RHD c.487-256T>G and RHD c.940-187_940-188del), while three other mutations (RHD c.149-682C>A, RHD c.802-37delA and RHD c.939 + 1151dup) had no effect on splicing compared to wild type.
CONCLUSIONS: All identified RHD variants contain at least one mutation that probably affects splicing to generate D-negative phenotype. Hence, ethnic RhD antigen background must be considered when developing transfusion and obstetric strategies.

Sensorineural hearing loss (SNHL) is the most prevalent genetic sensory defect in humans, affecting about 1 in 1000 newborns around the world. Non-syndromic SNHL accounts for nearly 70% of hereditary hearing loss and 80% of SNHL cases show an autosomal recessive mode of inheritance (ARNSHL). In the present study, we applied targeted-exome sequencing to a family with a single proband affected by congenital sensorineural hearing loss. 127 known genes were sequenced to find the causative mutation. One novel homozygous donor splice site mutation, c.4596 + 1G > A (IVS12 + 1G > A) was found in MYO15A gene. Analysis of this mutation within the family showed that the mutation segregates with hearing loss. New DNA sequencing technologies could lead to identification of the disease causing variants especially in highly heterogeneous disorders such as hearing loss.

Point mutations are the most common cause of inherited diseases. Bioinformatics tools can help to predict the pathogenicity of mutations found during genetic screening, but they may work less well in determining the effect of point mutations in non-coding regions. In silico analysis of intronic variants can reveal their impact on the splicing process, but the consequence of a given substitution is generally not predictable. The aim of this study was to functionally test five intronic variants (MYBPC3-c.506-2A>C, MYBPC3-c.906-7G>T, MYBPC3-c.2308+3G>C, SCN5A-c.393-5C>A, and ACTC1-c.617-7T>C) found in five patients affected by inherited cardiomyopathies in the attempt to verify their pathogenic role. Analysis of the MYBPC3-c.506-2A>C mutation in mRNA from the peripheral blood of one of the patients affected by hypertrophic cardiac myopathy revealed the loss of the canonical splice site and the use of an alternative splicing site, which caused the loss of the first seven nucleotides of exon 5 (MYBPC3-G169AfsX14). In the other four patients, we generated minigene constructs and transfected them in HEK-293 cells. This minigene approach showed that MYBPC3-c.2308+3G>C and SCN5A-c.393-5C>A altered pre-mRNA processing, thus resulting in the skipping of one exon. No alterations were found in either MYBPC3-c.906-7G>T or ACTC1-c.617-7T>C. In conclusion, functional in vitro analysis of the effects of potential splicing mutations can confirm or otherwise the putative pathogenicity of non-coding mutations, and thus help to guide the patient\'s clinical management and improve genetic counseling in affected families.