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The aim of this study was to identify the genetic basis of two female siblings - born to consanguineous Sudanese parents - diagnosed clinically as having the rare condition of 25-hydroxylase deficiency (vitamin D-dependent rickets type 1B). The initial diagnosis was established based on clinical data, laboratory and radiological findings retrospectively. Primers for all exons (5) of human CYP2R1 (NM_024514) were generated followed by Sanger sequencing on exons 1–5 for both girls and their parents. Homozygosity for a point mutation (c.85C > T) was detected, leading to a nonsynonymous variant at position 29 in exon 1, resulting in a premature stop codon (p.Q29X). This is a previously unknown variant that leads to a severely truncated protein and predicted to be among the 0.1 % most deleterious genomic variants(CADD score 36). To our knowledge, this family represents the first case series from Sudan with a confirmed CYP2R1 gene mutation and the 6th world-wide. With the lack of genetic facilities, diagnosis should be suspected by the persistently low 25 hydroxyvitamin D level in spite of proper treatment and after ruling out liver disease and malabsorption. Patients in this case series showed healing of rickets when treated with high doses of 1,25-dihydroxyvitamin D3 (1,25(OH)D3; calcitriol) and oral calcium.

Background Autosomal recessive non-syndromic hearing loss (ARNSHL) is a highly heterogeneous genetic disease. PDZD7 is a new ARNSHL associated gene. Until now, nine PDZD7 biallelic mutation families with ARNSHL have been reported. Here we report a case of Chinese patient with ARNSHL linked to novel mutations in PDZD7 genes. Method The pathogenic mutations were detected by whole exome sequencing for hereditary deafness-related genes of both the proband and his parents. We used kinship detection, mutational hazard prediction, genotype-phenotype correlation analysis and variation screening for potential pathogenic mutations. Re-sequencing was used to confirm the mutations by Sanger sequence. Real time quantitative PCR (RT-qPCR) was used to analyze the PDZD7 gene expression. Population-based screening for variation frequency, evolutionary conservation comparisons, pathogenicity evaluation, and protein structure prediction were conducted to assess the pathogenicity of the novel mutations of PDZD7 gene. Results We determined three variants of the PDZD7 gene that contributed to the deafness of the patient (PDZD7 c.192G > A, p. Met64Ile; c.1648C > T p. Gln550* and c.2341_2352delCGCAGCCGCAGCp. Arg781_Ser 784del). Pathogenic analysis in accordance with the ACMG/AMP Standards and Guidelines identified two novel mutations as Likely Pathogenic. The expression level of PDZD7 gene in the patient was decreased compared to the normal control (P Conclusion Three mutations in PDZD7 gene linked to ARNSHL were identified in a Chinese pedigree. The findings expand not only our knowledge of genetic causes of ARNSHL, but also PDZD7 genes mutation spectrum of the disease. They will aid personalized genetic counseling, molecular diagnostics and clinical management of this condition.

Maple syrup urine disease (MSUD) is a rare autosomal recessive genetic metabolic disease, with a high incidence rate in infants. We analyzed the data of molecular genetic analysis of five infants whose metabolism screening suspected MSUD and described their clinical symptoms. Further, we performed next-generation sequencing and Sanger sequencing to determine the genetic causes of the disease. Bioinformatics tools were used to predict the pathogenicity of novel mutations by performing structural modeling. All the five infants showed symptoms before one year of age and had elevated plasma leucine and valine levels. Among them, four infants presented an obvious increase in the urine lactic acid level. We identified the genetic cause of the disease in four infants and analyzed the pathogenicity of six novel mutations, viz., two mutations in BCKDHA (p.Gly180Asp and p.Arg265Gln), three in BCKDHB (p.Tyr169Cys, p.Ala331Thr, and p.Gly336Ser), and one in DBT (p.Leu69Arg), using in silico analysis. We also reviewed previously reported mutations in Chinese patients and summarized their genotypic and phenotypic characteristics. Our study has confirmed or corrected the clinical diagnosis and enriched the mutation spectrum of BCKDHA, BCKDHB, and DBT. We suggest blood and urine metabolism screening combined with next generation sequencing to diagnose MSUD, especially in infants, to achieve early diagnosis and early treatment.

Background Arrhythmogenic cardiomyopathy (ACM) is an inherited heart muscle disease, which is mainly caused by desmosomal mutations. Sarcomere variants were the primary genetic basis of hypertrophic cardiomyopathy (HCM) and were recently detected in arrhythmogenic cardiomyopathy (ACM). Our aim is to seek potential pathogenic variants of sarcomere genes in our ACM cohort and describe their characteristics. Methods We performed targeted sequencing of 14 sarcomere genes in 84 patients with ACM and set strict criteria to identify potential pathogenic variants. Clinical screening was performed on all available family members of the patients carrying sarcomere variants and specific variants were tested in screened family members by Sanger sequencing. Results We identified 6 sarcomere variants in 6 (7%) patients, which were all definite ACM. Sarcomere variants were detected in NEBL, MYH7, MYH6 and TNNI3, with low prevalence in controls and predicted pathogenic in silico. Among these patients, three had previous detected PKP2 variants. Patients with sarcomere variants all experienced major arrhythmic cardiac event (MACE) with the average age of the first documented MACE being 41.2 ± 11.0 years. Pedigrees analysis showed none of the sarcomere variants carriers among the family members were affected, indicating very low penetrance. Conclusions We detected some sarcomere variants in our ACM cohort. Although those patients with sarcomere variants had severe arrhythmic burden, family co-segregation analysis didn't strongly support a primary role in the pathogenesis of ACM.

Introduction Congenital insensitivity to pain with anhidrosis (CIPA) is a rare autosomal recessive disorder resulting from NTRK1 mutation. Over 105 NTRK1 mutations have been reported in CIPA patients worldwide. The causative NTRK1 mutations lead to loss of function of the TrkA protein, an important ligand for nerve growth factor (NGF), and therefore induce various clinical phenotypes associated with neuron maturation defects. Materials and methods Three patients from unrelated families with CIPA were subjected to detailed clinical examinations. Blood samples were collected from all the patients and their available family members, as well as 200 healthy volunteers. Sanger sequencing for all the exons and splicing sites of NTRK1 was performed on all samples. The phenotype-genotype relationship and genetic epidemiology of Chinese CIPA patients were also analysed. Results A total of four different NTRK1 mutations [c.851-33T>A, c.44G>A (p.Trp15*), c.287+2dupT, c.1549G>C (p.Gly517Arg)] were identified in these families, and c.1549G>C (p.Gly517Arg) was a novel mutation that had not been reported previously. The ‘mild’ manifestations observed in patients with c.851-33T>A indicated this mutation as a ‘mild’ mutation. After reviewing studies reporting mutations in Chinese CIPA patients, we speculate the mutation c.851-33T>A is one of the founder mutations in the Chinese population. Conclusions Our research expanded the spectrum of the NTRK1 mutations associated with CIPA patients, provided additional clues relating to the phenotype-genotype relationship in CIPA, and summarized the features of the genetic epidemiology of CIPA in the Chinese ethnic group.

Hearing loss is a common disease, of which genetic factors are the main cause. The incidence of mild or moderate postlingual deafness in children is not high, and the impact on life and learning is not as severe as that of prelingual deafness. This leads to insufficient attention to the disorder in the clinic. To date, only a few disease-causing genes have been reported. This report describe a case of novel heterozygous mutations in OTOGL that causes nonsyndromic mild sensorineural hearing loss. Basic information, imaging examinations, audiological examination, and vestibular function tests of the proband were collected. Blood samples of the proband's family were collected and analyzed by whole exome sequencing and Sanger sequencing. A pedigree diagram was drawn and the genetic patterns were analyzed. The proband is a 16-year-old female student with mild sensorineural hearing loss. High-resolution CT of the inner ear and vestibular function tests showed no abnormalities. The age of onset was approximately 4 years old. Except for hearing loss, no lesions were seen in other organs. The parents of the proband were not close relatives and had normal hearing. Two novel heterozygous mutations were found in the OTOGL gene. The c.5038del (p.D1680Ifs*6) variant was inherited from the father, and the c.2770C > T (p.R924X) variant from the mother. They enriched the mutation spectrum of OTOGL, which provides the basis for gene function research and genetic consultation.

Idiopathic hypogonadotropic hypogonadism (IHH) is characterized by low levels of gonadotropins and delayed or absent sexual development. Most of the patients are diagnosed in late adolescence or early adulthood. Determining the diagnosis of IHH in prepubertal patients can be challenging. Making a timely, correct diagnosis has important clinical implications. Here we aimed to identify the genetic cause of IHH in three prepubertal siblings from a Chinese Han family and give appropriate treatment advice. Using whole exome sequencing (WES), we identified a novel homozygous GNRHR mutation (NM_000406; c.364C>T, p.L122F) in two prepubertal boys with cryptorchidism and micropenis. Sanger sequencing showed that their younger asymptomatic sister also had the homozygous GNRHR mutation. This mutation was inherited from the father and the mother. Immunofluorescence analysis showed that in permeabilized cells, expression of the mutant receptor on the cell membrane was significantly lower than that of wild-type. Calcium mobilization assays demonstrated that c.364C>T in the GNRHR gene is a complete loss-of-function mutation that caused IHH. These results may contribute to the genetic diagnosis of the three prepubertal siblings with IHH. According to this diagnosis, timely hormonal treatment can be given for the three prepubertal patients to induce pubertal development, especially for the asymptomatic female.

The kallikrein-related peptidase 15 (KLK15) gene is a member of the largest cluster of serine proteases in the human genome. Exhibiting trypsin-like activity, KLK15 is most likely involved in the activation of prostate-specific antigen (PSA; also known as KLK3), an established biomarker for the diagnosis and screening of prostate cancer. High mRNA expression levels of KLK15 have already been reported in ovarian and prostate cancer, in contrast with breast cancer, where KLK15 has been proposed as a biomarker of favorable prognosis. In this study, we exploited the next-generation sequencing (NGS) technology along with 3' rapid amplification of cDNA ends (3' RACE) to discover alternative KLK15 splice variants. Extensive computational analysis of the obtained NGS data revealed the existence of novel splice junctions, thus supporting the existence of novel KLK15 transcripts. Six novel KLK15 splice variants were identified and verified by Sanger sequencing. Two of them (KLK15 v.11 and v.12) contain an open reading frame and are hence predicted to encode two novel KLK15 protein isoforms. Expression analysis of each KLK15 splice variant in sixteen cDNA pools from malignant cell lines and in normal cell lines (HEK293, HaCaT, and BJ cells) revealed very different expression profiles of particular KLK15 transcripts. Moreover, the new KLK15 splice variants were shown to be expressed in breast, ovarian, prostate, urinary bladder, colon, and renal tissue specimens. Due to the prominent clinical value of KLK15 mRNA expression, the novel KLK15 transcripts appear as candidate cancer biomarkers for diagnostic and/or prognostic purposes and, therefore, merit further investigation.

Hirschsprung's disease (HSCR) is a complex disorder with multiple pathogenic gene mutations. Protocadherin alpha 9 (PCDHA9) was identified as a potential candidate gene for HSCR by whole-exome sequencing in a Chinese family. Sanger sequencing in 298 HSCR cases revealed two sporadic Chinese patients with a novel missence PCDHΑ9 mutation (NM_031857; c.1280C > T[p.Ala427Val]) and one sporadic Chinese patient with another novel missence PCDHΑ9 mutation (c.1425C > G[p.Phe475Leu]).The silico predictions and 3D modeling suggest the deleterious effect of identified mutations on protein function. Immunohistochemistry analysis showed PCDHΑ9 was predominantly expressed in the myenteric plexus of human colon tissues. For mouse embryos, PCDHΑ9 was expressed in the stomach but rarely seen in the intestine during E10.5-12.5, then obviously expressed in the intestinal mucosa at E13.5 and extensively expressed in intestinal muscularis and mucosa at E14.5. Moreover, the down-regulation of PCDHΑ9 in the SH-SY5Y cell line promoted the proliferation and migration rate but inhibited the apoptotic rate. In summary, PCDHΑ9 is potentially related to HSCR and the clustered protocadherins (Pcdhs) may involve in the enteric nervous system (ENS) ontogeny.

Background Rothmund-Thomson syndrome (RTS) is a rare autosomal recessive disorder mainly characterized by cutaneous poikiloderma, sparse hair, short stature and skeletal defects. Deleterious mutations in the RecQ-like DNA helicase type 4 (RECQL4) gene have been detected in approximately two-thirds of RTS cases. Methods Three Chinese patients from two unrelated families were enrolled for clinical evaluation. Targeted next-generation sequencing (NGS) using a custom panel consisting of 705 short-stature-related genes was performed for the probands. Variants detected by NGS were confirmed by Sanger sequencing and examined in family members. Results The probands presented with characteristic features of severe growth delay, poikiloderma mostly on the face, buttocks and extremities, sparse or absent hair, eyelashes, and eyebrows, forearm reduction defects, small hands with hypoplasia of the middle phalanx (little finger) in one of the probands, epicanthus, hypertelorism, and dental abnormalities. In addition, novel auricle features and other rare facial features, including narrow palpebral fissure, depressed nasal bridge, and small chin were exhibited. Four novel RECQL4 variants were identified, including three pathogenic frameshift variants, c.1724_1725delAC, p.His575fs*7; c.2421dupT, p.Asp808*; c.1770_1807del, p.Pro591fs*2, and one likely pathogenic missense variant, c.691G>A, p.Gly231Ser. Conclusion Our study expands the mutational spectrum of RECQL4 gene and reveals novel phenotypes observed in Chinese RTS patients.

Genome flanking regions surrounding transcription start sites (TSSs) are critical for the regulation of gene expression, containing many translational regulatory elements. To investigate whether critical single nucleotide polymorphisms (SNPs) exist around TSSs in the dairy goat genome, we performed high throughput DNA sequencing to compare two dairy goat groups with discrepant litter sizes. After genome mapping, SNP calling, and annotation, we screened the SNPs within 2kb scales surrounding annotated TSSs in high fecundity (HF) and low fecundity (LF) groups, respectively. We attempted to identify distinct SNPs and motifs near the TSSs in both groups. The SNPs near the TSSs most were consistent; 318 new SNPs were uncovered in the HF group, of which 305 were heterozygote SNPs, 13 were homozygote SNPs, and majority of which were distributed on chromosome 2 and 29. After validation by Sanger sequencing we found that a SNP in CHI16: 27612330 C>A in the PSEN2 gene presented an A/A genotype in the HF group and an A/A or A/C genotype in the LF group. In conclusion, our study provides insightful information into the dairy goat genomic variations surrounding TSSs, which may contribute to enhanced litter size. Based on comparison studies of SNPs exist around transcription start sites between high fecundity group and low fecundity group. Our finding provides insights concerning the goat litter size phenotypic and will promote future goat breeding.

Previous studies focus on developing high sensitive PCR-related technologies to detect EGFR gene mutation in malignant pleural effusion (MPE) of non-small cell lung cancer patient (NSCLC) instead of improving the quality of clinical samples themselves. We therefore hypothesized that the enrichment of tumor cells in MPE could improve the quality of MPE for the more accurate detection of EGFR gene mutation in the patients with NSCLC. MPE were collected from 28 patients with NSCLC. The tumor cells in MPE were firstly enriched by the depletion of leukocytes with bi-antibodies and identified by multiple flow cytometry. CastPCR was performed to detect the deletions in exon 19 (DelEGFR19) and point mutations in 20 (T790M, c.2369C > T) and 21 (L858R, c.2573 T > G) of EGFR. All the detected mutations were confirmed their presence by Sanger sequencing. Before enrichment, the tumor cells in MPE were detected in 46.4% (13/28) of samples and the median value of the percentage of the tumor cells was only 0.64% including 3 (10.7%, 3/28) of samples more than 10% on it. After enrichment, the tumor cells were detected in all samples (100%) and the median value of the percentage of tumor cells was increased to 40.8% including only one (3.6%, 1/28) less than 10% on it. EGFR gene mutations were detected in 28.6% (8/28) and 42.9% (12/28) of the samples before and after enrichment, respectively. The sensitivity on the detection rate was relatively increased by 44.4%. Moreover, the mutations can be confirmed their presence by Sanger sequencing in 6 samples before enrichment (75%, 6/8) and 11 samples (91.7%, 11/12) after enrichment. It is valuable to improve the quality of the sample by the enrichment of the tumor cells from MPE for the following genetic analysis.

The detection of mutant DNA is critical for precision medicine, but low-frequency DNA mutation is very hard to be determined. CRISPR/Cas9 is a robust tool for in vivo gene editing, and shows the potential for precise in vitro DNA cleavage. Here we developed a DNA mutation detection system based on CRISPR/Cas9 that can detect gene mutation efficiently even in a low-frequency condition. The system of CRISPR/Cas9 cleavage in vitro showed a high accuracy similar to traditional T7 endonuclease I (T7E1) assay in estimating mutant DNA proportion in the condition of normal frequency. The technology was further used for low-frequency mutant DNA detection of EGFR and HBB somatic mutations. To the end, Cas9 was employed to cleave the wild-type (WT) DNA and to enrich the mutant DNA. Using amplified fragment length polymorphism analysis (AFLPA) and Sanger sequencing, we assessed the sensitivity of CRISPR/Cas9 cleavage-based PCR, in which mutations at 1%-10% could be enriched and detected. When combined with blocker PCR, its sensitivity reached up to 0.1%. Our results suggested that this new application of CRISPR/Cas9 system is a robust and potential method for heterogeneous specimens in the clinical diagnosis and treatment management.

Syndromic craniosynostosis is a group of multiple conditions with high heterogeneity, and many rare syndromes still remain to be characterized. To identify and analyze causative genetic variants in nine unrelated probands mainly manifested as syndromic craniosynostosis, we reviewed the relevant medical information of the patients and performed the whole exome sequencing, further verified with Sanger sequencing and parental background. Bioinformatics analysis was used to evaluate the potential deleterious or benign effect of each genetic variant through evolutionary conservation alignment, multi-lines of computer predication and the allele frequency in population dataset (control and patient). The Standards and guidelines from American College of Medical Genetics and Genomics was used to classify and interpret the pathogenicity for each genetic variant. All the nine probands were found to carry the possibly causative variants, among which three variants including two missense mutations (c.3385C > T in IFT122 gene, c.3581A > G in SMC1A gene) and a frameshift mutation (c.434dupA in TWIST1 gene) have never been reported in patients before. We suggested Cornelia de Lange syndrome caused by SMC1A variant is a neglected syndromic craniosynostosis. Our study not only expanded genotypic and phenotypic spectrum of the rare syndromes, but also confirmed that there existed an underlying genetic mechanism. We emphasized that deliberate selection of both the potential candidates and comprehensive detection methods for genetic analysis is important to increase the genetic diagnosis yield of syndromic craninosynostosis.

RNA editing is being recognized as an important post-transcriptional mechanism that may have crucial roles in introducing genetic variation and phenotypic diversity. Despite microRNA editing recurrence, defining its biological relevance is still under extended debate. To better understand microRNA editing function and regulation we performed an exhaustive characterization of the A-to-I site-specific patterns in mir-376a-1, a mammalian microRNA which RNA editing is involved in the regulation of development and in disease. Thorough an integrative approach based on high-throughput small RNA sequencing, Sanger sequencing and computer simulations we explored mir-376a-1 editing in samples from various individuals and primate species including human placenta and macaque, gorilla, chimpanzee and human brain cortex. We observed that mir-376a-1 editing is a common phenomenon in the mature and primary microRNA molecules and it is more frequently detected in brain than in placenta. Primary mir-376a-1 is edited at three positions, -1, +4 and +44. Editing frequency estimations and in silico simulations indicated that editing was not equally recurrent along the three mir-376a-1 sites, nevertheless no epistatic interactions among them were observed. Particularly, the +4 site, located in the seed region of the mature miR-376a-5p, reached the highest editing frequency in all samples. Secondary structure predictions revealed that the +4 position was the one that conferred the highest stability to the mir-376a-1 hairpin. We suggest that molecular stability might partially explain the editing recurrence observed in certain microRNAs and that editing events conferring new functional regulatory roles in particular tissues and species could have been conserved along evolution, as it might be the case of mir-376a-1 in primate brain cortex.

Hereditary hearing loss (HHL) is a common genetically heterogeneous disorder, which follows Mendelian inheritance in humans. Because of this heterogeneity, the identification of the causative gene of HHL by linkage analysis or Sanger sequencing have shown economic and temporal limitations. With recent advances in next-generation sequencing (NGS) techniques, rapid identification of a causative gene via massively parallel sequencing is now possible. We recruited a Korean family with three generations exhibiting autosomal dominant inheritance of hearing loss (HL), and the clinical information about this family revealed that there are no other symptoms accompanied with HL. To identify a causative mutation of HL in this family, we performed whole-exome sequencing of 4 family members, 3 affected and an unaffected. As the result, A novel splicing mutation, c.763+1G>T, in the solute carrier family 17, member 8 (SLC17A8) gene was identified in the patients, and the genotypes of the mutation were co-segregated with the phenotype of HL. Additionally, this mutation was not detected in 100 Koreans with normal hearing. Via NGS, we detected a novel splicing mutation that might influence the hearing ability within the patients with autosomal dominant non-syndromic HL. Our data suggests that this technique is a powerful tool to discover causative genetic factors of HL and facilitate diagnoses of the primary cause of HHL.

Approximately 20% of children with idiopathic nephrotic syndrome do not respond to steroid therapy. More than 30 genes have been identified as disease-causing genes for the steroid-resistant nephrotic syndrome (SRNS). Few reports were from the Chinese population. The coding regions of genes commonly associated with SRNS were analyzed to characterize the gene mutation spectrum in children with SRNS in central China. The first phase study involved 38 children with five genes (NPHS1, NPHS2, PLCE1, WT1, and TRPC6) by Sanger sequencing. The second phase study involved 33 children with 17 genes by next generation DNA sequencing (NGS. 22 new patients, and 11 patients from first phase study but without positive findings). Overall deleterious or putatively deleterious gene variants were identified in 19 patients (31.7%), including four NPHS1 variants among five patients and three PLCE1 variants among four other patients. Variants in COL4A3, COL4A4, or COL4A5 were found in six patients. Eight novel variants were identified, including two in NPHS1, two in PLCE1, one in NPHS2, LAMB2, COL4A3, and COL4A4, respectively. 55.6% of the children with variants failed to respond to immunosuppressive agent therapy, while the resistance rate in children without variants was 44.4%. Our results show that screening for deleterious variants in some common genes in children clinically suspected with SRNS might be helpful for disease diagnosis as well as prediction of treatment efficacy and prognosis.

Idiopathic hypogonadotropic hypogonadism (IHH) is a rare genetic disease caused by low doses of hypothalamic gonadotropin-releasing hormone (GnRH), leading to absence or delayed sexual development. Kallmann syndrome (KS) is characterized by IHH with anosmia or hyposmia. Here, we identified a novel splice site variant (c. 726+2T>G) of ANOS1 gene in three siblings with KS from a Chinese Han family by whole-exome sequencing (WES). In this family, KS is classified as an X-linked recessive inheritance pattern. This mutation was inherited from the mother by Sanger sequencing. An in vitro functional experiment has identified the deleterious effect of this mutation on the transcriptional level of ANOS1 gene. Importantly, the effectiveness of timely hormone replacement therapy was evaluated on the three siblings. Hence, finding genetic causes could be helpful in the early diagnosis and timely treatment of KS.

Introduction Adams-Oliver syndrome (AOS) is a rare developmental disorder characterized by the combination of aplasia cutis congenita of the scalp vertex and terminal transverse limb defects. DOCK6 (Dedicator of cytokinesis 6) is one of the six identified AOS genes. Methods We performed targeted next-generation sequencing (NGS) of a child with an AOS phenotype. Sanger DNA sequencing further validated her lineal consanguinity. To explore the pathological features of the mutation, a minigene assay was used to investigate the effects of the mutation on splicing. Results Two compound heterozygous DOCK6 mutations (c.4106+2T>C and c.3063 C>G (p.Y1021*)) were identified in this family, and both mutations have not been reported previously. Sanger DNA sequencing indicated that the mutations were inherited maternally and paternally, respectively. The results of the minigene assay showed that the c.4106+2T>C mutation resulted in aberrant splicing and caused a four-nucleotide insertion in the transcript and a premature stop codon. Conclusions Our findings expanded the number of reported cases of this rare disease and the mutation spectrum of DOCK6 mutations, which can serve as the basis for prenatal diagnosis and genetic counseling.

This study aimed to investigate single-nucleotide polymorphisms (SNPs) associated with lobaplatin-induced thrombocytopenia in patients with advanced lung cancer in China. Thirty-nine patients who received lobaplatin-based chemotherapy in the 307 Hospitals of Chinese People's Liberation Army from April 2017 to March 2018 were enrolled as study subjects. Peripheral blood DNA was extracted, and 79 candidate SNP positions were selected. A Sanger sequencing platform was employed to measure genotypes for locating the SNP positions associated with lobaplatin-induced thrombocytopenia. Of the 79 candidate genes, SNPs rs342275 and rs7694379 were significantly associated with lobaplatin-induced decrease in platelet (PLT) count (P 0.05). SNPs rs342275, rs342293, rs11789898, and rs17824620 showed significant association with lobaplatin-induced lowest PLT counts (P 0.05). SNPs rs342275, rs342293, rs11789898, rs17824620, and rs7694379 can be used as predictors of thrombocytopenia induced by lobaplatin-based chemotherapy in patients with advanced lung cancer in China.

Heart fatty acid-binding protein (H-FABP) belongs to a family of intracellular fatty acid-binding proteins that are involved in the transport of long-chain fatty acids. Previous studies have indicated that H-FABP is significantly associated with intramuscular fat (IMF) content in pig. In this study, we compared the mRNA and protein expression of H-FABP between Tibetan pig (with high IMF) and Large White pig (with low IMF). The expression of H-FABP at both mRNA and protein levels in the tissues of backfat, longissimus dorsi muscle and liver was found to be significantly higher in TP than in LW. Single-nucleotide polymorphisms (SNPs) in a 2 kb region upstream of the start codon of the gene were screened using Sanger sequencing. We accordingly identified three SNPs (C-1375G, C-314T and T-158G) between the TP and LW populations and genotyped these based on PCR-restriction fragment length polymorphisms (PCF-RFLPs) analysis. The results showed that the C-1375G site might regulate H-FABP gene expression and thus be associated with fat deposition in pigs. Our study provides important data for further investigating the regulatory mechanism of H-FABP for fat deposition in pigs.

Introduction Although distinctive, distal renal tubular acidosis (dRTA) and Hereditary Spherocytosis (HS) shares a common protein, the anion exchanger1 (AE1) encoded by SLC4A1gene. In spite of this, the co-existence of dRTA and HS has rarely been observed. To date, 23 mutations have been identified in SLC4A1 gene causing both autosomal recessive (AR) and autosomal dominant (AD) forms of dRTA. Methods We have assessed the applicability of the High Resolution Melting curve (HRM) method for the detection of SLC4A1 (A858D) mutation in 12 Indian families having AR dRTA coupled with HS. The reliability of the HRM analysis was verified by comparing the results of the HRM method with those of conventional methods such as Polymerase Chain Reaction-Restriction Fragment-Length Polymorphism (PCR-RFLP) and Sanger sequencing thereby confirming the diagnosis. Results We here described the clinical, hematological and genetic data of 16 individuals from 12 families having AR dRTA coupled with HS. All patients carried homozygous SLC4A1 (A858D) mutation, whereas their family members had heterozygous A858D obtained by HRM analysis and confirmed by RFLP and Sanger sequencing. Conclusion Our data indicates that a missense mutation of A858D in SLC4A1 gene is the most common cause of dRTA coupled with HS in the Indian population. HRM analysis can be used as a rapid screening method for common SLC4A1 mutations that cause AR dRTA in the Indian population.

The human L-DOPA decarboxylase (DDC) is an enzyme that displays a pivotal role in metabolic processes. It is implicated in various human disorders, including hepatocellular and lung cancer. Several splice variants of DDC have previously been described, most of which encode for protein isoforms of this enzyme. In the present study, we used next-generation sequencing (NGS) technology along with nested touchdown PCR and Sanger sequencing to identify new splice variants bearing novel exons of the DDC gene, in hepatocellular and lung cancer cell lines. Using an in-house-developed algorithm, we discovered seven novel DDC exons. Next, we determined the structure of ten novel DDC transcripts, three of which contain an open reading frame (ORF) and probably encode for three previously unknown protein isoforms of this enzyme. Future studies should focus on the elucidation of their role in cellular physiology and cancer pathobiology.

To find the variant spectrum of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and evaluate its frequent variants in Chinese congenital absence of vas deferens (CAVD) patients. A total of 276 patients with azoospermia and CAVD (aged from 21 to 44 years old) were investigated from May 2013 to September 2019 in the Third Affiliated Hospital of Sun Yat-sen University. Additionally, 50 healthy, unrelated volunteers were recruited as controls (aged from 21 to 46 years old). The 5′-UTR, exons and their flanking side of the CFTR gene were sequenced by high-throughput sequencing technology. The results were compared with those retrieved from the Ensembl Genome Browser. In addition, all 13 novel variants were further confirmed independently by Sanger sequencing and evaluated in the bioinformatics web servers. A schematic of the variant spectrum of the CFTR gene, including 13 novel variants (12 in CAVD patients, one in the control group), is shown, and the frequent variants in Chinese CAVD patients were 5 T (27.54%), c.-8G > C (7.25%), p.Q1352H (5.98%), and p.I556V (3.08%). 5 T was found to be the most frequent variant. p.Q1352H had a significantly high allelic frequency in CAVD patients (P C and p.I556V had high allelic frequencies but showed no difference between patients and controls (P > 0.05). p.Q1352H is the most common and important missense variant in Chinese patients with CAVD, while the pathological effects of C.-8G > C and p.I556V may be weak after evaluation.

Genetic disorders have been a major concern for public health in China, especially in the rural regions. However, there is little information available about prevalence of many common single-gene disorders in Guizhou Province in the south western part of China. In the present study, we performed a molecular newborn screening for four genetic disorders, including beta-thalassemia (β-thal), glucose-6-phosphate dehydrogenase (G6PD) deficiency, phenylketonuria (PKU), and non-syndromic hearing loss and deafness (NSHL) in this region. A total of 515 newborns were genotyped using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) developed for screening the mutations causing these four disorders, and then confirmed by Sanger sequencing. The results showed that 48 out of 515 newborns were carriers of mutations related to these four diseases, with a frequency of 1 in 11 (9.32%). The carrier frequencies for each disease are: β-thal 2.72%; G6PD deficiency 1.94%; PKU 0.78% and NSHL 4.47%. The genotyping results by MALDI-TOF MS were concordant with Sanger sequencing results within 30 randomly selected samples. This is the first study that reveals carrier frequencies of these four diseases in Guizhou Province. These data provide valuable information for the genetic counseling and disease prevention in Guizhou and southwest China.

To establish a three-step programmed method to find gene mutations related to maturity onset diabetes of the young (MODY). Target region capture and next-generation sequencing (NGS) were performed using customized oligonucleotide probes designed to capture suspected genes for MODY in 11 probands with clinically diagnosed MODY. The suspected associations of certain genes with MODY were then confirmed by Sanger sequencing in the probands and their family members. Finally, to validate variants of one of the genes of interest (glucokinase, GCK) as pathogenic mutations, protein function editing by the variant genes was assessed. In the target region capture and NGS phase, a total of nine variants of seven genes (GCK, WFS1, SLC19A2, SH2B1, SERPINB4, RFX6, and GATA6) were identified in eight probands. Two heterozygous GCK mutations located on the same allele (p.Leu77Arg and p.Val101Met) were identified in a MODY family. Sanger sequencing was used to confirm the variants identified by NGS to be present in probands and their diabetic family members, but not in non-diabetic family members. Finally, enzyme kinetic and thermal stability analyses revealed that the p.Leu77Arg mutation or the p.Leu77Arg mutation in combination with the p.Val101Met mutation inactivates GCK function and stability, while mutation of p.Val101Met alone does not. The p.Leu77Arg but not p.Val101Met GCK mutation is therefore considered a pathogenic mutation associated with MODY. Genetic screening coupled with gene-editing protein function testing is an effective and reliable method by which causative gene mutations of MODY can be identified.

The mutation spectrum for the phenylalanine hydroxylase (PAH) gene was investigated in a cohort of 84 hyperphenylalaninemia (HPA) patients from Romania identified through newborn screening or neurometabolic investigations. Differential diagnosis identified 81 patients with classic PAH deficiency while 3 had tetrahydropterin-cofactor deficiency and/or remained uncertain due to insufficient specimen. PAH-genetic analysis included a combination of Sanger sequencing of exons and exon–intron boundaries, MLPA and NGS with genomic DNA, and cDNA analysis from immortalized lymphoblasts. A diagnostic efficiency of 99.4% was achieved, as for one allele (out of a total of 162 alleles) no mutation could be identified. The most prevalent mutation was p.Arg408Trp which was found in ~ 38% of all PKU alleles. Three novel mutations were identified, including the two missense mutations p.Gln226Lys and p.Tyr268Cys that were both disease causing by prediction algorithms, and the large genomic deletion EX6del7831 (c.509 + 4140_706 + 510del7831) that resulted in skipping of exon 6 based on PAH-cDNA analysis in immortalized lymphocytes. The genomic deletion was present in a heterozygous state in 12 patients, i.e. in ~ 8% of all the analyzed PKU alleles, and might have originated from a Romanian founder.

Background GAMT deficiency is an autosomal recessive disorder of creatine biosynthesis causing developmental delays or intellectual disability in untreated patients as a result of irreversible brain damage occurring prior to diagnosis. Normal neurodevelopmental outcome has been reported in patients treated from neonatal period highlighting the importance of early treatment. Methods Five hundred anonymized newborns from the National Newborn Screening Program of The Netherlands were included into this pilot study. Direct sequencing of the coding region of the GAMT gene was applied following DNA extraction. The disease causing nature of novel missense variants in the GAMT gene was studied by overexpression studies. GAA and creatine was measured in blood dot spots. Results We detected two carriers, one with a known common (c.327G>A) and one with a novel mutation (c.297_309dup (p.Arg105Glyfs*) in the GAMT gene. The estimated incidence of GAMT deficiency was 1:250,000. We also detected five novel missense variants. Overexpression of these variants in GAMT deficient fibroblasts did restore GAMT activity and thus all were considered rare, but not disease causing variants including the c.131G>T (p.Arg44Leu) variant. Interestingly, this variant was predicted to be pathogenic by in silico analysis. The variants were included in the Leiden Open Variation Database (LOVD) database (www.LOVD.nl/GAMT). The average GAA level was 1.14 μmol/L ± 0.45 standard deviations. The average creatine level was 408 μmol/L ± 106. The average GAA/creatine ratio was 2.94 ± 0.136. Conclusion The estimated incidence of GAMT deficiency is 1:250,000 newborns based on our pilot study. The newborn screening for GAMT deficiency should be implemented to identify patients at the asymptomatic stage to achieve normal neurodevelopmental outcome for this treatable neurometabolic disease. Biochemical investigations including GAA, creatine and GAMT enzyme activity measurements are essential to confirm the diagnosis of GAMT deficiency. According to availability, all missense variants can be assessed functionally, as in silico prediction analysis of missense variants is not sufficient to confirm the pathogenicity of missense variants.

Fabry disease (FD) was an X-linked lysosomal storage disorder resulting from a deficiency in glycosphingolipid catabolism caused by mutations in the α-galactosidase A gene GLA. Variant FD patients did not present with classical symptoms during childhood and were undiagnosed or misdiagnosed with other kidney diseases, such as chronic glomerulonephritis (CGN). In this study, we utilized exome sequencing and Sanger sequencing identified the variation p.E66Q of GLA completely co-segregated with the disease phenotype in a Chinese family, which previously been diagnosed as possible CGN. Female patients exhibited preferential X-chromosome inactivation (XCI) of the normal p.E66 allele, as indicated by XCI analysis. By measuring α-Gal A activity, we found that male patients in the pedigree had just little enzymatic activity while female patients had residual enzymatic activity. These patients were diagnosed with renal variant FD in subsequent clinical review. Our results directly implicated the GLA mutation p.E66Q as the genetic etiology of the Chinese renal variant FD pedigree.

Background We have previously demonstrated that cerebrospinal fluid-derived B cells from early relapsing–remitting multiple sclerosis (RRMS) patients that express a VH4 gene accumulate specific replacement mutations. These mutations can be quantified as a score that identifies such patients as having or likely to convert to RRMS. Furthermore, we showed that next generation sequencing is an efficient method for obtaining the sequencing information required by this mutation scoring tool, originally developed using the less clinically viable single-cell Sanger sequencing. Objective To determine the accuracy of MSPrecise, the diagnostic test that identifies the presence of the RRMS-enriched mutation pattern from patient cerebrospinal fluid B cells. Methods Cerebrospinal fluid cell pellets were obtained from RRMS and other neurological disease (OND) patient cohorts. VH4 gene segments were amplified, sequenced by next generation sequencing and analyzed for mutation score. Results The diagnostic test showed a sensitivity of 75% on the RRMS cohort and a specificity of 88% on the OND cohort. The accuracy of the test in identifying RRMS patients or patients that will develop RRMS is 84%. Conclusion MSPrecise exhibits good performance in identifying patients with RRMS irrespective of time with RRMS.

Background Osteogenesis imperfecta (OI) is a rare disease characterized by increased bone fragility and predisposition to fractures, bone deformities and other major signs such as dentinogenesis imperfecta, blue sclera and deafness. Over 90% of OI cases are caused by mutations in the COL1A1 and COL1A2 genes and the inheritance is autosomal dominant. Methods We present a case of a couple requesting genetic counseling, because the man was diagnosed with OI on a clinical and radiological basis and the woman was pregnant. Whole exomes sequencing (WES) was performed in order to identify the mutation (s), followed by prenatal diagnosis. Results WES identified a rare splicing mutation c.1155 + 1G > C in the COL1A1 gene recognized to be pathogenic and subsequently confirmed by next generation sequencing. The carrier state of the mutation was excluded for the fetus, so the pregnancy was further pursued and a healthy baby was born at term. Conclusions WES is a new and effective technique for detecting pathogenic variants in monogenic diseases and it is preferable to use such a technique in diseases with genetic heterogeneity especially when time does not allow another time-consuming diagnostic technique such classical Sanger sequencing. WES offers possibility to expand the global spectrum of OI pathogenic variants enabling the diagnosis of the disease.

Usher syndrome (USH) is a clinically common autosomal recessive disorder characterized by retinitis pigmentosa (RP) and sensorineural hearing loss with or without vestibular dysfunction. In this study, we identified a Hunan family of Chinese descent with two affected members clinically diagnosed with Usher syndrome type 3 (USH3) displaying hearing, visual acuity, and olfactory decline. Whole-exome sequencing (WES) identified a nonsense variant in ABHD12 gene that was confirmed to be segregated in this family by Sanger sequencing and exhibited a recessive inheritance pattern. In this family, two patients carried homozygous variant in the ABHD12 (NM_015600: c.249C>G). Mutation of ABHD12, an enzyme that hydrolyzes an endocannabinoid lipid transmitter, caused incomplete PHARC syndrome, as demonstrated in previous reports. Therefore, we also conducted a summary based on variants in ABHD12 in PHARC patients, and in PHARC patients showing that there was no obvious correlation between the genotype and phenotype. We believe that this should be considered during the differential diagnosis of USH. Our findings predicted the potential function of this gene in the development of hearing and vision loss, particularly with regard to impaired signal transmission, and identified a novel nonsense variant to expand the variant spectrum in ABHD12.

Autosomal recessive osteopetrosis (ARO) is a rare genetic bone disease characterized by dense and fragile bone, caused by a defect in osteoclasts responsible for the bone destruction. In this study, we aimed to investigate the mutations in TCIRG1 and SNX10 that are responsible for 50% and 4% of the cases, respectively. All amplicons were sequenced by Sanger sequencing following PCR amplification. As a result, six different mutations of the TCIRG1 gene were found in five of the twelve unrelated cases. These include two novel mutations, namely c.630 + 1G > T mutation and c.1778_1779delTG mutation of the gene which are identified as homozygous. A compound heterozygosity of known mutations c.649_674del26 and c.1372G > A and homozygous presence of the known c.2235 + 1G > A mutation were also observed in different patients. In addition, as a result of the prenatal testing in a family with osteopetrosis infant, the c.1674-1G > A mutation was detected as homozygous for the fetus. In TCIRG1, c.166C > T change, which is indicated as likely benign according to ClinVar database, was heterozygous. Several known polymorphisms; c.117 + 83 T > C, c.417 + 11A > G and c.714-19C > A in TCIRG1 gene; c.24 + 36 T > A and c.112-84G > A in SNX10 gene were also detected. In conclusion, our study revealed that five of the twelve cases carry at least one mutation of TCIRG1 gene. Further studies with more patients and other genes would help better understanding of genetic etiology of the disease.

Aims To diagnose a Chinese family with inherited ectopia lentis in a genetic method and analyze the genotype-phenotype correlation. Methods The phenotype of each family member was identified by detailed clinical examination. We used targeted next generation sequencing (NGS) to identify mutations in FBN1 in an efficient and accurate way. The mutation in FBN1 was confirmed in all affected family members by Sanger sequencing. Results A novel mutation c.385T>C (p.C129R) in FBN1 was identified in the proband by targeted NGS. The mutation was segregated in all affected family members and contributes to specific disease phenotypes. The same mutation was not found in other unaffected relatives and a 100 normal random population by Sanger sequencing. Conclusions Our study reports a novel mutation in FBN1 in a Chinese family and to diagnose this family as Marfan syndrome, we also expand the genotype-phenotype correlation of this disease.

Introduction Fructose-1,6-bisphosphatase deficiency (FBPase deficiency) is a rare inborn error of metabolism that affects gluconeogenesis. Ketotic hypoglycemia is the main symptom and can occur at any age, usually after long periods of fasting or during illness. The diagnosis may be achieved by measurement of the enzyme activity in a liver sample, but FBP1 analysis has become the most common approach. Aim To characterize the genotype of Southern Brazilian FBPase-deficient patients. Methodology The FBP1 gene of six unrelated patients (one had consanguineous parents) with previous diagnoses of FBPase deficiency (enzymatic, pts A, B, D, E; genetic through Next-Generation Sequencing-NGS, pt F; enzymatic and Sanger sequencing, pt C) was first analyzed through NGS. Pathogenic variants found in NGS were confirmed by Sanger sequencing. The pathogenicity of novel missense variants was evaluated through in silico analysis. Results Five patients (pt A, B, D, E, F) had their genotype identified by NGS, all of them being homozygous. In Pt C, NGS detected only one pathogenic variant. Among the 11 alleles analyzed, only three variants were found, two being novel: c.958G > A and c.986T > C. In silico analysis indicated the pathogenicity of both variants. Interestingly, the three variants seem to be linked to specific haplotypes, indicating that an endogamy effect may be acting on these alleles in the population of Southern Brazil. Conclusions Our data suggest that NGS is a good tool for the diagnosis of FBPase deficiency. Variants c.958G > A and c.986T > C are the most prevalent variants in the country.

Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) is an autosomal dominant entity characterized by eyelid malformations and caused by mutations in the forkhead box L2 (FOXL2) gene. Clinical and genetic analyses of large cohorts of BPES patients from different ethnic origins are important for a better characterization of FOXL2 mutational landscape. The purpose of this study is to describe the phenotypic features and the causal FOXL2 variants in a Mexican cohort of BPES patients. A total of 12 individuals with typical facial findings were included. Clinical evaluation included palpebral measurements and levator function assessment. The complete coding sequence of FOXL2 was amplified by PCR and subsequently analyzed by Sanger sequencing. A total of 11 distinct FOXL2 pathogenic variants were identified in our cohort (molecular diagnostic rate of 92%), including 5 novel mutations. Our results broaden the BPES-related mutational spectrum and supports considerable FOXL2 allelic heterogeneity in our population.

Autosomal Dominant Polycystic Kidney Disease (ADPKD), the most frequent genetic disorder of the kidneys, is characterized by a typical presenting symptoms include cysts development in different organs and a non-cysts manifestations. ADPKD is caused by mutations in PKD1 or PKD2 genes. In this study, we aimed to search for molecular causative defects among PKD1 and PKD2 genes. Eighteen patients were diagnosed based on renal ultrasonography and renal/extra-renal manifestations. Then, Sanger sequencing was performed for PKD1 and PKD2 genes. Multiplex Ligation dependent Probe Amplification method (MLPA) methods was performed for both PKD genes. Mutational analysis of the PKD2 gene revealed the absence of variants and no deletions or duplications of both PKD genes were detected. But three novels mutations i.e. p.S463C exon 7; c. c.11156+2T>C IVS38 and c.8161-1G>A IVS22 and two previously reported c.1522T>C exon 7 and c.412C>T exon 4 mutations in the PKD1 gene were detected. Bioinformatics tools predicted that the novel variants have a pathogenic effects on splicing machinery, pre-mRNA secondary structure and stability and protein stability. Our results highlighted molecular features of Tunisian patients with ADPKD and revealed novel variations that can be utilized in clinical diagnosis and in the evaluation of living kidney donor. To the best of our knowledge, this is the first report of Autosomal Polycystic Kidney Disease in Tunisia.

Introduction Calpainopathy is an autosomal recessive form of limb girdle muscular dystrophies (LGMDs) caused by mutations in the CAPN3 gene. CAPN3 is a Ca2+-dependent cystein protease consisting of 821 amino acids. LGMD is a highly heterogeneous disorder and mutation identification of this disease by Sanger sequencing of all genes is expensive and time consuming. Using autozygosity mapping is an effective approach to address this issue. Methods We used two sets of multiplex STR (Short tandem repeat) markers linked to CAPN3, DYSF, SGCA, SGCB, SGCG, SGCD genes following sequencing of the CAPN3 gene. In silico analysis and mutation detection in one hundred ethnically matched healthy individuals were carried out to determine the pathogenicity of novel mutations. Sequence variant interpretation was performed using the American College of Medical Genetics and Genomics (ACMG) guideline. Results Sixteen out of 50 families linked to the CAPN3 gene. In this study, mutations were found in 14 out of 16 families including 4 novel (c.1894A > T, c.567delG, c.2254-2256delAAC, and c.2373C > T) and 9 previously reported mutations consisting of 5 missense (c.2105C > T, c.2243G > A, c.1714C > T, c.291C > A, c.956C > T), 3 splice site (c.2380 + 2 T > G, c.946-2A > G, c.380G > A), and one indel (c.2257delinsAA) mutations. Discussion The c.2105C > T was found to be the most frequent mutation in this study. The results of this study revealed that most cases with splicing, frame shift and nonsense mutations experienced more severe clinical manifestations. Nonetheless, this should be confirmed by further studies on larger sample size.

To study the correlation between adenosine deaminase (ADA) and IFN-γ gene polymorphism and the risk of tuberculous pericarditis (TBP).Two-hundred and forty-five patients with TBP were enrolled as a study group, including 140 males and 105 females. According to the general information of TBP patients in the study group, 115 patients with non-tuberculous pericarditis were recruited as the control group. Four ADA single nucleotide polymorphisms (SNPs; rs121908715, rs79281338, rs121908723, and rs61737144) and the rs2069707 locus of the IFN-γ polymorphism were detected using PCR and Sanger sequencing.Carriers with the T allele at the rs121908715 locus of ADA had a higher risk of TBP (adjusted OR = 3.986, 95% CI = 1.858-8.718, p 0.001). There was a significantly higher risk of TBP in carriers with the G allele at the rs121908723 locus of ADA (adjusted OR = 2.334, 95% CI = 1.084-5.102, p = 0.018). The risk of TBP was higher in carriers with the G allele at the rs2069707 locus of IFN-γ (adjusted OR = 2.844, 95% CI = 1.399-5.853, p = 0.002). TB patients who carry both the T allele at the rs121908715 locus of ADA and the G allele of the IFN-γ gene, or who both carry the G allele at the rs126908723 locus of ADA and the G allele at the rs2069707 locus of IFN-γ have a higher risk of contracting TBP (adjusted OR = 11.034, 95% CI = 2.781-15.328, p 0.001 and adjusted OR = 10.315, 95% CI = 4.522-13.854, p 0.001).The ADA SNPs rs12190871 and rs121908723, and the rs2069707 locus SNPs of IFN-γ are risk factors for contracting TBP.

The human SR-related CTD associated factor 1 (SCAF1) gene is a new member of the human SR (Ser/Arg-rich) superfamily of pre-mRNA splicing factors, which has been discovered and cloned by members of our lab. SCAF1 interacts with the CTD domain of the RNA polymerase II polypeptide A and is firmly involved in pre-mRNA splicing. Although it was found to be expressed widely in multiple human tissues, its mRNA levels vary a lot. The significant relation of SCAF1 with cancer has been confirmed by many studies, since SCAF1 mRNA transcript was found to be overexpressed in breast and ovarian tumors, confirming its significant prognostic value as a cancer biomarker in both these human malignancies. In this study, we describe the discovery and cloning of fifteen novel transcripts of the human SCAF1 gene (SCAF1 v.2 - v.16), using nested PCR and NGS technology. In detail, extensive bioinformatic analysis revealed that these novel SCAF1 splice variants comprise a total of nine novel alternative splicing events between the annotated exons of the gene, thus producing seven novel SCAF1 transcripts with open-reading frames, which are predicted to encode novel SCAF1 isoforms and eight novel SCAF1 transcripts with premature termination codons that are likely long non-coding RNAs. Additionally, a novel 3’ UTR was discovered and cloned using nested 3’ RACE and was validated with Sanger sequencing. In order to validate the NGS findings as well as to investigate the expression profile of each novel transcript, RT-PCR experiments were carried out with the use of variant-specific primers. Since SCAF1 is implicated in many human malignancies, qualifying as a potential biomarker, the quantification of the presented novel transcripts in human samples may have clinical applications in different types of cancer.