Variable Number Of Tandem Repeats Region Research Articles

MUC1-associated autosomal dominant tubulointerstitial kidney disease: prevalence in kidney failure of undetermined aetiology and clinical insights from Danish families.

Frameshift variants in the variable number tandem repeat region of mucin-1 (MUC1) cause autosomal dominant tubulointerstitial kidney disease (ADTKD-MUC1) but are challenging to detect. We investigated the prevalence in patients with kidney failure of undetermined aetiology and compared Danish families with ADTKD-MUC1. We recruited patients with suspected kidney failure of undetermined aetiology at ≤50 years and excluded those with a clear-cut clinical or histopathological kidney diagnoses or established genetic kidney diseases identified thorough medical record review. MUC1 genotyping was performed by SNaPshot analysis, detecting the most common pathogenic cytosine duplication, followed by bioinformatics pipeline VNtyper analysis of short-read sequencing data. Of 172 recruited patients, 123 underwent SNaPshot analyses, which were abnormal in 5/123 patients (4%). Next, VNtyper genotyping was performed in all patients, including the five with abnormal SNaPshot analysis. VNtyper re-identified the common cytosine duplication in all five patients and revealed novel frameshift variants in two additional patients, while the analyses were normal in the remaining 116 patients. All patients carrying frameshift variants in MUC1 fulfilled ADTKD criteria and had a family history of kidney failure. A considerable inter- and intrafamilial variability of chronic kidney disease stage relative to age was observed in families with ADTKD-MUC1. ADTKD-MUC1 was identified in 7/123 patients (6%) in a selected cohort of kidney failure of undetermined aetiology ≤50 years, and VNtyper effectively identified all pathogenic MUC1 variants.

Nanopore sequencing with unique molecular identifiers enables accurate mutation analysis and haplotyping in the complex lipoprotein(a) KIV-2 VNTR.

Repetitive genome regions, such as variable number of tandem repeats (VNTR) or short tandem repeats (STR), are major constituents of the uncharted dark genome and evade conventional sequencing approaches. The protein-coding LPA kringle IV type-2 (KIV-2) VNTR (5.6kb per unit, 1-40 units per allele) is a medically highly relevant example with a particularly intricate structure, multiple haplotypes, intragenic homologies, and an intra-VNTR STR. It is the primary regulator of plasma lipoprotein(a) [Lp(a)] concentrations, an important cardiovascular risk factor. Lp(a) concentrations vary widely between individuals and ancestries. Multiple variants and functional haplotypes in the LPA gene and especially in the KIV-2 VNTR strongly contribute to this variance. We evaluated the performance of amplicon-based nanopore sequencing with unique molecular identifiers (UMI-ONT-Seq) for SNP detection, haplotype mapping, VNTR unit consensus sequence generation, and copy number estimation via coverage-corrected haplotypes quantification in the KIV-2 VNTR. We used 15 human samples and low-level mixtures (0.5 to 5%) of KIV-2 plasmids as a validation set. We then applied UMI-ONT-Seq to extract KIV-2 VNTR haplotypes in 48 multi-ancestry 1000 Genome samples and analyzed at scale a poorly characterized STR within the KIV-2 VNTR. UMI-ONT-Seq detected KIV-2 SNPs down to 1% variant level with high sensitivity, specificity, and precision (0.977 ± 0.018; 1.000 ± 0.0005; 0.993 ± 0.02) and accurately retrieved the full-length haplotype of each VNTR unit. Human variant levels were highly correlated with next-generation sequencing (R2 = 0.983) without bias across the whole variant level range. Six reads per UMI produced sequences of each KIV-2 unit with Q40 quality. The KIV-2 repeat number determined by coverage-corrected unique haplotype counting was in close agreement with droplet digital PCR (ddPCR), with 70% of the samples falling even within the narrow confidence interval of ddPCR. We then analyzed 62,679 intra-KIV-2 STR sequences and explored KIV-2 SNP haplotype patterns across five ancestries. UMI-ONT-Seq accurately retrieves the SNP haplotype and precisely quantifies the VNTR copy number of each repeat unit of the complex KIV-2 VNTR region across multiple ancestries. This study utilizes the KIV-2 VNTR, presenting a novel and potent tool for comprehensive characterization of medically relevant complex genome regions at scale.

Exploring Gene Polymorphisms Associated with Otitis Media through Variable Number Tandem Repeats (VNTR) Region

Background: Otitis is an inflammation and infection of the inner lining of the middle ear. The problem known as suppurative otitis media if there are droplets and holes in the tympanic membrane due to this inflammation and infection of the mucous membrane. The present study focused on specific genes suspected to be associated with otitis media in 60 subjects clinically diagnosed with OM The study focuses on two genes, MUC5B and IL-1RN, whose genes mutations by variable number of tandem repeat (VNTR) regionsMethods: Variable numbers of tandem repeats (VNTR) regions were used to genotype 60 patients with otitis media and 30 healthy individuals for genotypic polymorphisms for the MUC5B and IL-1RN genes by number of tandem repeats (VNTR) regions on a variable basis Blood samples were collected from participants, and three milliliters were placed directly into EDTA tubes. Subsequently, PCR products were detected by agarose gel electrophoresis and visualized by ethidium bromide staining. The size of amplified DNA fragments was determined by comparison with a 100-bp DNA ladder molecule size markerResults: About the same, the paper represents the association of gene polymorphisms with otitis media by genotyping a total of 60 patients as OM, and, on the other hand, 30 individuals were noted to be healthy. This was to determine genotypic polymorphisms for the two genes, that is, MUC5B and IL1RN, within the VNTR region. The results showed that the carriers of allele 2 in the genotypic position had a risk of nearly twofold getting otitis media.Conclusion: Polymorphism of the MUC5B and IL-1RN genes can be responsible for the severity of the inflammatory process in the middle ear, complicated by otitis media or hearing loss.Keywords: Otitis media; Gene polymorphism; MUC5B; IL-1RN; VNTR

#4410 NOVEL DOMINANT ALG5 VARIANT IN TWO UNRELATED FAMILIES WITH LATE-ONSET ADPKD AND ATYPICAL TUBULOINTERSTITIAL CHANGES

Abstract Background and Aims Autosomal dominant polycystic kidney disease (ADPKD) is a monogenic disorder renowned for its clinical and genetic heterogeneity. Recently, variants in the ALG5 gene have been described as a cause of atypical ADPKD and interstitial fibrosis through the disruption of polycystin 1 maturation and trafficking via underglycosylation. In this report, we investigated genetic cause of the disease in two unrelated Irish families displaying late-onset ADPKD phenotype with atypical tubulointerstitial changes. Method Routine clinical and radiological evaluations were carried out. Available kidney biopsy specimens were reassessed. Genetic testing was performed on probands and all relatives with cystic kidneys. First, a custom-targeted gene panel of 227 genes associated with kidney disease was used, but the causal variant was not identified. Then reading frame-changing variants in variable number tandem repeats region of MUC1 were excluded by single-molecule real-time sequencing. Whole exome sequencing revealed a variant in the ALG5 gene, which is not included in the initial custom gene panel. By targeted Sanger sequencing, segregation of the variant with the disease phenotype was confirmed in both investigated families. Multiple bioinformatic tools were employed to predict the effect on protein structure and functions. Results The clinical diagnosis was consistent in most of the 20 affected individuals with non-enlarged cystic kidneys and few or no liver cysts. All but 1 individual underwent radiological assessment; 7 had kidney and liver cysts, 7 had only kidney cysts, and 5 had no kidney cysts at ages 68.6±14.4, 55.1±15.8, 46.8±15.6 years, respectively. Polycystic liver phenotype (>20 cysts) was present in two individuals. Biopsy-proven extensive kidney interstitial fibrosis and cystic tubular dilation were evident in four affected individuals with available kidney samples. Of the 20 genetically-defined individuals, 4 had end-stage kidney failure at a mean age of 70.25±3.1 years. Ten individuals were CKD stage 3 or greater, while 6 were CKD stage 4 or lower at ages 43.2±12.6 and 63.1±7.5 years (P value 0.004). A novel heterozygous missense variant in the ALG5 gene (NM_013338.5, c.235C>T, p.Arg79Trp) was identified in affected members from investigated families. Genetic screening of 20 affected and 10 unaffected individuals from both pedigrees revealed segregation of the variant with the disease phenotype. The novel ALG5 variant was classified as likely pathogenic as it was absent in the Genome Aggregation Database (gnomAD), is located in a conserved region and is predicted to be deleterious on protein stability. Conclusion This study expands the clinical and genetic spectrum of the identified range of ADPKD, considering the pathogenic ALG5 variants. Establishing a precise diagnosis of atypical cystic and interstitial kidney disease is crucial, with essential implications including follow-up, genetic counselling, prognostication, and therapeutic interventions.

Clinical efficacy and safety of individualized pembrolizumab administration based on pharmacokinetic in advanced non-small cell lung cancer: An open-label, single-arm, exploratory clinical trial.

e21161 Background: Pembrolizumab with or without chemotherapy has become the standard therapy in advanced non-small cell lung cancer (NSCLC), with a fixed dose of 200mg every 3 weeks. We performed this study to explore the clinical efficacy and safety of pharmacokinetic (PK)-guided pembrolizumab administration in advanced NSCLC. Methods: In this prospective, open-label, single-arm exploratory study, we enrolled advanced NSCLC patients without sensitizing EGFR or ALK mutation in Sun Yat-Sen University Cancer Center. Eligible patients received pembrolizumab 200mg every 3 weeks with or without chemotherapy for four cycles, then for patients without progressive disease, pembrolizumab was administrated in new dose-intervals (T) until disease progression. We set the effective concentration (Ce) at 15μg/ml and new dose-intervals (T) was calculated according to steady state plasma-concentration (Css) of pembrolizumab using following equation: Css × 21D = Ce (15μg/ml) ×T. Primary endpoint was the progression-free survival (PFS), secondary endpoints were objective response rate (ORR), and safety. Besides, advanced NSCLC patients who regularly received pembrolizumab 200mg 3-weekly until disease progression and more than four cycles in our center were respectively enrolled as the history-controlled cohort. Patients with Css of pembrolizumab also underwent genetic polymorphism analysis of variable number of tandem repeats region (VNTR) in neonatal Fc receptor (FcRn). This study is registered at Clinicaltrials. gov, NCT05226728. Results: Between Nov 2019 and Dec 2020, total 58 patients were enrolled and received pembrolizumab 200mg every 3 weeks with or without chemotherapy, and 33 patients further received pembrolizumab in new adjusted dose-intervals according to Css after four cycles. The Css of pembrolizumab ranged from 11.01 to 61.21 μg/ml, 30 patients need prolonged intervals (22-80d) and 3 shortened intervals (15-20d). In PK-guided cohort, the median PFS was 15.1 months (95%CI 10.2-20.0 months) and ORR 57.6%, whereas the median PFS in history-controlled cohort was 7.7 months (95% CI 5.1-10.2 months) and ORR 48.2%. The immune-related adverse events occurred in 15.2% of patients in PK-guided cohort and 17.9% of patients in history-controlled cohort. The VNTR3/VNTR3 genotype of FcRn had significantly higher Css of pembrolizumab than VNTR2/VNTR3 ( p= 0.005). Conclusions: PK-guided pembrolizumab administration showed promising clinical efficacy and manageable toxicity, which provided an alternative rational therapeutic strategy of pembrolizumab in advanced NSCLC. Clinical trial information: NCT05226728.

MO034: Novel MUC1 variant identified by massively parallel sequencing explains interstitial kidney disease in a large Dutch family

Abstract BACKGROUND AND AIMS Chronic kidney disease (CKD) can be caused by a wide variety of systemic and renal disorders. In approximately 20% of patients, the cause of CKD remains unknown. Previous studies have shown that massively parallel sequencing (MPS) can be a valuable diagnostic tool in patients with unexplained CKD. Here, we describe a large family with kidney failure of unknown origin, in which we aimed to identify the primary renal disease diagnosis with MPS in a routine healthcare setting. METHOD We conducted MPS in a large family in the Netherlands including six generations with documented kidney failure at young age. Affected individuals had a bland sediment and kidney biopsy revealed interstitial fibrosis in several family members. Whole exome sequencing followed by variant filtering and analysis based on the CKD-Y (CKD-young) gene panel (v18.1, 141 genes) was performed by the Genome Diagnostics Section of the University Medical Center Utrecht. RESULTS MPS revealed a novel frameshift variant [c.326_350dup, p.(Ser119Profs*119), NM_001 204 286.1] in MUC1, which co-segregated with the renal phenotype in the family. This variant is positioned before the first variable number of tandem repeat (VNTR) domain of MUC1 and leads to a + 1 base frameshift in the open reading frame of the MUC1 mRNA. The predicted C-terminal amino-acid sequence of the resulting mutant protein is the same as the previously reported pathogenic cytosine insertions in the VNTR. Variants in MUC1 are associated with autosomal dominant tubulointerstitial kidney disease (ADTKD), which matches the clinical phenotype in this family. Immunohistochemical analysis in the index patient showed MUC1fs deposition in kidney tissue, confirming the diagnosis of ADTKD-MUC1. CONCLUSION We report a novel frameshift mutation in MUC1 identified with MPS. This study highlights that MUC1 variants positioned before the first VNTR region can lead to ADTKD and that genetic testing in patients with suspected ADTKD-MUC1 should also include regions outside of the VNTR. Additionally, this study confirms the relevance of MPS as a tool to screen adult patients with early-onset kidney failure.

Detecting MUC1 Variants in Patients Clinicopathologically Diagnosed With Having Autosomal Dominant Tubulointerstitial Kidney Disease

IntroductionAutosomal dominant tubulointerstitial kidney disease (ADTKD)-MUC1 is predominantly caused by frameshift mutations owing to a single-base insertion into the variable number tandem repeat (VNTR) region in MUC1. Because of the complexity of the variant hotspot, identification using short-read sequencers (SRSs) is challenging. Although recent studies have revealed the usefulness of long-read sequencers (LRSs), the prevalence of MUC1 variants in patients with clinically suspected ADTKD remains unknown. We aimed to clarify this prevalence and the genetic characteristics and clinical manifestations of ADTKD-MUC1 in a Japanese population using an SRS and an LRS.MethodsFrom January 2015 to December 2019, genetic analysis was performed using an SRS in 48 patients with clinically suspected ADTKD. Additional analyses were conducted using an LRS in patients with negative SRS results.ResultsShort-read sequencing results revealed MUC1 variants in 1 patient harboring a cytosine insertion in the second repeat unit of the VNTR region; however, deeper VNTR regions could not be read by the SRS. Therefore, we conducted long-read sequencing analysis of 39 cases and detected MUC1 VNTR variants in 8 patients (in total, 9 patients from unrelated families). With the inclusion of family-affected patients (n = 31), the median age at the development of end-stage kidney disease (ESKD) was 45 years (95% CI: 40–40 years).ConclusionIn Japan, the detection rate of MUC1 variants in patients with clinically suspected ADTKD was 18.8%. More than 20% of patients with negative SRS results had MUC1 variants detected by an LRS.

VNTR polymorphism in the breakpoint region of ABL1 and susceptibility to bladder cancer

BackgroundABL1 is primarily known as a leukemia-related oncogene due to translocation, but about 2.2% of ABL1 mutations have been identified in bladder cancer, and high expression in solid cancer has also been detected.MethodsHere, we used the NCBI database, UCSC genome browser gateway and Tandem repeat finder program to investigate the structural characterization of the ABL1 breakpoint region and to identify the variable number of tandem repeats (VNTR). To investigate the relationship between ABL1-MS1 and bladder cancer, a case-controlled study was conducted in 207 controls and 197 bladder cancer patients. We also examined the level of transcription of the reporter gene driven by the ABL1 promoter to determine if the VNTR region affects gene expression.ResultsIn our study, one VNTR was identified in the breakpoint region, the intron 1 region of ABL1, and was named ABL1-MS1. In the control group, only two common alleles (TR13, TR15) were detected, but an additional two rare alleles (TR14, TR16) were detected in bladder cancer. A statistically significant association was identified between the rare ABL1-MS1 allele and bladder cancer risk: P = 0.013. Investigating the level of transcription of the reporter gene driven by the ABL1 promoter, VNTR showed inhibition of ABL1 expression in non-cancer cells 293 T, but not in bladder cancer cells. In addition, ABL1-MS1 was accurately passed on to offspring according to Mendelian inheritance through meiosis.ConclusionsTherefore, the ABL1-MS1 region can affect ABL1 expression of bladder cancer. This study provides that ABL1-MS1 can be used as a DNA fingerprinting marker. In addition, rare allele detection can predict susceptibility to bladder cancer.

Alzheimer Disease Pathology-Associated Polymorphism in a Complex Variable Number of Tandem Repeat Region Within the MUC6 Gene, Near the AP2A2 Gene.

We found evidence of late-onset Alzheimer disease (LOAD)-associated genetic polymorphism within an exon of Mucin 6 (MUC6) and immediately downstream from another gene: Adaptor Related Protein Complex 2 Subunit Alpha 2 (AP2A2). PCR analyses on genomic DNA samples confirmed that the size of the MUC6 variable number tandem repeat (VNTR) region was highly polymorphic. In a cohort of autopsied subjects with quantitative digital pathology data (n = 119), the size of the polymorphic region was associated with the severity of pTau pathology in neocortex. In a separate replication cohort of autopsied subjects (n = 173), more pTau pathology was again observed in subjects with longer VNTR regions (p = 0.031). Unlike MUC6, AP2A2 is highly expressed in human brain. AP2A2 expression was lower in a subset analysis of brain samples from persons with longer versus shorter VNTR regions (p = 0.014 normalizing with AP2B1 expression). Double-label immunofluorescence studies showed that AP2A2 protein often colocalized with neurofibrillary tangles in LOAD but was not colocalized with pTau proteinopathy in progressive supranuclear palsy, or with TDP-43 proteinopathy. In summary, polymorphism in a repeat-rich region near AP2A2 was associated with neocortical pTau proteinopathy (because of the unique repeats, prior genome-wide association studies were probably unable to detect this association), and AP2A2 was often colocalized with neurofibrillary tangles in LOAD.

Effect of MUC1 length polymorphisms on the NLRP3 inflammasome response of human macrophages

Mucin 1 is a cell-membrane associated mucin, expressed on epithelial and immune cells that helps protect against pathogenic infections. In humans, MUC1 is highly polymorphic, predominantly due to the presence of a variable number tandem repeat (VNTR) region in the extracellular domain that results in MUC1 molecules of typically either short or long length. A genetic link is known between these MUC1 polymorphisms and inflammation-driven diseases, although the mechanism is not fully understood. We previously showed that MUC1 on murine macrophages specifically restricts activation of the NLRP3 inflammasome, thereby repressing inflammation. This study evaluated the effect of MUC1 VNTR polymorphisms on activity of the NLRP3 inflammasome in human macrophages, finding that long MUC1 alleles correlated with increased IL-1β production following NLRP3 inflammasome activation. This indicates that the length of MUC1 can influence IL-1β production, thus providing the first evidence of an immune-modulatory role of MUC1 VNTR polymorphisms in human macrophages.

Pharmacogenetic role of dopamine transporter (SLC6A3) variation on response to disulfiram treatment for cocaine addiction.

Disulfiram has been beneficial in treating cocaine addiction in several studies. Patients with two SLC6A3 (DAT1) rs28363170 10-repeat alleles who have with genetically high dopamine transporter (DAT) levels may benefit from increased dopamine levels resulting from disulfiram treatment. After stabilization for 2 weeks on methadone, 70 cocaine and opioid codependent patients were randomized into disulfiram and placebo groups for 12 weeks of treatment. We genotyped the SLC6A3 (DAT1) 40 bp 3'-untranslated region variable number tandem repeat variant and evaluated its role in moderating disulfiram efficacy for cocaine dependence. Among the 10,10-repeat genotype group, cocaine-positive urines dropped from 78% to 48% and from 80% to 75% among the 9-repeat carrier group in the disulfiram group (P = 0.0001, with an effect size of 0.09). No difference was observed in cocaine-positive urines in the placebo group between the 10,10-repeat genotype and the 9-allele carrier patients. We found that patients with genetically higher DAT levels had better treatment outcomes with disulfiram pharmacotherapy of cocaine dependence than those with lower DAT levels. (Am J Addict 2019;28:311-317).

Hytrosavirus genetic diversity and eco-regional spread in Glossina species

BackgroundThe management of the tsetse species Glossina pallidipes (Diptera; Glossinidae) in Africa by the sterile insect technique (SIT) has been hindered by infections of G. pallidipes production colonies with Glossina pallidipes salivary gland hypertrophy virus (GpSGHV; Hytrosaviridae family). This virus can significantly decrease productivity of the G. pallidipes colonies. Here, we used three highly diverged genes and two variable number tandem repeat regions (VNTRs) of the GpSGHV genome to identify the viral haplotypes in seven Glossina species obtained from 29 African locations and determine their phylogenetic relatedness.ResultsGpSGHV was detected in all analysed Glossina species using PCR. The highest GpSGHV prevalence was found in G. pallidipes colonized at FAO/IAEA Insect Pest Control Laboratory (IPCL) that originated from Uganda (100%) and Tanzania (88%), and a lower prevalence in G. morsitans morsitans from Tanzania (58%) and Zimbabwe (20%). Whereas GpSGHV was detected in 25–40% of G. fuscipes fuscipes in eastern Uganda, the virus was not detected in specimens of neighboring western Kenya. Most of the identified 15 haplotypes were restricted to specific Glossina species in distinct locations. Seven haplotypes were found exclusively in G. pallidipes. The reference haplotype H1 (GpSGHV-Uga; Ugandan strain) was the most widely distributed, but was not found in G. swynnertoni GpSGHV. The 15 haplotypes clustered into three distinct phylogenetic clades, the largest contained seven haplotypes, which were detected in six Glossina species. The G. pallidipes-infecting haplotypes H10, H11 and H12 (from Kenya) clustered with H7 (from Ethiopia), which presumably corresponds to the recently sequenced GpSGHV-Eth (Ethiopian) strain. These four haplotypes diverged the most from the reference H1 (GpSGHV-Uga). Haplotypes H1, H5 and H14 formed three main genealogy hubs, potentially representing the ancestors of the 15 haplotypes.ConclusionThese data identify G. pallidipes as a significant driver for the generation and diversity of GpSGHV variants. This information may provide control guidance when new tsetse colonies are established and hence, for improved management of the virus in tsetse rearing facilities that maintain multiple Glossina species.

Novel motif of variable number of tandem repeats in TPMTpromoter region and evolutionary association of variable number of tandem repeats with TPMT*3alleles.

SNPs in the gene for TPMT exemplify one of the most successful translations of pharmacogenomics into clinical practice. This study explains the correlation between common SNPs and variable number of tandem repeats (VNTR) in promoter of the gene. We determined VNTR polymorphisms, as well as TPMT*2 and TPMT*3 SNPs and TPMT activity in Slovenian and Italian individuals and lymphoblastoid cell lines. We observed a previously unreported VNTR allele, AB7C, in a TPMT*3A heterozygous individual. VNTRs with two (AB2C) and three or more (ABnC, n≥3) B motifs were statistically significant in complete linkage disequilibrium (D'=1, r2=1, p<0.0001) with the TPMT*3C and TPMT*3A alleles, respectively. The study provides insights into the stepwise evolution of TPMT*3 alleles from *3C to *3A, with increasing number of B motifs in the VNTR region.

Characterization of VNTRs Within the Entire Region ofSLC6A3and Its Association with Hypertension

The dopamine transporter SLC6A3 (DAT1) mediates uptake of dopamine into presynaptic terminals. In addition, in previous reports, hypertensive rats were associated with DAT gene, but the genetic association with SLC6A3 and hypertension is still unknown. We examined the distribution of variable number of tandem repeats (VNTRs) and conducted polymorphic analysis of the entire region of SLC6A3. Ten VNTR regions (MS1-10) were revealed throughout the intronic and UTRs; seven VNTR regions were newly isolated and three VNTRs were previously reported. Four VNTR regions (SLC6A3-MS1, -MS4, -MS8 [rs3836790], and -MS9 [rs28363170]) showed polymorphism and these loci were found to be transmitted through meiosis following Mendelian inheritance. These VNTR polymorphisms may be useful markers for paternity mapping and DNA fingerprinting. Furthermore, we also conducted a case-control study between the controls and essential hypertensive cases. Analysis of the genotypes of SLC6A3-MS8 (rs3836790) revealed that having an 8/6-repeat allele, which was only detected in hypertensive cases, was associated with hypertension (p < 0.05). Additional significant association was identified between the short 7-repeat allele of SLC6A3-MS9 (rs28363170) and the occurrence of hypertension (odds ratio 2.02; p < 0.05). These results revealed the genetic association between SLC6A3 and hypertension, and the specific VNTR alleles of SLC6A3 may be a risk factor for hypertension.

New PAH gene promoter KLF1 and 3′-region C/EBPalpha motifs influence transcription in vitro

Phenylketonuria (PKU) is a metabolic disease caused by mutations in the phenylalanine hydroxylase (PAH) gene. Although the PAH genotype remains the main determinant of PKU phenotype severity, genotype-phenotype inconsistencies have been reported. In this study, we focused on unanalysed sequences in non-coding PAH gene regions to assess their possible influence on the PKU phenotype. We transiently transfected HepG2 cells with various chloramphenicol acetyl transferase (CAT) reporter constructs which included PAH gene non-coding regions. Selected non-coding regions were indicated by in silico prediction to contain transcription factor binding sites. Furthermore, electrophoretic mobility shift assay (EMSA) and supershift assays were performed to identify which transcriptional factors were engaged in the interaction. We found novel KLF1 motif in the PAH promoter, which decreases CAT activity by 50% in comparison to basal transcription in vitro. The cytosine at the c.-170 promoter position creates an additional binding site for the protein complex involving KLF1 transcription factor. Moreover, we assessed for the first time the role of a multivariant variable number tandem repeat (VNTR) region located in the 3'-region of the PAH gene. We found that the VNTR3, VNTR7 and VNTR8 constructs had approximately 60% of CAT activity. The regulation is mediated by the C/EBPalpha transcription factor, present in protein complex binding to VNTR3. Our study highlighted two novel promoter KLF1 and 3'-region C/EBPalpha motifs in the PAH gene which decrease transcription in vitro and, thus, could be considered as PAH expression modifiers. New transcription motifs in non-coding regions will contribute to better understanding of the PKU phenotype complexity and may become important for the optimisation of PKU treatment.

Development and Validation of a Mass Spectrometry–Based Assay for the Molecular Diagnosis of Mucin-1 Kidney Disease

Mucin-1 kidney disease, previously described as medullary cystic kidney disease type 1 (MCKD1, OMIM 174000), is an autosomal dominant tubulointerstitial kidney disease recently shown to be caused by a single-base insertion within the variable number tandem repeat region of the MUC1 gene. Because of variable age of disease onset and often subtle signs and symptoms, clinical diagnosis of mucin-1 kidney disease and differentiation from other forms of hereditary kidney disease have been difficult. The causal insertion resides in a variable number tandem repeat region with high GC content, which has made detection by standard next-generation sequencing impossible to date. The inherently difficult nature of this mutation required an alternative method for routine detection and clinical diagnosis of the disease. We therefore developed and validated a mass spectrometry-based probe extension assay with a series of internal controls to detect the insertion event using 24 previously characterized positive samples from patients with mucin-1 kidney disease and 24 control samples known to be wild type for the variant. Validation results indicate an accurate and reliable test for clinically establishing the molecular diagnosis of mucin-1 kidney disease with 100% sensitivity and specificity across 275 tests called.

Synthesis of a Liposomal MUC1 Glycopeptide-Based Immunotherapeutic and Evaluation of the Effect of l-Rhamnose Targeting on Cellular Immune Responses.

Generation of a CD8(+) response to extracellular antigen requires processing of the antigen by antigen presenting cells (APC) and cross-presentation to CD8(+) T cell receptors via MHC class I molecules. Cross-presentation is facilitated by efficient antigen uptake followed by immune-complex-mediated maturation of the APCs. We hypothesize that improved antigen uptake of a glycopeptide sequence containing a CD8(+) T cell epitope could be achieved by delivering it on a liposome surface decorated with an immune complex-targeting ligand, an l-Rhamnose (Rha) epitope. We synthesized a 20-amino-acid glycopeptide TSAPDT(GalNAc)RPAPGSTAPPAHGV from the variable number tandem repeat region of the tumor marker MUC1 containing an N-terminal azido moiety and a tumor-associated α-N-acetyl galactosamine (GalNAc) at the immunogenic DTR motif. The MUC1 antigen was attached to Pam3Cys, a Toll-like receptor-2 ligand via copper(I)-catalyzed azido-alkyne cycloaddition (CuAAc) chemistry. The Rha-decorated liposomal Pam3Cys-MUC1-Tn 4 vaccine was evaluated in groups of C57BL/6 mice. Some groups were previously immunized to generate anti-Rha antibodies. Anti-Rha antibody expressing mice that received the Rha liposomal vaccine showed higher cellular immunogenicity compared to the control group while maintaining a strong humoral response.

TPMT gene expression is increased during maintenance therapy in childhood acute lymphoblastic leukemia patients in a TPMT gene promoter variable number of tandem repeat-dependent manner.

6-mercaptopurine influences in vitro TPMT gene expression in a TPMT promoter variable number of tandem repeats (VNTR)-dependent manner. We studied TPMT expression following 6-mercaptopurine and methotrexate administration in childhood acute lymphoblastic leukemia (ALL) patients and the pharmacogenomic potential of the VNTR architecture. TPMT gene expression was determined in childhood ALL patients at diagnosis (n = 57) and during the maintenance therapy (n = 27). A threefold increase of TPMT gene expression was obtained during maintenance therapy, modulated by the architecture of the VNTR region. The TPMT promoter genetic variants need to be considered at the very beginning of the maintenance therapy for childhood ALL patients. The TPMT promoter VNTR region may serve as a pharmacogenomic biomarker when introducing thiopurine therapy.

Association between the expression of Aggrecan and the distribution of Aggrecan gene variable number of tandem repeats with symptomatic lumbar disc herniation

To study the expression of Aggrecan and the relationship between the variable number of tandem repeats (VNTR) of Aggrecan and lumbar disc herniation (LDH). The disease group comprised of 74 patients already diagnosed with symptomatic LDH. The control group consisted of 15 patients restricted to spinal trauma and 113 healthy blood donors without symptoms of LDH who were not diagnosed with LDH. Disc tissue samples were obtained from surgical operations and blood samples were donated from all participants. The Aggrecan expression in isolated tissues was assessed by western blot using specific antibodies. The Aggrecan gene VNTR region was analyzed by PCR. The Aggrecan expression positive rate of control group was statistically and significantly higher (control group:86.67%, disease group:13.51%;χ(2) = 34.83, P < 0.05) than that of the disease group. Moreover, there was a statistically significant higher frequency of Allele 25 or Allele 21 in disease group compared to controls (A25disease group = 22.97%, A25control group = 12.11%, χ(2)A25 = 8.20, PA25 = 0.004; A21disease group = 6.76%, A21control group = 0.39%, χ(2)A21 = 14.35, PA21 = 0.000). Compared to the participants with 2 Alleles>25 repeats, subjects with 1 or 2 Alleles ≤ 25 repeats statistically and significantly over represented the disease group without the expression of Aggrecan (χ(2) = 5.69, P = 0.017). The findings suggest a relationship between Aggrecan and symptomatic LDH, where symptomatic LDH has a tendency of allele 21 and allele 25 repeats.In addition, an association between the distribution of Aggrecan gene VNTR polymorphism and the expression of Aggrecan is observed in symptomatic LDH.

Expression and characterization of a novel recombinant version of the secreted human mucin MUC5AC in airway cell lines.

Molecular manipulation and expression of mucins, large glycoproteins that provide the structural framework of mucus, are challenging due to mucins' size and numerous domains, including variable number tandem repeat (VNTRs) regions that are sites of O-glycosylation. Only individual human mucin domains have been expressed in mammalian cells. We produced recombinant versions of MUC5AC, a major secreted mucin in the respiratory tract, encoding the N-terminus, C-terminus, N- and C-termini together, and N- and C-termini interspersed with two native tandem repeat sequences (N+2TR+C) in both tracheal and bronchial cell lines. The latter protein contains all of the functional domains required for the biosynthesis and secretion of glycosylated mucin. The N-terminus protein was found in monomeric and higher molecular mass forms suggesting that secreted MUC5AC may form a branched netlike structure analogous to that described for MUC2. At the C-terminus, proteins underwent cleavage, polymerization, and glycosylation. Thus, they appear to undergo pivotal processing steps as predicted for native MUC5AC, which is analogous to that for other individual recombinant mucin domains. Secretion occurred when cells were grown on transwell filter inserts but not on plastic, indicating that the extracellular environment likely plays a role in mucin processing. The secreted N+2TR+C protein differed in molecular mass from the intracellular form, indicating that additional processing occurred. These recombinant proteins, expressed in different backgrounds, can potentially address the role of different mucin domains on MUC5AC processing and function as well as the role of MUC5AC in health and disease.