Tandem Repeat Research Articles

Chromosome analysis of 303 pregnancy losses in Mexico

Chromosome analysis of 303 pregnancy losses in Mexico

Analysis of PAH gene variants and prenatal diagnosis for 43 Chinese pedigrees affected with Phenylketonuria

To explore the characteristics of phenylalanine hydroxylase (PAH) gene variants and prenatal diagnosis for 43 Chinese pedigrees affected with Phenylketonuria (PKU). Forty three PKU pedigrees diagnosed at the First Affiliated Hospital of Zhengzhou University between 2019 and 2021 were selected as the study subjects. Variants of the PAH gene of the probands were screened by high-throughput sequencing, and candidate variants were verified by Sanger sequencing. Negative cases were further analyzed by multiplex ligation-dependent probe amplification (MLPA) to detect large fragment deletions and duplications of the PAH gene. For 43 women undergoing subsequent pregnancy, Sanger sequencing, MLPA, combined with short tandem repeats (STR) sequence-based linkage analysis, were carried out for prenatal diagnosis. Among the 86 alleles carried by the 43 probands, 78 nucleotide variants (90.70%) and 3 large deletions (3.49%) were found based on high-throughput sequencing and MLPA. The 81 mutant alleles had included 21 missense variants, 5 splice site variants, 4 nonsense variants, 2 microdeletions, 1 insertional variant and 2 large fragment deletions. Relatively common variants have included p.Arg243Gln (23.26%), p.Arg111Ter (8.14%), EX6-96A>G (6.98%), p.Val399Val (5.81%) and p.Arg413Pro (4.65%). Most of the variants were located in exons 7, 11, 3, 6 and 12. For the 43 families undergoing prenatal diagnosis, 9 fetuses (20.45%) were diagnosed with PKU, 20 (45.45%) were heterozygous carriers, and 15 (34.09%) did not carry the same pathogenic allele as the proband. All neonates were followed up till 6 months old, and the accuracy of prenatal diagnosis was 100%. The combination of high-throughput sequencing, Sanger sequencing, MLPA and linkage analysis can increase the diagnostic rate of PKU and attain accurate prenatal diagnosis.

Genome Sequencing for Diagnosing Rare Diseases

BackgroundGenetic variants that cause rare disorders may remain elusive even after expansive testing, such as exome sequencing. The diagnostic yield of genome sequencing, particularly after a negative evaluation, remains poorly defined.MethodsWe sequenced and analyzed the genomes of families with diverse phenotypes who were suspected to have a rare monogenic disease and for whom genetic testing had not revealed a diagnosis, as well as the genomes of a replication cohort at an independent clinical center.ResultsWe sequenced the genomes of 822 families (744 in the initial cohort and 78 in the replication cohort) and made a molecular diagnosis in 218 of 744 families (29.3%). Of the 218 families, 61 (28.0%) — 8.2% of families in the initial cohort — had variants that required genome sequencing for identification, including coding variants, intronic variants, small structural variants, copy-neutral inversions, complex rearrangements, and tandem repeat expansions. Most families in which a molecular diagnosis was made after previous nondiagnostic exome sequencing (63.5%) had variants that could be detected by reanalysis of the exome-sequence data (53.4%) or by additional analytic methods, such as copy-number variant calling, to exome-sequence data (10.8%). We obtained similar results in the replication cohort: in 33% of the families in which a molecular diagnosis was made, or 8% of the cohort, genome sequencing was required, which showed the applicability of these findings to both research and clinical environments.ConclusionsThe diagnostic yield of genome sequencing in a large, diverse research cohort and in a small clinical cohort of persons who had previously undergone genetic testing was approximately 8% and included several types of pathogenic variation that had not previously been detected by means of exome sequencing or other techniques. (Funded by the National Human Genome Research Institute and others.)

Mini-heterochromatin domains constrain the cis-regulatory impact of SVA transposons in human brain development and disease.

SVA (SINE (short interspersed nuclear element)-VNTR (variable number of tandem repeats)-Alu) retrotransposons remain active in humans and contribute to individual genetic variation. Polymorphic SVA alleles harbor gene regulatory potential and can cause genetic disease. However, how SVA insertions are controlled and functionally impact human disease is unknown. Here we dissect the epigenetic regulation and influence of SVAs in cellular models of X-linked dystonia parkinsonism (XDP), a neurodegenerative disorder caused by an SVA insertion at the TAF1 locus. We demonstrate that the KRAB zinc finger protein ZNF91 establishes H3K9me3 and DNA methylation over SVAs, including polymorphic alleles, in human neural progenitor cells. The resulting mini-heterochromatin domains attenuate the cis-regulatory impact of SVAs. This is critical for XDP pathology; removal of local heterochromatin severely aggravates the XDP molecular phenotype, resulting in increased TAF1 intron retention and reduced expression. Our results provide unique mechanistic insights into how human polymorphic transposon insertions are recognized and how their regulatory impact is constrained by an innate epigenetic defense system.

The role of retroelements of the human genome in the development of type 1 diabetes mellitus

Retroelements (retrotransposons and endogenous retroviruses) are a class of mobile genetic elements that move in the genome by inserting their own reverse transcribed transcripts. They serve as drivers of epigenetic regulation; therefore, individual characteristics of the distribution of retroelements in the genome influence the development of multifactorial diseases. Type 1 diabetes mellitus is a multifactorial disease with an immune response against pancreatic β cells. The role of heredity in the development of the disease is estimated at 88%, and the role of allelic variants of various genes is determined. There are also other specific types of diabetes mellitus, which account for more than 2% of cases of diabetes mellitus and are monogenic diseases with an autosomal dominant mode of inheritance due to germline mutations in the MODY genes, including HNF4A, GCK, HNF1A, HNF1B. Most patients with type 1 diabetes have the protein product and ribonucleic acid (RNA) of the insulin inhibitor HERV-W-Env, which is caused by abnormal expression of the human endogenous retrovirus (HERV). An assumption has been made about the role of retroelements in the development of type 1 diabetes mellitus. This is due to their involvement in the phylogenetic formation of the endocrine system, since in evolution retroelements turned out to be sources of regulatory sequences of hormone genes, nuclear hormone receptors and binding sites for them. Type 1 diabetes mellitus is associated with the integration of HERV into the HLA-DQ gene region, with allelic variants and sizes of VNTR variable tandem repeats (part of the SVA retroelements), which regulate the expression of the insulin gene and other hormones. For this reason, it is likely that the development of type 1 diabetes mellitus may be based on individual characteristics of the distribution of HERVs in the human genome and their dynamic changes in ontogenesis. HERVs also play a role in the etiopathogenesis of diabetes mellitus through the activation of an autoimmune response, the triggering factors of which are exogenous viral infections and stress. Thus, retroelements are involved in various mechanisms of the development of type 1 diabetes mellitus, which reflects their global regulatory influence on endocrine regulation.

Short Tandem Repeat Profile for Authentication of Immortal Murine Cancer Cell Line MH-22A

Short Tandem Repeat Profile for Authentication of Immortal Murine Cancer Cell Line MH-22A

Genetic etiology study in a large cohort with congenital insensitivity to pain with anhidrosis.

Pathogenic variations in the NTRK1 can cause congenital insensitivity to pain with anhidrosis (CIPA), a rare autosomal recessive inherited neuropathy. The precise diagnosis of CIPA relies on the identification of pathogenic genotypes. Therefore, it is essential to expand the NTRK1 variation spectrum and improve molecular diagnosis methods. In this study, 74 probands with typical manifestations of CIPA but unknown genotypes were recruited. A comprehensive molecular genetic analysis was performed to identify variations in the NTRK1, using techniques including Sanger and next-generation sequencing, bioinformatic analysis, quantitative polymerase chain reaction (qPCR), gap-PCR, short tandem repeat (STR) genotyping, and reverse-transcription PCR. In addition, functional assays were conducted to determine the pathogenicity of variants of uncertain significance (VUS) and further characterized changes in glycosylation and phosphorylation of 14 overexpressed mutant vectors with variants at different domains in the TrkA protein, which is encoded by NTRK1. A total of 48 variations in the NTRK1 were identified, including 22 novel ones. When combined with data from another 53 CIPA patients examined in our previous work, this study establishes the largest genotypic and phenotypic spectra of CIPA worldwide, including 127 CIPA families. Moreover, functional studies indicated that the pathogenicity of VUS mainly affected insufficient glycosylation in the extracellular domain and abnormal phosphorylation in the intracellular domain. This study not only provides important evidence for precise diagnosis of CIPA but also further enriches our understanding of the pathogenesis of this disease.

Influence of point mutations on PR65 conformational adaptability: Insights from molecular simulations and nanoaperture optical tweezers.

PR65 is the HEAT repeat scaffold subunit of the heterotrimeric protein phosphatase 2A (PP2A) and an archetypal tandem repeat protein. Its conformational mechanics plays a crucial role in PP2A function by opening/closing substrate binding/catalysis interface. Using in silico saturation mutagenesis, we identified PR65 "hinge" residues whose substitutions could alter its conformational adaptability and thereby PP2A function, and selected six mutations that were verified to be expressed and soluble. Molecular simulations and nanoaperture optical tweezers revealed consistent results on the specific effects of the mutations on the structure and dynamics of PR65. Two mutants observed in simulations to stabilize extended/open conformations exhibited higher corner frequencies and lower translational scattering in experiments, indicating a shift toward extended conformations, whereas another displayed the opposite features, confirmed by both simulations and experiments. The study highlights the power of single-molecule nanoaperture-based tweezers integrated with in silico approaches for exploring the effect of mutations on protein structure and dynamics.

Paramutation at the maize pl1 locus is associated with RdDM activity at distal tandem repeats.

Exceptions to Mendelian inheritance often highlight novel chromosomal behaviors. The maize Pl1-Rhoades allele conferring plant pigmentation can display inheritance patterns deviating from Mendelian expectations in a behavior known as paramutation. However, the chromosome features mediating such exceptions remain unknown. Here we show that small RNA production reflecting RNA polymerase IV function within a distal downstream set of five tandem repeats is coincident with meiotically-heritable repression of the Pl1-Rhoades transcription unit. A related pl1 haplotype with three, but not one with two, repeat units also displays the trans-homolog silencing typifying paramutations. 4C interactions, CHD3a-dependent small RNA profiles, nuclease sensitivity, and polyadenylated RNA levels highlight a repeat subregion having regulatory potential. Our comparative and mutant analyses show that transcriptional repression of Pl1-Rhoades correlates with 24-nucleotide RNA production and cytosine methylation at this subregion indicating the action of a specific DNA-dependent RNA polymerase complex. These findings support a working model in which pl1 paramutation depends on trans-chromosomal RNA-directed DNA methylation operating at a discrete cis-linked and copy-number-dependent transcriptional regulatory element.

De novo design of buttressed loops for sculpting protein functions.

In natural proteins, structured loops have central roles in molecular recognition, signal transduction and enzyme catalysis. However, because of the intrinsic flexibility and irregularity of loop regions, organizing multiple structured loops at protein functional sites has been very difficult to achieve by de novo protein design. Here we describe a solution to this problem that designs tandem repeat proteins with structured loops (9-14 residues) buttressed by extensive hydrogen bonding interactions. Experimental characterization shows that the designs are monodisperse, highly soluble, folded and thermally stable. Crystal structures are in close agreement with the design models, with the loops structured and buttressed as designed. We demonstrate the functionality afforded by loop buttressing by designing and characterizing binders for extended peptides in which the loops form one side of an extended binding pocket. The ability to design multiple structured loops should contribute generally to efforts to design new protein functions.

Binding of Akkermansia muciniphila to mucin is O-glycan specific

The intestinal anaerobic bacterium Akkermansia muciniphila is specialized in the degradation of mucins, which are heavily O-glycosylated proteins that constitute the major components of the mucus lining the intestine. Despite that adhesion to mucins is considered critical for the persistence of A. muciniphila in the human intestinal tract, our knowledge of how this intestinal symbiont recognizes and binds to mucins is still limited. Here, we first show that the mucin-binding properties of A. muciniphila are independent of environmental oxygen concentrations and not abolished by pasteurization. We then dissected the mucin-binding properties of pasteurized A. muciniphila by use of a recently developed cell-based mucin array that enables display of the tandem repeats of human mucins with distinct O-glycan patterns and structures. We found that A. muciniphila recognizes the unsialylated LacNAc (Galβ1-4GlcNAcβ1-R) disaccharide selectively on core2 and core3 O-glycans. This disaccharide epitope is abundantly found on human colonic mucins capped by sialic acids, and we demonstrated that endogenous A. muciniphila neuraminidase activity can uncover the epitope and promote binding. In summary, our study provides insights into the mucin-binding properties important for colonization of a key mucin-foraging bacterium.

Transcriptome sequencing of the endangered land snail Karaftohelix adamsi from the Island Ulleung: De novo assembly, annotation, valuation of fitness genes and SSR markers.

The Bradybaenidae snail Karaftohelix adamsi is endemic to Korea, with the species tracked from Island Ulleung in North Gyeongsang Province of South Korea. K. adamsi has been classified under the Endangered Wildlife Class II species of Korea and poses a severe risk of extinction following habitat disturbances. With no available information at the DNA (genome) or mRNA (transcriptome) level for the species, conservation by utilizing informed molecular resources seems difficult. In this study, we used the Illumina short-read sequencing and Trinity de novo assembly to draft the reference transcriptome of K. adamsi. After assembly, 13,753 unigenes were obtained of which 10,511 were annotated to public databases (a maximum of 10,165 unigenes found homologs in PANM DB). A total of 6,351, 3,535, 358, and 3,407 unigenes were ascribed to the functional categories under KOG, GO, KEGG, and IPS, respectively. The transcripts such as the HSP 70, aquaporin, TLR, and MAPK, among others, were screened as putative functional resources for adaptation. DNA transposons were found to be thickly populated in comparison to retrotransposons in the assembled unigenes. Further, 2,164 SSRs were screened with the promiscuous presence of dinucleotide repeats such as AC/GT and AG/CT. The transcriptome-guided discovery of molecular resources in K. adamsi will not only serve as a basis for functional genomics studies but also provide sustainable tools to be utilized for the protection of the species in the wild. Moreover, the development of polymorphic SSRs is valuable for the identification of species from newer habitats and cross-species genotyping.

Molecular mechanisms responsible KPC-135-mediated resistance to ceftazidime-avibactam in ST11-K47 hypervirulent Klebsiella pneumoniae

ABSTRACT Ceftazidime-avibactam resistance attributable to the bla KPC-2 gene mutation is increasingly documented in clinical settings. In this study, we characterized the mechanisms leading to the development of ceftazidime-avibactam resistance in ST11-K47 hypervirulent Klebsiella pneumoniae that harboured the bla KPC-135 gene. This strain possessed fimbriae and biofilm, demonstrating pathogenicity. Compared with the wild-type KPC-2 carbapenemase, the novel KPC-135 enzyme exhibited a deletion of Glu168 and Leu169 and a 15-amino acid tandem repeat between Val262 and Ala276. The bla KPC-135 gene was located within the Tn6296 transposon truncated by IS26 and carried on an IncFII/IncR-type plasmid. Compared to the bla KPC-2-positive cloned strain, only the MIC of ceftazidime increased against bla KPC-135-positive K. pneumoniae and wasn’t inhibited by avibactam (MIC 32 μg/mL), while clavulanic acid and vaborbactam demonstrated some inhibition. Kinetic parameters revealed that KPC-135 exhibited a lower Km and kcat/Km with ceftazidime and carbapenems, and a higher (∼26-fold) 50% inhibitory concentration with avibactam compared to KPC-2. The KPC-135 enzyme exerted a detrimental effect on fitness relative to the wild-type strain. Furthermore, this strain possessed hypervirulent determinants, which included the IncHI1B/FIB plasmid with rmpA2 and expression of type 1 and 3 fimbriae. In conclusion, we reported a novel KPC variant, KPC-135, in a clinical ST11-K47 hypervirulent K. pneumoniae strain, which conferred ceftazidime-avibactam resistance, possibly through increased ceftazidime affinity and decreased avibactam susceptibility. This strain simultaneously harboured resistance and virulence genes, posing an elevated challenge in clinical treatment.

Construction and Identification of a Human Colorectal Adenoma Epithelial Cell Line by SV40 Large T-Antigen Transfection.

Colorectal adenoma represents the critical step in the development of colorectal cancer. The establishment of an immortalized epithelial cell line of colorectal adenoma of human origin would provide a tool for studying the mechanism of precancerous lesions, screening the efficacy of novel drugs, and constructing in vivo disease models. Currently, there is no commercially available stable supply of epithelial cells from precancerous lesions. This study aimed to establish a natural LHPP low-expressing precancerous epithelial cell line by SV40-LT antigen gene transfection. Simian vacuolating virus 40(SV40), SV40-LT overexpressed lentivirus vector, was transfected into primary human colorectal adenomatous polyp epithelial cells. The transfected cells were screened, and the screened cells were amplified to obtain the epithelial cell line: IHCRA- CELL. The cells were identified by morphological observation, cell proliferation, Quantitative real-time PCR (qPCR), and Short Tandem Repeats (STR) experiments. Morphologically, the cells showed epithelial-like characteristics, such as polygon shape, desmosomes mitochondria, and strong positive keratin staining. There was no significant difference between the transfected cells and the primary cells. Through the STR identification experiment, no matching cell lines were found in the cell lines retrieval. We successfully established a natural LHPP low-expressing precancerous epithelial cell line by SV40-LT antigen gene transfection, which has been patented and is now preserved in the Chinese Typical Culture Preservation Center. It was verified that the transformed cells maintained the phenotype and biological characteristics of epithelial cells. This cell line can be used to study the mechanism of precancerous lesions, screen the efficacy of novel drugs, and construct in vivo disease models.

Assessing Privacy Vulnerabilities in Genetic Data Sets: Scoping Review

Background Genetic data are widely considered inherently identifiable. However, genetic data sets come in many shapes and sizes, and the feasibility of privacy attacks depends on their specific content. Assessing the reidentification risk of genetic data is complex, yet there is a lack of guidelines or recommendations that support data processors in performing such an evaluation. Objective This study aims to gain a comprehensive understanding of the privacy vulnerabilities of genetic data and create a summary that can guide data processors in assessing the privacy risk of genetic data sets. Methods We conducted a 2-step search, in which we first identified 21 reviews published between 2017 and 2023 on the topic of genomic privacy and then analyzed all references cited in the reviews (n=1645) to identify 42 unique original research studies that demonstrate a privacy attack on genetic data. We then evaluated the type and components of genetic data exploited for these attacks as well as the effort and resources needed for their implementation and their probability of success. Results From our literature review, we derived 9 nonmutually exclusive features of genetic data that are both inherent to any genetic data set and informative about privacy risk: biological modality, experimental assay, data format or level of processing, germline versus somatic variation content, content of single nucleotide polymorphisms, short tandem repeats, aggregated sample measures, structural variants, and rare single nucleotide variants. Conclusions On the basis of our literature review, the evaluation of these 9 features covers the great majority of privacy-critical aspects of genetic data and thus provides a foundation and guidance for assessing genetic data risk.

Insights into ribosomal DNA dominance and magnification through characterization of isogenic deletion alleles.

The major loci for the large primary ribosomal RNA (rRNA) genes (35S rRNAs) exist as hundreds to thousands of tandem repeats in all organisms and dozens to hundreds in Drosophila. The highly repetitive nature of the ribosomal DNA (rDNA) makes it intrinsically unstable, and many conditions arise from the reduction in or magnification of copy number, but the conditions under which it does so remain unknown. By targeted DNA damage to the rDNA of the Y chromosome, we created and investigated a series of rDNA alleles. We found that complete loss of rDNA leads to lethality after the completion of embryogenesis, blocking larval molting and metamorphosis. We find that the resident retrotransposons-R1 and R2-are regulated by active rDNA such that reduction in copy number derepresses these elements. Their expression is highest during the early first instar, when loss of rDNA is lethal. Regulation of R1 and R2 may be related to their structural arrangement within the rDNA, as we find they are clustered in the flanks of the nucleolus organizing region (NOR; the cytological appearance of the rDNA). We assessed the complex nucleolar dominance relationship between X- and Y-linked rDNA using a histone H3.3-GFP reporter construct and incorporation at the NOR and found that dominance is controlled by rDNA copy number as at high multiplicity the Y-linked array is dominant, but at low multiplicity the X-linked array becomes derepressed. Finally, we found that multiple conditions that disrupt nucleolar dominance lead to increased rDNA magnification, suggesting that the phenomena of dominance and magnification are related, and a single mechanism may underlie and unify these two longstanding observations in Drosophila.

#2606 Clinically boosted exome-sequencing is necessary for accurate and highly sensitive genetic diagnosis of ADTKD-MUC1

Abstract Background and Aims The human genome includes tandem repeats with variable length (VNTR) and a subset of these repeats have been associated with rare human diseases. Specific frameshift variants in the coding-VNTR of the MUC1 gene cause autosomal dominant tubulointerstitial kidney disease—MUC1 (ADTKD-MUC1). Calling variants from VNTR using short-read sequencing (SRS) is challenging due to poor read mappability, motif complexity (34*60-mer motifs are known up to now), variable repetition, and enormous motif sequence similarity. We have recently developed a computational pipeline called VNtyper, tailored for the precise detection of disease-causing variants within the MUC1 VNTR region using short-read sequencing data. This advancement allowed us to identify overlooked cases in a hereditary renal disease registry, leading to the diagnosis of at least 40 patients. Given that regular exome sequencing with low VNTR coverage proved inefficient for VNtyper, our focus shifted to boost the capture of MUC1 VNTR in exome. This improvement aims to enable the application of VNtyper in ADTKD-MUC1 diagnosis through exome sequencing. Method VNtyper utilizes two independent genotyping algorithms (Kestrel and code-adVNTR) along with MUC1 VNTR-specific reference sequence for the variant detection and is sensitive to the VNTR coverage. We employ Twist custom panels with v1 protocol for target enrichment in both our panel and exome sequencing. This procedure allows for the inclusion of spike-in probes without interfering with other targets. We designed an NTI panel that specifically targets 6 genes associated with ADTKD, including UMOD, MUC1, HNF1B, REN, SEC61A1, and DNAJB11. These genes were captured using a 1x tiling approach, whereas the MUC1 VNTR region was captured with 4x tiling approach. During the exome target enrichment process, we included the NTI probes as a spike-in to boost VNTR coverage. We conducted a routine exome sequencing on a total of 6 samples, consisting of 3 positive controls and 3 negative controls. Additionally, we conducted 16 boosted exome, using 3 negative controls and 13 MUC1 positive samples. Prior to using VNtyper on both exomes, we conducted initial quality controls to verify that there were no alterations in the coverages of other genes. Results The mean coverage of MUC1 VNTR in the regular exome was 72.1x, while in the boosted exome, it exhibited a significant increase to 144x. Adding spike in probes did not have significant effect on the coverage of the contig and there was no alteration in ratio of heterozygosity. As anticipated, in the regular exome, VNtyper was unsuccessful in identifying pathogenic variation in 2 out of 3 true positives. However, all negative controls tested negative. When we used the enhanced exome, we accurately identified all 13 true positive cases, while the negative control cases stayed negative. Through downsampling experiments (reducing read depth from 50% to 5% of the total) on panel data with a mean coverage of 700x, we determined that coverage below 100 should be considered as low coverage for VNTR genotyping using our pipeline. This underscores the critical importance of maintaining adequate coverage for accurate and reliable VNTR genotyping. Conclusion We have achieved successful enhancements in the accuracy and sensitivity of ADTKD diagnosis through the application of VNtyper on clinically boosted exome data. While VNtyper could identify MUC1-positive patients in regular exome data, the effectiveness is influenced by the number of repeats in both alleles and the specific motif in which the variation is present, which significantly decreases the sensitivity. In summary, the VNtyper pipeline demonstrates its ability to detect pathogenic variations in ADTKD-MUC1 on panel data (100% sensitivity), and the improvement in VNTR capture in exome significantly enhances the sensitivity of the ADTKD diagnosis.

A combination influenza mRNA vaccine candidate provided broad protection against diverse influenza virus challenge

Influenza viruses present a significant threat to global health. The production of a universal vaccine is considered essential due to the ineffectiveness of current seasonal influenza vaccines against mutant strains. mRNA technology offers new prospects in vaccinology, with various candidates for different infectious diseases currently in development and testing phases. In this study, we encapsulated a universal influenza mRNA vaccine. The vaccine encoded influenza hemagglutinin (HA), nucleoprotein (NP), and three tandem repeats of matrix protein 2 (3M2e). Twice-vaccinated mice exhibited strong humoral and cell-mediated immune responses in vivo. Notably, these immune responses led to a significant reduction in viral load of the lungs in challenged mice, and also conferred protection against future wild-type H1N1, H3N2, or H5N1 influenza virus challenges. Our findings suggest that this mRNA-universal vaccine strategy for influenza virus may be instrumental in mitigating the impact of future influenza pandemics.

#382 Unexpected diagnoses of ADTKD-MUC1 with the help of VNtyper

Abstract Background and Aims Autosomal dominant tubulointerstitial kidney disease (ADTKD) due to pathogenic variants in the MUC1 gene is difficult to diagnose since these variants lie in a large variable number tandem repeats (VNTR) and require specialized genetic testing, such as SNaPshot minisequencing. We recently developed a computational pipeline, VNtyper, for easier reliable detection of MUC1 VNTR pathogenic variants and have applied this tool to a large cohort of patients with various phenotypes of kidney disease. The aim of this study is to clinically describe patients in whom MUC1 pathogenic variants were unexpectedly detected with the help of VNtyper. Method We applied a computational pipeline, VNtyper, to a large cohort of patients with suspected hereditary kidney disease referred for genetic testing, regardless of their phenotype. This cohort included patients evaluated from 2017 to 2023 in the Molecular Genetics Department of Necker-Enfants Malades Hospital (Paris, France). Clinical characteristics were collected from patients in whom we detected new MUC1 VNTR pathogenic variants. Results 44 out of 3735 patients tested (1.2%) were newly diagnosed with a MUC1 pathogenic variant. Of these patients, ADTKD was clinically suspected in only 31 out of 44 patients (70%). In 2 patients, there was a pathogenic variant in another gene that could explain the phenotype, in addition to the MUC1 pathogenic variant. In fact, one 52-year-old patient had polycystic kidneys, nephrolithiasis, hematuria and normal kidney function. Her mother also had kidney cysts with absence of chronic kidney disease (CKD). This patient had a pathogenic variant in IFT140 gene (c.2399+1G>T) which could explain her autosomal dominant polycystic kidney disease (ADPKD) phenotype. Whether the MUC1 variant contributes to her disease remains unclear, especially seeing as her kidney function is normal at the age of 52 years. On the other hand, one patient had kidney cysts discovered at 3 days of age. A heterozygous HNF1B deletion was detected which could explain the phenotype. The following detection of a pathogenic MUC1 variant was thus an incidental finding and seemed de novo, although parents declined to get tested. These incidental findings can be challenging, especially when it comes to counselling and follow-up. There was no family history of kidney disease in 7 out of 44 patients (16%), and in one patient, we were able to confirm de novo disease since her parents were tested and were negative. It was unfortunately not possible to get DNA from the parents of the other patients. This is the first case of proven de novo ADTKD-MUC1 described to our knowledge. De novo variants in ADTKD-MUC1 are not surprising since the 7C stretch found in VNTR is a hotspot for mutagenesis. Interestingly, these patients seemed to have a more severe clinical presentation with an early onset of disease (median age at diagnosis 28 years [18, 32]). However, this could be due to a selection bias. In fact, young age of onset of CKD may have prompted clinicians to refer these patients for genetic testing despite negative family history, whereas older patients with possible ADTKD-MUC1 and no family history of kidney disease might be presumed to have another etiology of CKD. One way to confirm this would be to test a large cohort of patients with CKD from unknown etiology with no family history of CKD. Considering the nonspecific clinical manifestations of ADTKD-MUC1 and the phenotypic variability, it has previously been suggested that the most important clue to diagnosis is the presence of CKD in first-degree family. The possibility of de novo cases makes the clinical diagnosis of ADKTD-MUC1 even more challenging. Conclusion With VNtyper, we were able to diagnose new cases of ADTKD-MUC1 in a large cohort of patients with various phenotypes, with a significant prevalence of 1.2%. ADTKD-MUC1 was not suspected in 30% of these patients who would have maybe never been diagnosed otherwise. This is also the first study in which we describe a proven de novo case of ADTKD-MUC1. Pathogenic variants in the MUC1 gene should thus be in the differential diagnosis of all unspecified CKD, even if there is no family history since de novo variants are also possible.

#276 ADTKD genetic diagnostics in Europe: where do we stand and what is needed?

Abstract Background and Aims We are the first European patient organization to pursue the goal of raising awareness of the monogenic kidney disease ADTKD (autosomal dominant tubulointerstitial kidney disease) and supporting its research (www.adtkd.de/en-gb). To date, causative mutations are known in at least five disease genes (UMOD, MUC1, REN, HNF1B, SEC61A1) [1] and one can expect that even more disease genes exist. There are already strong international collaborations to define the clinical course of ADTKD [2] and to evaluate a targeted therapy in clinical trials [3]. Clinical registries form the basis for the inclusion of patients, preferably at an early stage of the disease. Such a registry already exists in the USA and is currently being established in Europe (ADTKD-Net) as part of the European Joint Program on Rare Diseases (EJP RD). Due to its non-specific and variable clinical characteristics, ADTKD is often misdiagnosed or not diagnosed at all. Experts therefore suspect a high number of unreported cases. There are also challenges in genetic diagnostics. While next generation sequencing (NGS) panels have been established for ADTKD, they are usually unable to detect pathogenic MUC1 variants due to their specific location in the genome. This is the insertion of an additional cytosine (dupC) in a VNTR (Variable Number of Tandem Repeats) domain of the MUC1 gene, which cannot be detected by NGS and therefore requires special procedures such as SnaPshot Minisequencing [4] or VNtyper [5]. For instance, there are only two laboratories that can perform this specific MUC1 testing in Germany to date. The aim of this study is to assess the status quo of ADTKD diagnostics in Europe. We hypothesize that there is a lack of appropriate structures to adequately and reliably diagnose ADTKD, notably ADTKD-MUC1. The study is intended to help identify deficits and obstacles in order to address these in a targeted manner. Methods We used Survey Monkey to create a digital survey, which we sent by email to around 50 European ADTKD experts. They were asked to answer 7 key questions (Fig. 1). For the selection of suitable ADTKD experts, we use various sources such as publications on ADTKD in PubMed, the Expert Finder and the European Patient Advocacy Groups (ePAG) in the European Reference Network for Rare Kidney Diseases (ERKNet) and our own network. Our aim is to cover at least the 30 largest countries in Europe in terms of population. Results Results will be presented at the ERA conference. Conclusion With our survey we would like to substantiate the hypothesis that the possibilities of molecular diagnostics of ADTKD in Europe are currently insufficient. Based on our results, short and long-term measures should be taken to remedy potential deficits so that as many patients as possible can be identified and have access to therapeutic options in the future. Another interesting aspect is the awareness of ADTKD among European nephrologists. This could be a future activity of our patient organization.