Intronic Deletion Research Articles

Analysis of MET genetic aberrations in Chinese solid tumor patients.

e15031 Background: Mesenchymal-epithelial transition (MET) gene encodes a member of the receptor tyrosine kinase family of proteins and is a proto-oncogene. MET gene takes part in various signaling pathways such as PI3K/AKT, Ras/MAPK, and JAK/STAT which are associated with tumor development and progression. This study aimed to reveal MET aberrated characteristics in Chinese solid tumor patients and explore the occurrence of MET oncogenic variants in solid tumors. Methods: This study retrospectively analyzed MET alterations in 1856 solid tumor patients in China from laboratory of Simcere Diagnosis (Nanjing, China) during 2019-2021. Next-generation sequencing (NGS) was performed to detect gene mutations in tumor or blood samples of the patients. Results: We screened out 1856 patients with MET gene alterations from 45717 patients with solid tumor. The high frequency of MET gene alterations was lung cancer (72.8%), colorectal cancer (10.7%), gastric cancer (4.9%), and liver cancer (2.7%). MET gene variants included missense (61.7%), copy number variation (copy number≥5) (15.4%), truncation (7.9%), splicing site variant (7.2%),fusion (1.7%),and in-frame insertion/deletions (0.9%). Among them, MET amplification was detected in 285 patients, and 93 patients carried high-level MET amplification (copy number≥10). High-level MET amplification was most frequently detected in lung cancer (41, 44.1%), gastric cancer (17, 18.3%) and liver cancer (14, 15.1%). MET exon 14 skipping was detected in 287 solid tumor patients (15.5%), of which 277 patients were lung cancer, the remaining were liver cancer, colorectal cancer, gastric cancer, osteosarcoma, melanoma, bladder cancer and glioma. We analyzed MET exon 14 alterations and classified them into 4 subgroups according to the alteration types and locations. The most common alterations were at splice donor sites (215, 74.91%), followed by the splice acceptor sites (35, 12.20%), Y1003 site (5, 1.74%), and other types (32, 11.15%). Other types included insertion or deletion in MET intron 13 or 14, for example, c.3028+21_3028+65del, c.2888-20_2888-11del, c.2888-27_2888-9del, etc. 7 lung cancer patients carried MET exon 14 skipping and MET amplification simultaneously. 36 patients carried MET gene fusions, and mostly were lung cancer (22, 61.1%). The breakpoints often located in exon 2 of MET (4, 11.1%). Conclusions: We discovered MET alterations in multiple types of solid tumors in this study. We found classic MET exon 14 skipping and MET high-level amplification in solid tumors except for lung cancer. It suggested that the activation of MET-driven signaling pathway was ubiquitous in pan-cancer, which might benefit from MET inhibitors.

Identification and Characterization of Variants in Intron 6 of the LPL Gene Locus among a Sample of the Kuwaiti Population.

Lipoprotein lipase (LPL) is responsible for the hydrolysis of lipoproteins; hence defective LPL is associated with metabolic disorders. Here, we identify certain intronic insertions and deletions (InDels) and single nucleotide polymorphisms (SNPs) in intron 6 of the LPL gene and investigate their associations with different phenotypic characteristics in a cohort of the general Kuwaiti population. Two specific regions of intron 6 of the LPL gene, which contain InDels, were amplified via Sanger sequencing in 729 subjects. Genotypic and allelic frequencies were estimated, and genetic modeling was used to investigate genetic associations of the identified variants with lipid profile, body mass index (BMI), and risk of coronary heart disease (CHD). A total of 16 variants were identified, including 2 InDels, 2 novel SNPs, and 12 known SNPs. The most common variants observed among the population were rs293, rs274, rs295, and rs294. The rs293 “A” insertion showed a significant positive correlation with elevated LDL levels, while rs295 was significantly associated with increased BMI. The rs274 and rs294 variants showed a protective effect of the minor allele with decreased CHD prevalence. These findings shed light on the possible role of LPL intronic variants on metabolic disorders.

Intron mutations and early transcription termination in Duchenne and Becker muscular dystrophy.

DMD pathogenic variants for Duchenne and Becker muscular dystrophy are detectable with high sensitivity by standard clinical exome analyses of genomic DNA. However, up to 7% of DMD mutations are deep intronic and analysis of muscle-derived RNA is an important diagnostic step for patients who have negative genomic testing but abnormal dystrophin expression in muscle. In this study, muscle biopsies were evaluated from 19 patients with clinical features of a dystrophinopathy, but negative clinical DMD mutation analysis. Reverse transcription-polymerase chain reactionor high-throughput RNA sequencingmethods identified 19 mutations with one of three pathogenic pseudoexon types: deep intronic point mutations, deletions or insertions, and translocations. In association with point mutations creating intronic splice acceptor sites, we observed the first examples of DMD pseudo 3'-terminal exon mutations causing high efficiency transcription termination within introns. This connection between splicing and premature transcription termination is reminiscent of U1 snRNP-mediating telescripting in sustaining RNA polymerase II elongation across large genes, such as DMD. We propose a novel classification of three distinct types of mutations identifiable by muscle RNA analysis, each of which differ in potential treatment approaches. Recognition and appropriate characterization may lead to therapies directed toward full-length dystrophin expression for some patients.

C1: Targeting mutational landscape of TP53 gene in patients diagnosed with oral cancers living in Senegal

INTRODUCTION: Looking for the mutational landscape on a genomic scale has provided in the last few years unprecedented perspectives for understanding molecular pathogenesis of cancer progression, response to treatment and genetic predisposition. In oral cancers, numerous genomic studies have been devoted to alterations in the TP53 gene and their clinical significance in carcinogenesis. Herein, we screened for genomic variants of TP53 predisposing to oral cancers in Senegalese patients. MATERIALS AND METHODS: Eighty-eight patients with confirmed diagnostic were recruited after informed consent from Stomatology Unit of Aristide le Dantec Hospital. Blood samples were collected from each patient to perform DNA extraction, PCR amplification of all coding exons of TP53 followed by Sanger Sequencing of PCR products. Nucleotidic sequences were analyzed with Genalys software. RESULTS: Mean age at diagnosis was estimated at 51.9 ± 17.98 years with a sex-ratio of 0.76. The majority of patients (64/88) had a negative alcohol-tobacco status. Tumor histology showed that 90% of patients had squamous cell carcinoma localised preferentially on the tongue (22.7%), gum (20.5%) and cheek mucosa (19.3%). Mutation screening of TP53 showed that 52.27% of the patients carried at least one mutation. Eleven genomic variants were identified, seven already reported in mutation databases and four new variants. The most recurrent variants was p.Pro72Arg (rs1042522; Arginine allelic frequency estimated at 31.26%) and a 16 bp deletion in intron 3 (rs59758982; allelic frequency estimated at 26.25%). Linkage disequilibrium (LD) calculation has shown that these two variants are in strong LD but tests of association with controls show that they could not predispose towards oral cancers. CONCLUSION: The results of the present investigation suggest that the two most common variants found in Senegalese patients are not predisposing to oral cancers. Further comprehensive investigations on reported and new variants found in this study should be conducted to highlight their functional impacts and hypothetic oncogenic associated function.

Molecular Dissection of Structural Variations Involved in Antithrombin Deficiency

Inherited antithrombin deficiency, the most severe form of thrombophilia, is predominantly caused by variants in SERPINC1. Few causal structural variants have been described, usually detected by multiplex ligation-dependent probe amplification or cytogenetic arrays, which only define the gain or loss and the approximate size and location. This study has done a complete dissection of the structural variants affecting SERPINC1 of 39 unrelated patients with antithrombin deficiency using multiplex ligation-dependent probe amplification, comparative genome hybridization array, long-range PCR, and whole genome nanopore sequencing. Structural variants, in all cases only affecting one allele, were deleterious and caused a severe type I deficiency. Most defects were deletions affecting exons of SERPINC1 (82.1%), but the whole cohort was heterogeneous, as tandem duplications, deletion of introns, or retrotransposon insertions were also detected. Their size was also variable, ranging from 193 bp to 8 Mb, and in 54% of the cases involved neighboring genes. All but two structural variants had repetitive elements and/or microhomologies in their breakpoints, suggesting a common mechanism of formation. This study also suggested regions recurrently involved in structural variants causing antithrombin deficiency and found three structural variants with a founder effect: the insertion of a retrotransposon, duplication of exon 6, and a 20-gene deletion. Finally, nanopore sequencing was determined to be the most appropriate method to identify and characterize all structural variants at nucleotide level, independently of their size or type.

The comparative studies of complete chloroplast genomes in Actinidia (Actinidiaceae): novel insights into heterogenous variation, clpP gene annotation and phylogenetic relationships.

The genus Actinidia, also called kiwifruit, is characterized with abundant balanced nutritional metabolites, including exceptionally high vitamin C content. However, the traditional classification could not fully reflect the actual Actinidia species' relationships, which need further revision through more accurate approaches. Compared to the nuclear genome, the chloroplast genome has simple heredity characteristics, conserved genome structure and small size, suitable for deciphering complicated species' phylogenetic relationships. Here, the genome-wide comprehensive comparative analyses were performed over 29 independent chloroplast genomes' sequences derived from 25 Actinidia taxa. The average genome size is 156,673.38bp, with an average 37.20% GC content. The long repeat sequences rather than SSRs (simple sequence repeats) in Actinidia were revealed to be the causal agent leading to the chloroplast genome size expansion. The clpP gene sequences with exon merge and intron deletion were annotated in all the 29 chloroplast genomes tested, which has been previously reported to be lost in Actinidia species. Comprehensive sequence analyses indicated the distinct variation at the clpP gene locus was Actinidiaceae-specific, emerging after the Actinidiaceae-other Ericales species divergence. Four highly divergent sequences (i.e., rps16 ~ trnQ-UUG, rps4 ~ trnT-UGU, petA ~ psbJ, and rps12 ~ psbB) evolved in the LSC (large single-copy) and SSC (small single-copy) regions embodying rps12 ~ psbB (including clpP gene and its up/downstream noncoding sequence) were identified as variation hot spots in Actinidia species. Based on either LSC region alone, combined sequences of LSC and SSC or the whole chloroplast genome sequences, three identical phylogenetic trees of the 25 Actinidia taxa with relatively improved resolution were reconstructed, consistently supporting the reticulate evolutionary lineage in Actinidia. Our findings could help to better understand the evolution characteristics of chloroplast genomes and phylogenetic relationships among Actinidia species.

Loss of circRNAs from the crh-1 gene extends the mean lifespan in Caenorhabditis elegans.

Accumulation of circular RNAs (circRNAs) during aging occurs on a genome‐wide level for multiple organisms, but its significance is unknown. Generating circRNA loss‐of‐function mutants is difficult because the vast majority of these RNAs are comprised of exons shared with protein‐coding mRNAs. In Caenorhabditis elegans, most circRNAs were previously found to accumulate during aging. Two of the most abundant, age‐accumulating circRNAs are generated from exon 4 of the crh‐1 gene (circ‐crh‐1). Here, we found that the biogenesis of circ‐crh‐1 was regulated by the double‐stranded RNA‐binding protein ADR‐1. We identified Reverse Complementary Match (RCM) sequences in introns flanking circ‐crh‐1. Using CRISPR‐Cas9, we deleted the downstream RCM and found that this completely eliminated expression of the circRNA without affecting linear mRNA expression from the crh‐1 gene. Remarkably, worms lacking circ‐crh‐1 exhibited a significantly longer mean lifespan. Lifespan was partially restored to wild type by expression of circ‐crh‐1 in neural tissues. Widespread transcriptome alterations in circ‐crh‐1 mutants were identified using RNA‐Seq. Moving forward, intronic RCM deletion using CRISPR should be a widely applicable method to identify lifespan‐regulating circRNAs in C. elegans.

Genotypic and allelic frequency of a mutation in the NHEJ1 gene associated with collie eye anomaly in dogs in Italy.

BackgroundA 7.8‐kb deletion in intron 4 of the NHEJ1 canine gene is associated with Collie Eye Anomaly (CEA). This deletion has been described in sheep‐herding breeds related to the collie lineage and in several other dog breeds. A genetic test based on this association can distinguish three genotypes: normal, carrier and affected. The present study is a retrospective investigation of the presence of the CEA allele frequencies in selected breeds from the Italian dog population over a 10‐year time span.MethodsGenotype data, for the 7.8 kb deletion in intron 4 of the NHEJ1 gene, from 496 dogs belonging to Border collie (BC, n = 334), Shetland Sheepdog (SS, n = 74), Australian Shepherd (AS, n = 52), Nova Scotia Duck Tolling Retriever (NS, n = 20) and Rough Collie (RC, n = 16) were analysed. The genetic frequency of CEA allele was estimated in breeds with higher observations (BC, SS and AS).ResultsHealthy carriers were 50%, 45%, 29.6%, 17.3% and 12.5% in SS, NS, BC, AS and RC, respectively. The affected recessive homozygotes were 81.3%, 10.8% and 1.5% in RC, SS and BC, respectively. The CEA allelic frequencies were 0.36, 0.16 and 0.087 in SS, BC and AS, respectively.ConclusionThe results support the usefulness of this type of genetic analysis to optimize the care of dogs where the CEA mutation is present, including assessing the health risk to susceptible dogs within a breed and to provide an objective basis for breeding programmes.

Divergent variant patterns among 19 patients with Rubinstein-Taybi syndrome uncovered by comprehensive genetic analysis including whole genome sequencing.

Rubinstein-Taybi syndrome (RSTS) is characterized by dysmorphic facial features, broad thumbs, and intellectual disability. CREB-binding protein (CREBBP) or E1A-binding protein P300 (EP300) are causative genes. To elucidate the underlying genetic and genomic architecture related to the RSTS phenotype, we performed comprehensive genetic analysis targeting CREBBP and/or EP300 in 22 clinically diagnosed patients. During the 11-year study period, we used several analysis methods including high-resolution melting, array-based comparative genomic hybridization, panel-based exome sequencing, whole exome sequencing, and whole genome sequencing (WGS). We identified the causative variants in 19 patients (86.3%), but they were variable and complex, so we must combine multiple analysis methods. Notably, we found genetic alterations in the non-coding regions of two patients (10.5%, 2/19): scattered deletions including a partial 5'-untranslated region of CREBBP in one patient (all coding exons were intact), and a deep 229-bp intronic deletion in another patient, resulting in a splicing error. Furthermore, we identified rare clinical findings: two patients with an EP300 variant showed abnormal development of the neural tube, and one patient with a CREBBP variant had anorectal atresia with a cloaca. Our findings expand the allelic heterogeneity of RSTS, underscore the utility of comprehensive genetic analysis, and suggest that WGS may be a practical diagnostic strategy.

A novel multiplex polymerase chain reaction assay for the genotypic survey of the non-homologous end-joining factor 1 gene associated with Collie eye anomaly in Thailand.

Background and Aim:Collie eye anomaly (CEA) is a hereditary and congenital ocular disorder, which affects several dog breeds, including Collies, Collie-related breeds, and other purebreds. An intronic deletion of 7799-bp in the non-homologous end-joining factor 1 (NHEJ1) gene has been identified as the genetic defect causing CEA. This study aimed to investigate the prevalence of CEA based on NHEJ1 genotyping assay in Thailand.Materials and Methods:We clarified the prevalence of CEA in 224 dogs from five purebred dog breeds using a novel multiplex polymerase chain reaction (PCR)-based technique and confirmed the genotypic status with direct DNA sequencing.Results:The highest frequency of the mutated NHEJ1 allele was 83.3% for Rough Collies, followed by 7.8% for Border Collies, 5.1% for Australian Shepherds, and 2.8% for Shetland Sheepdogs. The heterozygous mutated NHEJ1 genotype detected for Rough Collies, Border Collies, Australian Shepherds, and Shetland Sheepdogs was 33.3%, 15.6%, 10.3%, and 3.3%, respectively. The homozygous mutated NHEJ1 genotype was detected only in Rough Collies and Shetland Sheepdogs, accounting for 66.7% and 1.1%, respectively. Thai Ridgeback Dogs were not affected by this mutation.Conclusion:This study describes, for the 1st time, the genotypic survey of the NHEJ1 gene associated with CEA in dogs in Thailand. In addition, we successfully developed a new multiplex PCR assay with high accuracy, reproducibility, and cost-efficiency and validated its usefulness for determining NHEJ1 genotypes.

South Asian-Specific MYBPC3 Δ25bp Deletion Carriers Display Hypercontraction and Impaired Diastolic Function Under Exercise Stress.

Background: A 25-base pair (25bp) intronic deletion in the MYBPC3 gene enriched in South Asians (SAs) is a risk allele for late-onset left ventricular (LV) dysfunction, hypertrophy, and heart failure (HF) with several forms of cardiomyopathy. However, the effect of this variant on exercise parameters has not been evaluated.Methods: As a pilot study, 10 asymptomatic SA carriers of the MYBPC3Δ25bp variant (52.9 ± 2.14 years) and 10 age- and gender-matched non-carriers (NCs) (50.1 ± 2.7 years) were evaluated at baseline and under exercise stress conditions using bicycle exercise echocardiography and continuous cardiac monitoring.Results: Baseline echocardiography parameters were not different between the two groups. However, in response to exercise stress, the carriers of Δ25bp had significantly higher LV ejection fraction (%) (CI: 4.57 ± 1.93; p < 0.0001), LV outflow tract peak velocity (m/s) (CI: 0.19 ± 0.07; p < 0.0001), and higher aortic valve (AV) peak velocity (m/s) (CI: 0.103 ± 0.08; p = 0.01) in comparison to NCs, and E/A ratio, a marker of diastolic compliance, was significantly lower in Δ25bp carriers (CI: 0.107 ± 0.102; p = 0.038). Interestingly, LV end-diastolic diameter (LVIDdia) was augmented in NCs in response to stress, while it did not increase in Δ25bp carriers (CI: 0.239 ± 0.125; p = 0.0002). Further, stress-induced right ventricular systolic excursion velocity s' (m/s), as a marker of right ventricle function, increased similarly in both groups, but tricuspid annular plane systolic excursion increased more in carriers (slope: 0.008; p = 0.0001), suggesting right ventricle functional differences between the two groups.Conclusions: These data support that MYBPC3Δ25bp is associated with LV hypercontraction under stress conditions with evidence of diastolic impairment.

Aluelement in the RNA binding motif protein, X-linked 2 (RBMX2) gene found to be linked to bipolar disorder.

ObjectiveWe have used long-read single molecule, real-time (SMRT) sequencing to fully characterize a ~12Mb genomic region on chromosome Xq24-q27, significantly linked to bipolar disorder (BD) in an extended family from a genetic sub-isolate. This family segregates BD in at least four generations with 24 affected individuals.MethodsWe selected 16 family members for targeted sequencing. The selected individuals either carried the disease haplotype, were non-carriers of the disease haplotype, or served as married-in controls. We designed hybrid capture probes enriching for 5-9Kb fragments spanning the entire 12Mb region that were then sequenced to screen for candidate structural variants (SVs) that could explain the increased risk for BD in this extended family.ResultsAltogether, 201 variants were detected in the critically linked region. Although most of these represented common variants, three variants emerged that showed near-perfect segregation among all BD type I affected individuals. Two of the SVs were identified in or near genes belonging to the RNA Binding Motif Protein, X-Linked (RBMX) gene family—a 330bp Alu (subfamily AluYa5) deletion in intron 3 of the RBMX2 gene and an intergenic 27bp tandem repeat deletion between the RBMX and G protein-coupled receptor 101 (GPR101) genes. The third SV was a 50bp tandem repeat insertion in intron 1 of the Coagulation Factor IX (F9) gene.ConclusionsAmong the three genetically linked SVs, additional evidence supported the Alu element deletion in RBMX2 as the leading candidate for contributing directly to the disease development of BD type I in this extended family.

A deletion containing a CTCF-element in intron 8 of the Bbs7 gene is partially responsible for juvenile obesity in the Berlin Fat Mouse

The Berlin Fat Mouse Inbred (BFMI) line is a model for juvenile obesity. Previous studies on crosses between BFMI and C57Bl/6N (B6N) have identified a recessive defect causing juvenile obesity on chromosome 3 (jObes1). Bbs7 was identified as the most likely candidate gene for the observed effect. Comparative sequence analysis showed a 1578 bp deletion in intron 8 of Bbs7 in BFMI mice. A CTCF-element is located inside this deletion. To investigate the functional effect of this deletion, it was introduced into B6N mice using CRISPR/Cas9. Two mice containing the target deletion were obtained (B6N Bbs7emI8∆1 and Bbs7emI8∆2) and were subsequently mated to BFMI and B6N to generate two families suitable for complementation. Inherited alleles were determined and body composition was measured by quantitative magnetic resonance. Evidence for a partial complementation (13.1–15.1%) of the jObes1 allele by the CRISPR/Cas9 modified B6N Bbs7emI8∆1 and Bbs7emI8∆2 alleles was found. Mice carrying the complementation alleles had a 23–27% higher fat-to-lean ratio compared to animals which have a B6N allele (P(Bbs7emI8∆1) = 4.25 × 10–7; P(Bbs7emI8∆2) = 3.17 × 10–5). Consistent with previous findings, the recessive effect of the BFMI allele was also seen for the B6N Bbs7emI8∆1 and Bbs7emI8∆2 alleles. However, the effect size of the B6N Bbs7emI8∆1 and Bbs7emI8∆2 alleles was smaller than the BFMI allele, and thus showed only a partial complementation. Findings suggest additional variants near Bbs7 in addition to or interacting with the deletion in intron 8.

596 Multidisciplinary in-depth investigation in a young athlete suffering from syncope caused by myocardial bridge

Abstract Aims Laboratory medicine along with genetic investigations are taking on an increasingly important role in monitoring athlete’s health conditions. Acute or intense exercise can lead to metabolic imbalances, muscle injuries, or can point up cardiovascular disorders. Methods and results This study aimed to monitor the health status of a basketball player, through an integrated approach including biochemical and genetic investigations and advanced imaging techniques, to shed light on the causes of recurrent syncope he experienced during exercise. Biochemical analyses showed that the athlete had abnormal iron, ferritin, and bilirubin levels. Coronary computed tomographic angiography highlighted the presence of an intramyocardial bridge, suggesting this may be the cause of the observed syncopes. The athlete was excluded from competitive activity. To understand if this cardiac malformation could be caused by an inherited genetic condition, both array-CGH and whole exome sequencing were performed. Array-CGH showed two intronic deletions involving MACROD2 and COMMD10 genes, which could be related to the congenital heart defect; the whole exome sequencing highlighted the genotype compatible with the Gilbert syndrome. However, no clear pathogenic mutations related to the patient’s cardiological phenotype were detected, even after applying machine learning methods. Conclusions This case highlights the importance and the need to provide exhaustive personalized diagnostic work-up for the athletes to cover the cause of their malaise, safeguarding their health. This multidisciplinary approach can be useful to create ad personam training and treatments, thus avoiding the appearance of diseases and injuries which, if underestimated, can become irreversible disorders and sometimes can lead to the death of the athlete.

Deletions in VANGL1 are a risk factor for antibody-mediated kidney disease

SummaryWe identify an intronic deletion in VANGL1 that predisposes to renal injury in high risk populations through a kidney-intrinsic process. Half of all SLE patients develop nephritis, yet the predisposing mechanisms to kidney damage remain poorly understood. There is limited evidence of genetic contribution to specific organ involvement in SLE.1,2 We identify a large deletion in intron 7 of Van Gogh Like 1 (VANGL1), which associates with nephritis in SLE patients. The same deletion occurs at increased frequency in an indigenous population (Tiwi Islanders) with 10-fold higher rates of kidney disease compared with non-indigenous populations. Vangl1 hemizygosity in mice results in spontaneous IgA and IgG deposition within the glomerular mesangium in the absence of autoimmune nephritis. Serum transfer into B cell-deficient Vangl1+/− mice results in mesangial IgG deposition indicating that Ig deposits occur in a kidney-intrinsic fashion in the absence of Vangl1. These results suggest that Vangl1 acts in the kidney to prevent Ig deposits and its deficiency may trigger nephritis in individuals with SLE.

Reed Syndrome in Eighteen Patients: A Genodermatosis Where Piloleiomyomas May Be the Diagnostic Clue

Introduction: Hereditary leiomyomatosis and renal cell cancer (HLRCC) is caused by germline heterozygous mutations of the fumarate hydratase (FH) gene. Materials and Methods: A retrospective observational study was conducted, aimed at characterizing the clinical features, histopathology, and genetic mutations in eighteen patients with confirmed HLRCC diagnosis. Results: FH gene mutations were identified in the seven families studied, including a previously undescribed mutation. All index cases of the families included were suspected on skin manifestations. Thirteen of the 18 patients (72%) presented cutaneous leiomyomas. The chief complaint was pain, with complete but transient response to botulinum toxin in one. No evidence of malignant transformation was observed. Uterine leiomyomas were present in seven of the eight women studied (88%). There was no evidence of renal cell carcinoma in any of the patients in the study. The most frequently found mutations were missense type (43%), followed by large rearrangements (24%), intronic deletions (14%) and nonsense (14%). A novel mutation not previously described in the literature is presented. Conclusions: HLRCC is a rare disease but it is also probably underdiagnosed. Dermatologists have an essential role in its diagnosis, by recognizing the clinical characteristics of the syndrome and investigating the family history.

Extensive allele mining discovers novel genetic diversity in the loci controlling frost tolerance in barley

Key messageExome sequencing-based allele mining for frost tolerance suggests HvCBF14 rather than CNV at Fr-H2 locus is the main responsible of frost tolerance in barley.Wild relatives, landraces and old cultivars of barley represent a reservoir of untapped and potentially important genes for crop improvement, and the recent sequencing technologies provide the opportunity to mine the existing genetic diversity and to identify new genes/alleles for the traits of interest. In the present study, we use frost tolerance and vernalization requirement as case studies to demonstrate the power of allele mining carried out on exome sequencing data generated from > 400 barley accessions. New deletions in the first intron of VRN-H1 were identified and linked to a reduced vernalization requirement, while the allelic diversity of HvCBF2a, HvCBF4b and HvCBF14 was investigated by combining the analysis of SNPs and read counts. This approach has proven very effective to identify gene paralogs and copy number variants of HvCBF2 and the HvCBF4b-HvCBF2a segment. A multiple linear regression model which considers allelic variation at these genes suggests a major involvement of HvCBF14, rather than copy number variation of HvCBF4b-HvCBF2a, in controlling frost tolerance in barley. Overall, the present study provides powerful resource and tools to discover novel alleles at relevant genes in barley.

Whole Genome Sequencing of Diamond Blackfan Anemia Syndrome Patients Detects Mutations That Alter mRNA Splicing

Diamond Blackfan anemia syndrome (DBAS) is a rare, heritable bone marrow failure syndrome characterized by severe macrocytic anemia, congenital anomalies and predisposition to cancer, most often diagnosed during infancy. More than 98% of DBAS patients with a molecular diagnosis have mutations in a gene encoding one of the ~80 ribosomal proteins (RP) leading to haploinsufficiency. A molecular diagnosis in a patient with DBAS is critical for a definitive diagnosis, the identification of compatible related transplant donors, and developing reproductive strategies for families. Targeted sequencing of RP genes, single nucleotide polymorphism comparative genome hybridization (SNP array) to detect >30 kb deletions (Farrar et al. Blood. 2011) and exome sequencing (WES) (Ulrisch et al. Am J Hum Genet. 2018) has identified RP mutations in ~80% of patients, leaving ~20% of patients with DBAS without a molecular diagnosis.Targeted sequencing and WES focus on only coding sequences. We hypothesized that remaining 20% of DBAS mutations were in the non-coding regions of RP genes, such as promoters or introns. To test this hypothesis, we collected DNA with informed consent for whole genome sequencing (WGS) analysis from 14 patients with no molecular diagnosis after targeted sequencing, SNP array or WES. On average, we aligned ~3.2x10 7 paired end reads of 250 base pairs for each patient (~65X coverage). We focused our analysis on the sequences in and around the RP genes. To identify deletions, we used a suite of detection tools: DELLY, GRIDSS, MANTA, and LUMPY. More than 90% of deletions identified by any 2 of these tools were confirmed by PCR.We identified 5 deletions in the introns of RP genes, ranging from 11 to 467 base pairs in length, which we hypothesized disrupted splicing of the nascent RNA transcript. To test this, we created minigenes in which we replaced exon 2 of a gamma globin gene with either the WT or mutant RP exon. All wild type exons spliced normally. A 467 base pair deletion in RPL27 exon 3 was sufficient to prevent the correct splicing of that intron. Examination of the eCLIP data for RNA binding proteins revealed that spliceosome complex proteins (including SF3B1, SF3B4 and EFTUD2) and Dead-box RNA helicases bind in the deleted region. A 28 base pair deletion in exon 3 of RPL6 removes a polypyrimidine tract that is a critical part of the 3' splice junction consensus sequence, which we presume is also deleterious. The other 3 intronic deletions did not disrupt splicing.We also identified 2 causative point mutations. A point mutation 5 bases into intron 1 of the RPS26 gene changes a base in the 5' splice donor consensus sequence, which activated a cryptic splice donor in the 5' untranslated region. This aberrant splice removes the ATG initiation codon causing an untranslatable RNA. In another patient, we identified a mutation in exon 1 of the RPS27 gene, judged to be a benign amino acid change. This mutation disrupted splicing.by activating a cryptic splice donor site in the 5' untranslated region which removes the ATG initiation codon and causes a frame shift.We were referred two patients with possible duplications of the RPL35a gene. To identify duplications, we employed MinION long read single molecule sequencing. We had an average read length of ~ 6-10kb with the longest read being 1.3Mb. Overall coverage was >85X. We used minimap2 to align the reads to the reference human genome and used SNIFFLES to call the variants. One patient was the parent of DBAS-affected patient with no history of anemia. In this patient, we identified a duplication of 400 kb that included the entire RPL35a region along with genes on either side. We conclude that this duplication is not likely to cause DBA. The second patient was diagnosed with DBAS. In this patient, we identified a duplication of 4 kb including exons 1 and 2 of RPL35a We conclude that this duplication disrupts the RPL35a gene and is a likely cause of DBA.Whole genome sequencing of 15 DBAS patients identified 5 likely causative mutations in RP genes, confirming that most genetically undiagnosed cases of DBAS will involve known genes encoding RP. We conclude that the pipeline for obtaining a molecular diagnosis for DBAS from targeted sequencing, SNP array, and exome sequencing to whole genome sequencing. DisclosuresVlachos: Novartis: Membership on an entity's Board of Directors or advisory committees. Lipton: Celgene: Membership on an entity's Board of Directors or advisory committees.

A quantitative trait variant in Gabra2 underlies increased methamphetamine stimulant sensitivity.

Psychostimulant (methamphetamine, cocaine) use disorders have a genetic component that remains mostly unknown. We conducted genome-wide quantitative trait locus (QTL) analysis of methamphetamine stimulant sensitivity. To facilitate gene identification, we employed a Reduced Complexity Cross between closely related C57BL/6 mouse substrains and examined maximum speed and distance traveled over 30 min following methamphetamine (2 mg/kg, i.p.). For maximum methamphetamine-induced speed following the second and third administration, we identified a single genome-wide significant QTL on chromosome 11 that peaked near the Cyfip2 locus (LOD = 3.5, 4.2; peak = 21 cM [36 Mb]). For methamphetamine-induced distance traveled following the first and second administration, we identified a genome-wide significant QTL on chromosome 5 that peaked near a functional intronic indel in Gabra2 coding for the alpha-2 subunit of the GABA-A receptor (LOD = 3.6–5.2; peak = 34–35 cM [66–67 Mb]). Striatal cis-expression QTL mapping corroborated Gabra2 as a functional candidate gene underlying methamphetamine-induced distance traveled. CRISPR/Cas9-mediated correction of the mutant intronic deletion on the C57BL/6J background to the wild-type C57BL/6NJ allele was sufficient to reduce methamphetamine-induced locomotor activity toward the wild-type C57BL/6NJ-like level, thus validating the quantitative trait variant (QTV). These studies show the power and efficiency of Reduced Complexity Crosses in identifying causal variants underlying complex traits. Functionally restoring Gabra2 expression decreased methamphetamine stimulant sensitivity and supports preclinical and human genetic studies implicating the GABA-A receptor in psychostimulant addiction-relevant traits. Importantly, our findings have major implications for studying psychostimulants in the C57BL/6J strain—the gold standard strain in biomedical research.

Hyper-IgE Syndrome due to an Elusive Novel Intronic Homozygous Variant in DOCK8.

Rare, biallelic loss-of-function mutations in DOCK8 result in a combined immune deficiency characterized by severe and recurrent cutaneous infections, eczema, allergies, and susceptibility to malignancy, as well as impaired humoral and cellular immunity and hyper-IgE. The advent of next-generation sequencing technologies has enabled the rapid molecular diagnosis of rare monogenic diseases, including inborn errors of immunity. These advances have resulted in the implementation of gene-guided treatments, such as hematopoietic stem cell transplant for DOCK8 deficiency. However, putative disease-causing variants revealed by next-generation sequencing need rigorous validation to demonstrate pathogenicity. Here, we report the eventual diagnosis of DOCK8 deficiency in a consanguineous family due to a novel homozygous intronic deletion variant that caused aberrant exon splicing and subsequent loss of expression of DOCK8 protein. Remarkably, the causative variant was not initially detected by clinical whole-genome sequencing but was subsequently identified and validated by combining advanced genomic analysis, RNA-seq, and flow cytometry. This case highlights the need to adopt multipronged confirmatory approaches to definitively solve complex genetic cases that result from variants outside protein-coding exons and conventional splice sites.