Bp Duplication Research Articles

Using long‐read adaptive nanopore sequencing to decipher a novel pathogenic duplication in the PRPH2 gene in patients with macular dystrophy

Purpose: Detailed genetic characterization of three individuals from two families with macular dystrophy caused by exon 2 duplication of the PRPH2 gene.Methods: Two of the affected members (P1, P2) from one family were treated in the Helsinki University Hospital, Finland and one (P3) at Loma Linda University Eye Institute, CA, USA. The patients underwent comprehensive ophthalmic examination, and their genomic DNA was subjected to macular (P2) and retinal dystrophy (P3) gene panels or targeted genetic testing (P1). Additionally, Oxford nanopore adaptive sequencing was used for characterizing and mapping the breakpoints of the identified partial duplication of the PRPH2 gene.Results: Based on the ophthalmic examination, the mother (P1) of the Finnish family was diagnosed with butterfly‐shaped pattern dystrophy (BPD) and the daughter (P2) with vitelliform macular dystrophy. P3 (male) from the USA was diagnosed with BPD in the right eye and central areolar choroidal dystrophy in the left eye. Exome‐based genetic testing indicated that all patients are heterozygous for the same 482 bp duplication c.(581+1_582‐1)_(828+1_829‐1)dup between introns 1 and 2 of the PRPH2 gene, duplicating exon 2 (NM_000322.5). In‐depth analysis of the patients DNA samples by long‐read adaptive nanopore sequencing revealed the duplication to be ∼4kb in size. For all patients the 3’ breakpoint (BP) is located directly upstream from an intronic alu‐element (AluYM1). In the Finnish family the 5’BP resides in a novel alu‐element (AluYa5) lacking from the hg38 reference genome. The P3 lacks the novel‐alu element found in the Finnish family and instead the 5’BP resides in a known alu‐element (AluSq), located some 300bp upstream.Conclusions: We report here that the macular dystrophy in the two families is caused by duplication involving exon 2 of the PRPH2 gene. The alu‐elements at the breakpoints indicate non‐allelic homologous recombination as a possible mechanism for the duplication event.

Using long‐read adaptive nanopore sequencing to decipher a novel pathogenic duplication in the PRPH2 gene in patients with macular dystrophy

Purpose: Detailed genetic characterization of three individuals from two families with macular dystrophy caused by exon 2 duplication of the PRPH2 gene.Methods: Two of the affected members (P1, P2) from one family were treated in the Helsinki University Hospital, Finland and one (P3) at Loma Linda University Eye Institute, CA, USA. The patients underwent comprehensive ophthalmic examination, and their genomic DNA was subjected to macular (P2) and retinal dystrophy (P3) gene panels or targeted genetic testing (P1). Additionally, Oxford nanopore adaptive sequencing was used for characterizing and mapping the breakpoints of the identified partial duplication of the PRPH2 gene.Results: Based on the ophthalmic examination, the mother (P1) of the Finnish family was diagnosed with butterfly‐shaped pattern dystrophy (BPD) and the daughter (P2) with vitelliform macular dystrophy. P3 (male) from the USA was diagnosed with BPD in the right eye and central areolar choroidal dystrophy in the left eye. Exome‐based genetic testing indicated that all patients are heterozygous for the same 482 bp duplication c.(581+1_582‐1)_(828+1_829‐1)dup between introns 1 and 2 of the PRPH2 gene, duplicating exon 2 (NM_000322.5). In‐depth analysis of the patients DNA samples by long‐read adaptive nanopore sequencing revealed the duplication to be ∼4kb in size. For all patients the 3’ breakpoint (BP) is located directly upstream from an intronic alu‐element (AluYM1). In the Finnish family the 5’BP resides in a novel alu‐element (AluYa5) lacking from the hg38 reference genome. The P3 lacks the novel‐alu element found in the Finnish family and instead the 5’BP resides in a known alu‐element (AluSq), located some 300bp upstream.Conclusions: We report here that the macular dystrophy in the two families is caused by duplication involving exon 2 of the PRPH2 gene. The alu‐elements at the breakpoints indicate non‐allelic homologous recombination as a possible mechanism for the duplication event.

Precise template-independent correction of c.1278insTATC frameshift mutation in Tay-Sachs disease restores full gene function, while gene reframing yields negligible recovery

Tay-Sachs disease is a fatal neurodegenerative disorder caused by HEXA mutations inactivating the metabolic enzyme HexA. The most common mutation is c.1278insTATC, a tandem 4 base pair (bp) duplication disrupting HEXA expression by frameshift. In an engineered cell model, we explore the use of CRISPR/Cas9 for therapeutic editing of c.1278insTATC. Within genomic microduplications, the microhomology-mediated end joining (MMEJ) pathway is favoured to repair double-stranded breaks with collateral deletion of one repeat. Protospacer adjacent motif (PAM) constraints on Cas9 endonuclease activity prevented cleavage at the duplication centre – the optimal position for MMEJ initiation. Rather, cleavage 1 bp from the c.1278insTATC duplication centre spontaneously reconstructed the wildtype sequence at ∼14.7% frequency, with concomitant restoration of normal cellular HexA activity. As an alternative to perfect correction, short insertions/deletions were serially introduced to restore an open reading frame across a 19 bp sequence encompassing c.1278insTATC. Frame-restored variants did not recover significant HexA function, presumably due to structural incompatibility of incurred amino acid insertions. Hence, precise correction of c.1278insTATC is the only therapeutically relevant outcome achieved in this study, with MMEJ highlighted as a potential template-free CRISPR/Cas9 modality to that end.

Correction: A 20 bp Duplication in Exon 2 of the Aristaless-Like Homeobox 4 Gene (ALX4) Is the Candidate Causative Mutation for Tibial Hemimelia Syndrome in Galloway Cattle

Correction: A 20 bp Duplication in Exon 2 of the Aristaless-Like Homeobox 4 Gene (ALX4) Is the Candidate Causative Mutation for Tibial Hemimelia Syndrome in Galloway Cattle

DNA sequences and distinct mechanisms for ura4-595 and ura4-294 alleles of S. pombe.

The ura4 gene of the fission yeast Schizosaccharomyces pombe supports both positive and negative selection; consequently, this gene is widely employed as a powerful tool to study diverse biological processes. Here we report the DNA sequences of two functionally null alleles, ura4-595 and ura4-294 . The ura4-595 allele has a four bp duplication of bp +63 to +66 (5'-CAAG-3') within the ORF and the ura4-294 allele has a nonsynonymous substitution (G to A) at bp +679. We infer that these alleles arose, respectively, by DNA polymerase template slipping and by nucleotide misincorporation (likely via cytosine deamination).

Identification and characterisation of de novo germline structural variants in two commercial pig lines using trio-based whole genome sequencing

BackgroundDe novo mutations arising in the germline are a source of genetic variation and their discovery broadens our understanding of genetic disorders and evolutionary patterns. Although the number of de novo single nucleotide variants (dnSNVs) has been studied in a number of species, relatively little is known about the occurrence of de novo structural variants (dnSVs). In this study, we investigated 37 deeply sequenced pig trios from two commercial lines to identify dnSVs present in the offspring. The identified dnSVs were characterised by identifying their parent of origin, their functional annotations and characterizing sequence homology at the breakpoints.ResultsWe identified four swine germline dnSVs, all located in intronic regions of protein-coding genes. Our conservative, first estimate of the swine germline dnSV rate is 0.108 (95% CI 0.038–0.255) per generation (one dnSV per nine offspring), detected using short-read sequencing. Two detected dnSVs are clusters of mutations. Mutation cluster 1 contains a de novo duplication, a dnSNV and a de novo deletion. Mutation cluster 2 contains a de novo deletion and three de novo duplications, of which one is inverted. Mutation cluster 2 is 25 kb in size, whereas mutation cluster 1 (197 bp) and the other two individual dnSVs (64 and 573 bp) are smaller. Only mutation cluster 2 could be phased and is located on the paternal haplotype. Mutation cluster 2 originates from both micro-homology as well as non-homology mutation mechanisms, where mutation cluster 1 and the other two dnSVs are caused by mutation mechanisms lacking sequence homology. The 64 bp deletion and mutation cluster 1 were validated through PCR. Lastly, the 64 bp deletion and the 573 bp duplication were validated in sequenced offspring of probands with three generations of sequence data.ConclusionsOur estimate of 0.108 dnSVs per generation in the swine germline is conservative, due to our small sample size and restricted possibilities of dnSV detection from short-read sequencing. The current study highlights the complexity of dnSVs and shows the potential of breeding programs for pigs and livestock species in general, to provide a suitable population structure for identification and characterisation of dnSVs.

Analysis of neurotransmitters validates the importance of the dopaminergic system in autism spectrum disorder.

The reasons behind the cardinal symptoms of communication deficits and repetitive, stereotyped behaviors that characterize autism spectrum disorder (ASD) remain unknown. The dopamine (DA) system, which regulates motor activity, goal-directed behaviors, and reward function, is believed to play a crucial role in ASD, although the exact mechanism is still unclear. Investigations have shown an association of the dopamine receptorD4 (DRD4) with various neurobehavioral disorders. We analyzed the association between ASD and four DRD4 genetic polymorphisms, 5' flanking 120-bp duplication (rs4646984), rs1800955 in the promoter, exon 1 12bp duplication (rs4646983), and exon 3 48bp repeats. We also examined plasma DA and its metabolite levels, DRD4 mRNA expression, and correlations of the studied polymorphisms with these parameters by case-control comparative analyses. The expression of DA transporter (DAT), which is important in regulating the circulating DA level, was also evaluated. A significantly higher occurrence of rs1800955 "T/TT" was observed in the probands. ASD traits were affected by rs1800955 "T" and the higher repeat alleles of the exon 3 48bp repeats, rs4646983 and rs4646984. ASD probands exhibited lower DA and norepinephrine levels together with higher homovanillic acid levels than the control subjects. DAT and DRD4 mRNA expression were down-regulated in the probands, especially in the presence of DAT rs3836790 "6R" and rs27072 "CC" and DRD4 rs4646984 higher repeat allele and rs1800955 "T". This pioneering investigation revealed a positive correlation between genetic variants, hypodopaminergic state, and impairment in socio-emotional and communication reciprocity in Indian subjects with ASD, warranting further in-depth analysis.

Commentary on A Patient with Coarse Facial Features and Molecular Odyssey: Lessons Learned and Best Practice.

The authors describe a 6-year-old female with macrocephaly, limited joint mobility, short stature, and normal cognitive development. Biochemical genetic studies demonstrated elevated urinary dermatan sulfate and decreased N-acetylgalactosamine-6-sulfatase (arylsulfatase B) activity. This collection of clinical features and biochemical abnormalities is specific for mucopolysaccharidosis (MPS) type VI (Maroteaux–Lamy syndrome). Targeted NGS analysis and whole-exome analysis identified a heterozygous pathogenic missense variant in ARSB. MPS VI is an autosomal recessive disorder, therefore biallelic variants would be required to cause an enzyme deficiency as well as the accumulation of urinary dermatan sulfate, necessitating a search for a second variant. Further dosage analysis and whole-genome sequencing did not identify a second variant in ARSB. Subsequent WGS analysis by a different laboratory identified an additional novel, heterozygous 97 bp duplication in ARSB, and this was confirmed by Sanger sequencing. The implementation of NGS into clinical laboratories has revolutionized genetic testing. Even early on, reports surfaced about missed variants leading to continued improvements in reagents and analysis pipelines (1). Indels of >50 bp are often missed by short-read sequencing and the integration of long-read analyses into clinical testing will likely improve the identification of these difficult variants. Missed variants can cause delays in treatment or limit familial testing. Additionally, in this case, if a molecular-first approach had been used and biochemical testing not initiated, the patient may have been considered a carrier.

Evaluating the clinical significance of FLT3 mutation status in Syrian newly diagnosed acute myeloid leukemia patients with normal karyotype

The FMS-like tyrosine kinase-3 internal tandem duplication (FLT3-ITD) is one of the most prevalent mutations, affecting between 20 and 30 percent of cases in patients with acute myeloid leukemia (AML). The Patients with a FLT3-ITD mutation have a poor prognosis. In the present study, we investigated the FLT3 (ITD-TKD) mutations in 100 newly adult Syrian patients with AML-Normal karyotype (NK). Our results revealed that prevalence of FLT3-ITD mutation was 24%. Interestingly, 20 patients had a typical duplication mutation and four patients had different mutations. From those four mentioned patients, two of them carried a 39 base pair (bp) duplication in different location: (c.1838_1877dup39, p.591–603dup) and (c.1836_1874 dup 39, p.591–603dup), the third patient, showed FLT3-ITD duplication and a newly insertion together, this insertion was not demonstrated before: (c.1842_1865dup24, c.1865_1866insGAA). Finally, the fourth patient exhibited a duplication of 21bp (c.1855_1875dup21, p.597–603dup). In addition, statistically significant differences were observed for the relation between the presence of FLT3-ITD mutation and lactate dehydrogenase (LDH) level, overall survival (OS), relapse, and event free survival (EFS). We demonstrated that our patients with FLT3-ITD mutation had a poor prognosis. Also, the frequency of FLT3-TKD mutation was low 2% and no compound between the two mutations was found, as individuals showed to carry the two mutations were not detected. These findings are likely useful for a better understanding of molecular leukemogenetic steps in AML-NK patients and may be beneficial for clinical relevance for risk grouping, study design and choice of therapy in Syrian population.

Heterozygous pathogenic variants involving CBFB cause a new skeletal disorder resembling cleidocranial dysplasia

BackgroundCleidocranial dysplasia (CCD) is a rare skeletal dysplasia with significant clinical variability. Patients with CCD typically present with delayed closure of fontanels and cranial sutures, dental anomalies, clavicular hypoplasia or...

Design and validation of a codominant molecular marker for fruit acidity in muskmelon (Cucumis melo)

AbstractAn experiment was conducted to analyse a germplasm panel of 92 Cucumis sp. accessions predominantly from India for fruit acidity and validate the involvement of ‘CmpH’ locus for this trait through design of suitable marker and association analysis. Among 92 accessions, 28 accessions were found to be sour with fruit pH < 5.0 and titratable acidity value >0.20%. We designed a primer pair ‘Dupl‐12’ flanking the causal polymorphism of 12 bp duplication in the earlier reported candidate gene ‘CmpH’ for fruit acidity. It amplified two types of alleles (180 and 192 bp) depending upon the absence or presence of 12 bp duplication and was codominant in nature. This was used to perform association analysis in the germplasm panel and was found to be significantly associated with titratable acidity (R2 = 73.43%) and pH (R2 = 70.76%), validating the role of ‘CmpH’ gene for fruit acidity. ‘Dupl‐12’ marker can therefore be employed for marker‐assisted transfer of acidity into elite backgrounds.

Bi-allelic FRA10AC1 variants in a multisystem human syndrome.

We read the recent article by von Elsner et al.1 with interest. The authors described five individuals from three families with homozygous FRA10AC1 variants, neurodevelopmental delay, intellectual disability, brain abnormalities, microcephaly, growth impairment and craniofacial dysmorphisms. Here, we report findings that support the conclusions reached by von Elsner and colleagues, and provide further insights into this novel disorder. Parents of all participating individuals provided informed consent according to the Declaration of Helsinki and The Central Manchester NHS Research Ethics Committees approved this study (02/CM/238). We ascertained two sisters born to consanguineous parents with global developmental delay, growth impairment, congenital malformations and facial dysmorphism overlapping that of Kabuki syndrome2,3 (OMIM 147920) (Fig. 1A and Table 1). In two affected and one unaffected sibling we performed autozygosity mapping as described previously4 and identified two regions of >2 Mb length that were homozygous only in the affected individuals (Supplementary Table 1). Whole exome sequencing of affected individuals as described previously,5 followed by segregation studies of candidate variants within the linked regions revealed a nonsense FRA10AC1 NM_145246.5:c.328C > T (p.Arg110Ter) variant as the likely cause for the phenotype (Fig. 1B). In gnomAD, there are no homozygous predicted loss of function (pLOF) FRA10AC1 variants, although heterozygous pLOF variants are present at a frequency of ∼1/2000. Next, we identified an individual in the DECIPHER database (257391) with a ∼13 kb homozygous deletion involving a part of FRA10AC1 and no other known gene (Fig. 1A and B). Characterization of the deletion by PCR and Sanger sequencing of genomic DNA (Supplementary material) revealed the variant to be an in-del consisting of a 12 721 bp deletion (Chr10:93 695 674–93 708 393; GRCh38.p12) and a 10 bp duplication. The 5′ breakpoint of the variant was in the intergenic region between FRA10AC1 and LGI1. The 3′ breakpoint was in the intron 4 of FRA10AC1. The duplicated sequence (TTAGTACACA) was a repeat of the first 10 bp of sequence downstream of the variant. The deletion results in loss of the 5′ untranslated region and first four exons of FRA10AC1. Reverse transcription-PCR of RNA extracted from fibroblasts from this patient, revealed absence of FRA10AC1 transcripts (Fig. 1C). Although the individual was ascertained due to her genotype, reverse phenotyping6 revealed her clinical features to overlap significantly with those of the affected individuals in Family 1 (Table 1).1 Collectively, these data provide evidence that bi-allelic pLOF FRA10AC1 variants cause a human developmental disorder.

Establishment of a human induced pluripotent stem cell line, KMUGMCi001-A, from a patient bearing a heterozygous c.772 + 3_772 + 4dup mutation in the ACVRL1 gene leading Telangiectasia, hereditary hemorrhagic, type 2 (HHT2)

Telangiectasia, hereditary hemorrhagic, type 2 (HHT2) is a rare autosomal dominant disease caused by a mutated ACVRL1 gene (Letteboer et al., 2005). The peripheral blood mononuclear cells (PBMCs) from a patient carrying a heterozygous 2 bp duplication in intron 6 of the ACVRL1 gene, NG_009549.1(NM_000020.2):c.772 + 3_772 + 4dup, were reprogrammed using episomal vectors. The inserted mutation in ACVRL1 will causes the abnormal splicing, which will be associated with HHT2. The cell line will enable proper in vitro disease modelling of HHT2(Roman and Hinck, 2017).

Recurrent mutations in topoisomerase IIα cause a previously undescribed mutator phenotype in human cancers

Topoisomerases nick and reseal DNA to relieve torsional stress associated with transcription and replication and to resolve structures such as knots and catenanes. Stabilization of the yeast Top2 cleavage intermediates is mutagenic in yeast, but whether this extends to higher eukaryotes is less clear. Chemotherapeutic topoisomerase poisons also elevate cleavage, resulting in mutagenesis. Here, we describe p.K743N mutations in human topoisomerase hTOP2α and link them to a previously undescribed mutator phenotype in cancer. Overexpression of the orthologous mutant protein in yeast generated a characteristic pattern of 2- to 4-base pair (bp) duplications resembling those in tumors with p.K743N. Using mutant strains and biochemical analysis, we determined the genetic requirements of this mutagenic process and showed that it results from trapping of the mutant yeast yTop2 cleavage complex. In addition to 2- to 4-bp duplications, hTOP2α p.K743N is also associated with deletions that are absent in yeast. We call the combined pattern of duplications and deletions ID_TOP2α. All seven tumors carrying the hTOP2α p.K743N mutation showed ID_TOP2α, while it was absent from all other tumors examined (n = 12,269). Each tumor with the ID_TOP2α signature had indels in several known cancer genes, which included frameshift mutations in tumor suppressors PTEN and TP53 and an activating insertion in BRAF. Sequence motifs found at ID_TOP2α mutations were present at 80% of indels in cancer-driver genes, suggesting that ID_TOP2α mutagenesis may contribute to tumorigenesis. The results reported here shed further light on the role of topoisomerase II in genome instability.

Epidermal growth factor receptor exon 20 insertion variants in non-small cell lung cancer patients.

Epidermal growth factor receptor (EGFR) exon 20 insertions occur rarely among different cancer types, with the highest frequency reported among non-small-cell lung cancer (NSCLC) patients, particularly adenocarcinomas (ADCs). Exon 20 insertions fall back in the tyrosine kinase domain, and can be clustered into two principal groups represented by in frame insertions and three to 21 bp (corresponding to 1-7 amino acids) duplications within amino acids 762 and 774. The identification of these alterations is key for an adequate management of NSCLC patients due to the possibility to treat these patients with specific targeted therapies. Next generation sequencing (NGS) technology, able to detect several hotspot gene mutations for different patients simultaneously, is the best detection approach due to its higher sensitivity and specificity compared to other techniques. Here we reviewed the principal biological characteristics, the main detection technologies and treatment options for NSCLC patients harbouring EGFR exon 20 insertions.

TP53 Intronic Polymorphisms and Risk of Esophageal Cancer in Southern Thai Population

Objective: The study aimed to determine the frequency of intron 3 16 base pair (bp) duplication polymorphism and intron 6 G to C substitution (G>C) of the TP53 gene and to evaluate the association these two intronic variants with the risk of esophageal cancer (EC).Material and Methods: A case-control study was conducted to evaluate the frequency and association of cancer. Cases were patients with squamous cell carcinoma of esophagus and controls were age and sex-matched non-cancer patients. Blood samples were also obtained from healthy blood donors. Polymerase chain reaction (PCR) was used to detect intron 3 16 bp duplication and PCR-restriction fragment length polymorphism was applied to detect intron 6 G>C. Logistic regression was used for the analysis.Results: Heterozygous intron 3 16 bp duplication (Del/Ins) was found in 10.1% (31/308) of blood donors, 9.3% (28/302) of controls and 8.6% (26/301) of EC cases. Intron 6 G>C was found in 0.3% (1/308) of blood donors, in 2.6% (8/310) of controls and 3.9% (12/307) of EC cases. Both variants displayed no significant association with risk of esophageal cancer (odd ratio (OR) = 1.16 [95% confidence interval (CI) = 0.64–2.11] for intron 3 16 bp duplication and OR = 0.81 [95% CI = 0.47-4.61] for intron 6 G>C.Conclusion: Southern Thai population have low frequency of intron 3 16 bp duplication polymorphism and intron 6 G>C variant, both of which are not likely to be associated with esophageal cancer susceptibility.

GRIDSS2: comprehensive characterisation of somatic structural variation using single breakend variants and structural variant phasing

GRIDSS2 is the first structural variant caller to explicitly report single breakends—breakpoints in which only one side can be unambiguously determined. By treating single breakends as a fundamental genomic rearrangement signal on par with breakpoints, GRIDSS2 can explain 47% of somatic centromere copy number changes using single breakends to non-centromere sequence. On a cohort of 3782 deeply sequenced metastatic cancers, GRIDSS2 achieves an unprecedented 3.1% false negative rate and 3.3% false discovery rate and identifies a novel 32–100 bp duplication signature. GRIDSS2 simplifies complex rearrangement interpretation through phasing of structural variants with 16% of somatic calls phasable using paired-end sequencing.

A homozygous duplication of the FGG exon 8-intron 8 junction causes congenital afibrinogenemia. Lessons learned from the study of a large consanguineous Turkish family.

Congenital afibrinogenemia is the most severe congenital fibrinogen disorder, characterized by undetectable fibrinogen in circulation. Causative mutations can be divided into two main classes: null mutations with no protein production at all and missense mutations producing abnormal protein chains that are retained inside the cell. The vast majority of cases are due to single base pair mutations or small insertions or deletions in the coding regions or intron-exon junctions of FGB, FGA and FGG. Only a few large rearrangements have been described, all deletions involving FGA. Here we report the characterization of a 403 bp duplication of the FGG exon 8-intron 8 junction accounting for congenital afibrinogenemia in a large consanguineous family from Turkey. This mutation, which had escaped detection by Sanger sequencing of short polymerase chain reaction (PCR) amplicons of coding sequences and splice sites, was identified by studying multiple alignments of reads obtained from whole exome sequencing of a heterozygous individual followed by PCR amplification and sequencing of a larger portion of FGG. Because the mutation duplicates the donor splice site of intron 8, we predicted that the impact of the mutation would be on FGG transcript splicing. Analysis of mRNA produced by cells transiently transfected with normal or mutant minigene constructs showed that the duplication causes production of several aberrant FGG transcripts generating premature truncating codons.

Effects of a Common Eight Base Pairs Duplication at the Exon 7-Intron 7 Junction on Splicing, Expression, and Function of OCT1.

Organic cation transporter 1 (OCT1, SLC22A1) is localized in the sinusoidal membrane of human hepatocytes and mediates hepatic uptake of weakly basic or cationic drugs and endogenous compounds. Common amino acid substitutions in OCT1 were associated with altered pharmacokinetics and efficacy of drugs like sumatriptan and fenoterol. Recently, the common splice variant rs35854239 has also been suggested to affect OCT1 function. rs35854239 represents an 8 bp duplication of the donor splice site at the exon 7-intron 7 junction. Here we quantified the extent to which this duplication affects OCT1 splicing and, as a consequence, the expression and the function of OCT1. We used pyrosequencing and deep RNA-sequencing to quantify the effect of rs35854239 on splicing after minigene expression of this variant in HepG2 and Huh7 cells and directly in human liver samples. Further, we analyzed the effects of rs35854239 on OCT1 mRNA expression in total, localization and activity of the resulting OCT1 protein, and on the pharmacokinetics of sumatriptan and fenoterol. The 8 bp duplication caused alternative splicing in 38% (deep RNA-sequencing) to 52% (pyrosequencing) of the minigene transcripts when analyzed in HepG2 and Huh7 cells. The alternatively spliced transcript encodes for a truncated protein that after transient transfection in HEK293 cells was not localized in the plasma membrane and was not able to transport the OCT1 model substrate ASP+. In human liver, however, the alternatively spliced OCT1 transcript was detectable only at very low levels (0.3% in heterozygous and 0.6% in homozygous carriers of the 8 bp duplication, deep RNA-sequencing). The 8 bp duplication was associated with a significant reduction of OCT1 expression in the human liver, but explained only 9% of the general variability in OCT1 expression and was not associated with significant changes in the pharmacokinetics of sumatriptan and fenoterol. Therefore, the rs35854239 variant only partially changes splicing, causing moderate changes in OCT1 expression and may be of only limited therapeutic relevance.

Evolution of immunogenetic components used to form ultralong VH CDR3 antibodies in Bovidae

Abstract Cattle has a restricted repository of immunoglobulin heavy chain gene segments with only one family of V, 4 J and 16 D segments functional. Among these segments are a V and D that, together, encode a heavy chain with an ultralong CDR3 region which manifests as a “stalk” supporting a “knob” domain of which only the knob has antigen binding capability. This antibody structure has boundless therapeutic potential, including for treatment of HIV and other antigens with veiled epitopes. Upon the discovery of this ultralong antibody in Bos taurus, speculation of its origin began. The components that should facilitate ultralong CDR3H antibodies appear in more species than just Bos taurus; they expand throughout the bovine subfamily as evidence by the presence of a V motif “TTVHQ” crested by an 8 base-pair (bp) duplication, and a comparatively long D segment. Several closely-related species encode the ultralong V segments gene in their germline IgH locus. Phylogenetically bookending the cluster that is genomically equipped for the ultralong cattlebody are the species Bos taurus and Bison bison with Bos mutus, indicus, and grunniens in between. The added length of the D segment is less consistent throughout the group as none of the species encode as long of segment as Bos taurus at 50 amino acids. Two species studied, Bos mutus and Bos grunniens, have 15 and 22 amino acids, respectively, encoded by their longest D segment, and Bison bison has 23. Though we do know the germline encodes the ultralong antibody, it is imperative to know if the gene rearrangement is actually expressed and employed in the mature repertoire.