Dinucleotide Deletion Research Articles

Ovine KRTAP36-2: A New Keratin-Associated Protein Gene Related to Variation in Wool Yield.

Keratin-associated proteins (KAPs) are structural components of wool fibres. High-glycine/tyrosine (HGT)-KAPs are a subset of the KAP family, and their abundance in fibres varies. In this study, we report the discovery of an ovine HGT-KAP gene to which we assigned the name KRTAP36-2. Polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) analyses revealed four variants of this gene in a screening population of 170 sheep from a variety of breeds. The DNA sequencing of the variants revealed four single-nucleotide polymorphisms (SNPs) and a dinucleotide deletion. Three of these SNPs were in the coding region, and one of these was non-synonymous and potentially led to the amino acid substitution p.Cys27Gly near the middle of the protein. The remaining SNP was located near the putative TATA box, and the di-nucleotide deletion was near the putative transcription initiation site. The effect of this variation in KRTAP36-2 was investigated in 274 Southdown × Merino lambs that were the progeny of five sires. Variation was only found to be associated with wool yield, that is, the proportion of the greasy fleece that remained as clean fleece upon scouring (expressed as a percentage). This may have some value in increasing wool production.

Нарушения усвоения сахарозы подтверждают своеобразие генетической истории эскимосов

Introduction. A dinucleotide deletion of AG at the rs781470490 locus of the SI gene (SI delAG variant) causes dysfunction of the sucrase-isomaltase enzyme and the inability to digest sugar. The deletion frequency in the world is 0.0021%, while among the Inuit (Eskimo) it reaches 17–20%. Research hypothesis: the SI delAG variant is common in all the populations of indigenous northerners. The study aimed to ascertain the prevalence of the SI delAG variant in the Cis-Ural, Siberian, and Far East populations of the Russian Federation and compare it to that of the Inuit people. Materials and methods. DNA genotyping and the SI delAG variant frequency analysis were carried out in the samples from study groups of Russians, Komi, Mansi, Khanty, Nenets, Shors, Evenks, Evens, Koryaks, Chukchi (n =613). Corresponding data for the Canadian and Greenlandic Inuit (n =391) were used for comparison. Results and discussion. The deletion in samples from the Chukchi, Koryak, and Even study groups occurred in 14, 7, and 4%, respectively; there was no the deletion found in the samples of the other Siberian and Cis-Ural groups. The carriage of the SI delAG variant decreases with the distance from the area of the Greenlandic Inuit. The Inuit, Chukchi, and Koryak groups form a distinct cluster. The Paleo-Eskimo do not carry the deletion. Minimal amounts of sugar-containing products appeared in the diet of the Greenlandic and Canadian Inuit at the end of the 19th century, 200 years later than in the diet of aboriginal northerners of Eurasia. Conclusions. There was no SI delAG variant found in the samples of the indigenous people of West Eurasia high latitude regions. The deletion emerged in Neo-Eskimo population 1.2–2 thousand years ago and has been retained in the small numbered isolate due to their way of existence and diet. In the populations of the northwest coast of the Sea of Okhotsk and the Chukchi Peninsula, the SI delAG variant was brought by the gene flow from the Inuit people. @ 2023. This work is licensed under a CC BY 4.0 license.

Comparative Analysis of Potential Determinants of Resistance to Aminoglycosides in <i>Burkholderia pseudomallei</i> Strains with Different Level of Sensitivity to Gentamicin

The aim of the study was to identify and compare potential determinants of aminoglycoside resistance in gentamicin susceptible Burkholderia pseudomallei strains.Materials and methods. A bioinformatics analysis of whole genome shotgun sequences of three B. pseudomallei strains having different levels of sensitivity to gentamicin was carried out.Results and discussion. B. pseudomallei is intrinsically resistant to aminoglycosides. Such strains, as a rule, are not taken into account in the classical scheme of isolation and identification. At the same time, there were no significant differences in the clinical manifestations of melioidosis during infection with gentamicin-resistant and sensitive strains. In B. pseudomallei strains of different sequence types (ST70, ST948, and ST1566), point missense mutations were found in the genes of three efflux pumps of the RND family: AmrAB-OprA, BpeAB-OprB, BpeEF-OprC, and one with unknown functions, as well as in the gene aminoglycoside-6’-N-acetyltransferase AAC(6’)-III. All three strains had amino acid substitutions in the AmrA periplasmic linker: ARG160SER, Arg116Gln and Gly237Arg, Thr317Lys, respectively. In moderately sensitive strains (ST948 and ST1566), an identical Val222Met substitution was found in the repressor of the AmrAB-OprA operon, AmrR. It is likely that the intermediate level of sensitivity to gentamicin in the studied strains is mediated by the constitutive expression of the AmrAB-OprA operon, which partially compensates for the structural defects. It is also possible that a dinucleotide deletion in the AAC (6’)-III aminoglycoside-6’-N-acetyltransferase gene, as well as detected mutations in the homologues of the periplasmic linker (BPSL2234) of an uncharacterized efflux operon of the RND family, are involved in the loss of resistance to gentamicin.

Своеобразие субэтнических групп ненцев по генетическим детерминантам метаболизма сахарозы, трегалозы и лактозы

Introduction. The diet of indigenous northerners is changing significantly on account of the rapid growth in volume and variety of consummated food sugars. Concurrently, an arrow of northern aboriginal groups are known to have an increased percentage of genetically determined disorders of disaccharide metabolism. The study aimed to assess the polymorphism of genes and alleles that determine production or activity of sucrase-isomaltase, trehalase and lactase enzymes in the groups of Nenets of Western Siberia and European Arctic. Materials and methods. The genotyping of the samples of biomaterial from 236 unrelated individuals formed the basis of the study. We analyzed the genotype and allele frequencies of the SI (rs781470490), TREH (rs2276064) and LCT (rs4988235) genes in the groups of Forest, Gydan, Yamal and European Tundra Nenets. Results. There were not a single sample with the AG dinucleotide deletion at the rs781470490 locus of the sucrase-isomaltase gene (SI gene) found. The TREH allele distributions appeared to be extremely close in all the sub-ethnic groups of Nenets. The frequency of the associated with trehalase deficiency A*TREH allele in the Nenets groups is at 0.25-0.26, which is significantly higher than in the non-arctic populations of Russia (0.01-0.06). We have confirmed a high prevalence in the Nenets populations of the CC*LCT variant of the lactase gene, which determines the limited production of the enzyme. The frequencies of the genotype vary in the Nenets sub-groups from 0.90 in the Gydan and European Tundra Nenets to 0.70 in the Forest (the difference from the European Tundra group is significant at the level of p = 0.06) and 0.65 in the Yamal Nenets (significantly differ from both the Gydan and European Tundra groups, p < 0.05). Conclusion. The difference in metabolism-related gene complexes can be found not only between peoples, but even between sub-ethnic groups.

Genetic and molecular characterization of determinant of six-rowed spike of barley carrying vrs1.a4.

Decisive role of reduced vrs1 transcript abundance in six-rowed spike of barley carrying vrs1.a4 was genetically proved and its potential causes were preliminarily analyzed. Six-rowed spike 1 (vrs1) is the major determinant of the six-rowed spike phenotype of barley (Hordeum vulgare L.). Alleles of Vrs1 have been extensively investigated. Allele vrs1.a4 in six-rowed barley is unique in that it has the same coding sequence as Vrs1.b4 in two-rowed barley. The determinant of row-type in vrs1.a4 carriers has not been experimentally identified. Here, we identified Vrs1.b4 in two-rowed accessions and vrs1.a4 in six-rowed accessions from the Qinghai-Tibet Plateau at high frequency. Genetic analyses revealed a single nuclear gene accounting for row-type alteration in these accessions. Physical mapping identified a 0.08-cM (~ 554-kb) target interval on chromosome 2H, wherein Vrs1 was the most likely candidate gene. Further analysis of Vrs1 expression in offspring of the mapping populations or different Vrs1.b4 and vrs1.a4 lines confirmed that downregulated expression of vrs1.a4 causes six-rowed spike. Regulatory sequence analysis found a single 'TA' dinucleotide deletion in vrs1.a4 carriers within a 'TA' tandem-repeat-enriched region ~ 1kb upstream of the coding region. DNA methylation levels did not correspond to the expression difference and therefore did not affect Vrs1 expression. More evidence is needed to verify the causal link between the 'TA' deletion and the downregulated Vrs1 expression and hence the six-rowed spike phenotype.

The role of VvMYBA2r and VvMYBA2w alleles of the MYBA2 locus in the regulation of anthocyanin biosynthesis for molecular breeding of grape (Vitis spp.) skin coloration.

SummaryIn grape, MYBA1 and MYBA2 at the colour locus are the major genetic determinants of grape skin colour, and the mutation of two functional genes (VvMYBA1 and VvMYBA2) from these loci leads to white skin colour. This study aimed to elucidate the regulation of grape berry coloration by isolating and characterizing VvMYBA2w and VvMYBA2r alleles. The overexpression of VvMYBA2r up‐regulated the expression of anthocyanin biosynthetic genes and resulted in higher anthocyanin accumulation in transgenic tobacco than wild‐type (WT) plants, especially in flowers. However, the ectopic expression of VvMYBA2w inactivated the expression of anthocyanin biosynthetic genes and could not cause obvious phenotypic modulation in transgenic tobacco. Unlike in VvMYBA2r, CA dinucleotide deletion shortened the C‐terminal transactivation region and disrupted the transcriptional activation activity of VvMYBA2w. The results indicated that VvMYBA2r positively regulated anthocyanin biosynthesis by forming the VvMYBA2r‐VvMYCA1‐VvWDR1 complex, and VvWDR1 enhanced anthocyanin accumulation by interacting with the VvMYBA2r‐VvMYCA1 complex; however, R44L substitution abolished the interaction of VvMYBA2w with VvMYCA1. Meanwhile, both R44L substitution and CA dinucleotide deletion seriously affected the efficacy of VvMYBA2w to regulate anthocyanin biosynthesis, and the two non‐synonymous mutations were additive in their effects. Investigation of the colour density and MYB haplotypes of 213 grape germplasms revealed that dark‐skinned varieties tended to contain HapC‐N and HapE2, whereas red‐skinned varieties contained high frequencies of HapB and HapC‐Rs. Regarding ploidy, the higher the number of functional alleles present in a variety, the darker was the skin colour. In summary, this study provides insight into the roles of VvMYBA2r and VvMYBA2w alleles and lays the foundation for the molecular breeding of grape varieties with different skin colour.

Clinical, Immunological, and Molecular Profile of Chronic Granulomatous Disease: A Multi-Centric Study of 236 Patients From India.

BackgroundChronic granulomatous disease (CGD) is an inherited defect in phagocytic respiratory burst that results in severe and life-threatening infections in affected children. Single center studies from India have shown that proportion of autosomal recessive (AR) CGD is more than that reported from the West. Further, affected patients have high mortality rates due to late referrals and difficulties in accessing appropriate treatment. However, there is lack of multicentric collaborative data on CGD from India.ObjectiveTo describe infection patterns, immunological, and molecular features of CGD from multiple centers in India.MethodsA detailed proforma that included clinical and laboratory details was prepared and sent to multiple centers in India that are involved in the care and management of patients with inborn errors of immunity. Twelve centers have provided data which were later pooled together and analyzed.ResultsOf the 236 patients analyzed in our study, X-linked and AR-CGD was seen in 77 and 97, respectively. Male female ratio was 172:64. Median age at onset of symptoms and diagnosis was 8 and 24 months, respectively. Common infections documented include pneumonia (71.6%), lymphadenitis (31.6%), skin and subcutaneous abscess (23.7%), blood-stream infection (13.6%), osteomyelitis (8.6%), liver abscess (7.2%), lung abscess (2.9%), meningoencephalitis (2.5%), splenic abscess (1.7%), and brain abscess (0.9%). Forty-four patients (18.6%) had evidence of mycobacterial infection. Results of molecular assay were available for 141 patients (59.7%)—CYBB (44.7%) gene defect was most common, followed by NCF1 (31.9%), NCF2 (14.9%), and CYBA (8.5%). While CYBA variants were documented only in Southern and Western parts of India, a common dinucleotide deletion in NCF2 (c.835_836delAC) was noted only in North Indian population. Of the 174 patients with available outcome data, 67 (38.5%) had expired. Hematopoietic stem cell transplantation was carried out in 23 patients, and 12 are doing well on follow-up.ConclusionsIn India, proportion of patients with AR-CGD is higher as compared to Western cohorts, though regional differences in types of AR-CGD exist. Clinical profile and mortality rates are similar in both X-linked and AR-CGD. However, this may be a reflection of the fact that milder forms of AR-CGD are probably being missed.

A novel, pathogenic dinucleotide deletion in the mitochondrial MT-TY gene causing myasthenia-like features

Mitochondrial DNA (mtDNA)-related diseases often pose a diagnostic challenge and require rigorous clinical and laboratory investigation. Pathogenic variants in the mitochondrial tRNA gene MT-TY, which encodes the tRNATyr, are a rare cause of mitochondrial disease. Here we describe a novel m.5860delTA anticodon variant in the MT-TY gene in a patient who initially presented with features akin to a childhood onset myasthenic syndrome. Using histochemical, immunohistochemical and protein studies we demonstrate that this mutation leads to severe biochemical defects of mitochondrial translation, which is reflected in the early onset and progressive phenotype. This case highlights the clinical overlap between mtDNA-related diseases and other neuromuscular disorders, and demonstrates the potential pitfalls in analysis of next generation sequencing results, given whole exome sequencing of a blood DNA sample failed to make a genetics diagnosis. Muscle biopsy remains an important requirement in the diagnosis of mitochondrial disease and in establishing the pathogenicity of novel mtDNA variants.

Development of an Antisense Oligonucleotide-Mediated Exon Skipping Therapeutic Strategy for Mucolipidosis II: Validation at RNA Level.

Lysosomal storage disorders (LSDs) are a group of rare inherited metabolic diseases caused by the malfunction of the lysosomal system, which results in the accumulation of undergraded substrates inside the lysosomes and leads to severe and progressive pathology. Despite there currently being a broad understanding of the molecular defects behind LSDs, curative therapies have been approved for only few of these diseases, whereas existing treatments are still mostly symptomatic with several limitations. Mucolipidosis type II alpha/beta (ML II) is one of most severe LSDs, which is caused by the total deficiency of the GlcNAc-1-phosphotransferase, a key enzyme for the formation of specific targeting signals on lysosomal hydrolases to lysosomes. GlcNAc-1-phosphotransferase is a multimeric enzyme complex encoded by two genes: GNPTAB and GNPTG. One of the most frequent ML II causal mutation is a dinucleotide deletion on exon 19 of GNPTAB (c.3503_3504del) that leads to the generation of a truncated protein, loss of GlcNAc-1-phosphotransferase activity, and missorting of multiple lysosomal enzymes. Presently, there is no therapy available for ML II. In this study, we explored the possibility of an innovative therapeutic strategy for ML II based on the use of antisense oligonucleotides (AOs) capable to induce the skipping of GNPTAB exon 19 harboring the most common disease-causing mutation, c.3503_3504del. The approach confirmed the ability of specific AOs for RNA splicing modulation, thus paving the way for future studies on the therapeutic potential of this strategy.

A Dinucleotide Deletion in the CD24 Gene Is a Potential Risk Factor for Colorectal Cancer

CD24 is a sialoglycoprotein anchored to the cell surface via glycosylphosphatidylinositol and is involved in intracellular signaling processes. It plays an important role in the early stages of the multistep process of colorectal carcinogenesis. Several single nucleotide polymorphisms in the CD24 gene are reported to exert a diverse effect on cancer risk. We aimed to elucidate whether CD24 TG/del genetic variants are associated with susceptibility to colorectal cancer (CRC). The study included 179 subjects, 36 with CRC (prior to surgery) and 143 healthy control subjects. Deoxyribonucleic acid was purified from peripheral blood leukocytes, and by using restriction fragment length polymorphism analysis, the CD24 gene was genotyped for the specific genetic variant, TG deletion. Additionally, CD24 protein expression levels were determined by Western blotting analysis. The incidence of the TG/del was higher among the CRC patients compared with healthy controls, 14% and 10%, respectively (P = .54). CD24 protein levels were significantly higher among CRC patients. There were no significant differences in CD24 expression between CRC patients at different stages of the disease or between patients who carry the mutation and those who did not. CD24 genetic variant might be of clinical value for risk assessment as part of cancer prevention programs. Further study on larger populations is needed to validate the importance of this dinucleotide deletion in CRC development. Overexpression of CD24 protein occurs early along the multistep process of CRC carcinogenesis, and a simple blood sample based on CD24 expression on peripheral blood leukocytes can contribute to early diagnosis.

Genome-wide mutagenesis resulting from topoisomerase 1-processing of unrepaired ribonucleotides in DNA

Ribonucleotides are the most common non-canonical nucleotides incorporated into DNA during replication, and their processing leads to mutations and genome instability. Yeast mutation reporter systems demonstrate that 2–5 base pair deletions (Δ2–5bp) in repetitive DNA are a signature of unrepaired ribonucleotides, and that these events are initiated by topoisomerase 1 (Top1) cleavage. However, a detailed understanding of the frequency and locations of ribonucleotide-dependent mutational events across the genome has been lacking. Here we present the results of genome-wide mutational analysis of yeast strains deficient in Ribonucleotide Excision Repair (RER). We identified mutations that accumulated over thousands of generations in strains expressing either wild-type or variant replicase alleles (M644G Pol ε, L612M Pol δ, L868M Pol α) that confer increased ribonucleotide incorporation into DNA. Using a custom-designed mutation-calling pipeline called muver (for mutationes verificatae), we observe a number of surprising mutagenic features. This includes a 24-fold preferential elevation of AG and AC relative to AT dinucleotide deletions in the absence of RER, suggesting specificity for Top1-initiated deletion mutagenesis. Moreover, deletion rates in di- and trinucleotide repeat tracts increase exponentially with tract length. Consistent with biochemical and reporter gene mutational analysis, these deletions are no longer observed upon deletion of TOP1. Taken together, results from these analyses demonstrate the global impact of genomic ribonucleotide processing by Top1 on genome integrity.

Adduct Fluorescence as a Tool to Decipher Sequence Impact on Frameshift Mutations Mediated by a C-Linked C8-Biphenyl-Guanine Lesion.

Aromatic chemicals can undergo metabolic activation to afford electrophilic species that react at the C8-site of 2'-deoxyguanosine (dG) to generate bulky C8-dG adducts as a basis of initiating carcinogenesis. These DNA lesions have served as models to understand the mechanism of frameshift mutagenesis, especially within CG-dinucleotide repeat sequences, such as NarI (5'-GGCXCC-3', where X = C8-dG adduct), however there is still limited capacity to predict the likelihood of mutation arising within particular contexts, and hence chemistry-based strategies are needed for probing relationships between nucleic acid sequence and structure with replication errors. In the NarI sequence, certain C8-dG adducts may trigger in the course of DNA synthesis the formation of a slipped mutagenic intermediate (SMI) that contains a two nucleotide (XC) bulge in the template strand that can form upstream of the polymerase active site. This distortion facilitates polymerization but affords a GC dinucleotide deletion product (-2 frameshift mutation). In the current study, incorporating the fluorescent C-linked 4-fluorobiphenyl-dG (FBP-dG) adduct into two 22-mer templates containing CG-dinucleotide repeats ( NarI: 3'-CXCGGC-5' and CG3: 3'-CXCGCG-5', X = FBP-dG) and performing primer extension reactions using DNA polymerase I, Klenow fragment exo- (Kf-) revealed a dramatic sequence-based difference in polymerase bypass efficiency. Primer extension past FBP-dG within the NarI sequence was strongly blocked, whereas Kf- extended the primer past FBP-dG within a CG3 template to afford a full-length product and the GC dinucleotide deletion. To model the nucleotide insertion steps in the fully paired (FP) versus the slipped mutagenic (SM) translesion pathways, adducted template:primer duplexes were constructed and characterized by UV thermal denaturation and fluorescence spectroscopy. The emission intensity of the FBP-dG lesion exhibits sensitivity to SMI formation (turn-on) versus a FP duplex (turn-off), permitting insight into adduct base-pairing within the template:primer duplexes. This fluorescence sensitivity provides a rationale for sequence impact on -2 frameshift mutations mediated by the C-linked FBP-dG lesion.

Novel Diagnostic Tool for p47phox-Deficient Chronic Granulomatous Disease Patient and Carrier Detection

Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by mutations of the phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Autosomal recessive p47phox-deficient CGD (p47phox CGD) is the second most frequent form of the disease in western countries, and more than 94% of patients have a disease-causing dinucleotide deletion (ΔGT) in the neutrophil cytosolic factor 1 (NCF1) gene. The ΔGT mutation is most likely transferred onto the NCF1 from one of its two pseudogenes co-localized on the same chromosome. The presence of NCF1 pseudogenes in healthy individuals makes the genetic diagnostics of ΔGT p47phox CGD challenging, as it requires the distinction between ΔGT in NCF1 and in the two pseudogenes. We have developed a diagnostic tool for the identification of p47phox CGD based on PCR co-amplification of NCF1 and its pseudogenes, followed by band intensity quantification of restriction fragment length polymorphism products. The single-day, reliable p47phox CGD diagnostics allow for robust discrimination of homozygous ΔGT p47phox CGD patients from heterozygous carriers and healthy individuals, as well as for monitoring gene therapy efficacy.

Key Value of RNA Analysis of MYBPC3 Splice-Site Variants in Hypertrophic Cardiomyopathy.

MYBPC3 splicing errors are a common cause of hypertrophic cardiomyopathy (HCM). Variants affecting essential splice-site dinucleotides inhibit splicing, whereas the impact of variants at conserved flanking nucleotides is less clear. We evaluated the contribution of MYBPC3 splice-site variants in a large cohort of patients with HCM and assessed the impact on splicing with RNA analysis. Patients attending a specialized multidisciplinary clinic, with a clinical diagnosis of HCM and genetic testing of at least 46 cardiomyopathy-associated genes, were included. Patients with variants in MYBPC3 splice sites with in silico-predicted effects on splicing were selected. RNA was extracted from fresh venous blood or paraffin-embedded myocardial tissue of the patients, amplified, and sequenced. Variants were classified for pathogenicity using the American College of Medical Genetics and Genomics guidelines. We found 29 rare MYBPC3 splice-site variants in 56 of 557 (10%) unrelated HCM probands. Three variants were not predicted to alter RNA splicing, and 13 essential splice dinucleotide, nonsense, and short insertion or deletion variants were not further assessed. RNA analysis was performed on 9 variants (c.654+5G>C, c.772G>A, c.821+3G>T, c.927-9G>A, c.1090G>A, c.1624G>A, c.1624+4A>T, c.3190+5G>A, and c.3491-3C>G), and RNA splicing errors were confirmed for 7. Four variants in 4 families resulted in clinically meaningful reclassifications. After RNA analysis, 4 of 56 (7%) families with MYBPC3 splice-site variants were reclassified from uncertain clinical significance to likely pathogenic. RNA analysis of splice-site variants can assist in understanding pathogenicity and increase the diagnostic yield of genetic testing in HCM.

The BRCA1 and BRCA2 Genes in Early-Onset Breast Cancer Patients.

Approximately 5-10% of breast cancers are attributable to genetic susceptibility. Mutations in the BRCA1 and BRCA2 genes are the best known genetic factors to date. The goal of this study was to determine the structure and distribution of haplotypes of the BRCA1 and BRCA2 genes in early-onset breast cancer patients. We enrolled 70 patients diagnosed with early-onset breast cancer. A total of 21 SNPs (11 on BRCA1 and 10 on BRCA2) and 1 dinucleotide deletion on BRCA1 were genotyped using nested allele-specific PCR methods. Linkage disequilibrium (LD) analysis was conducted, and haplotypes were deduced from the genotype data. Two tightly linked LD blocks were observed on each of the BRCA1 and BRCA2 genes. Variant-free haplotypes (TAT-AG for BRCA1 and ATA-AAT for BRCA2) were observed at a frequency of more than 50% on each gene along with variable frequencies of derived haplotypes. The variant 3'-subhaplotype CGC displayed strong LD with 5'-subhaplotypes GA, AA, and GG on BRCA1 gene. Haplotypes ATA-AGT, ATC-AAT, and ATA-AAC were the variant haplotypes frequent on BRCA2 gene. Although the clinical significance of these derived haplotypes has not yet been established, it is expected that some of these haplotypes, especially the less frequent subhaplotypes, eventually will be shown to be indicative of a predisposition to early-onset breast cancer.

The frequency of inactive sucrase-isomaltase variant in indigenous populations of Northeast Asia

The AG dinucleotide deletion at the rs781470490 locus of the sucrase-isomaltase gene (SI gene), common in the Eskimo people of Canada and Greenland, was screened in the indigenous populations of Northeast Asia (among Chukchi, Koryaks, Evens, and Evenks). Deletion was detected at the frequency of 14.3% (95% confidence interval (CI): 4.7–33.6%) in Chukchi, 7.3% (95% CI: 3.4–14.3%) in Koryaks, and 3.5% (95% CI: 1.3–8.5%) in Evens. Since the deletion at the rs781470490 locus leads to premature termination of the sucrase-isomaltase synthesis and, accordingly, to the appearance of an inactive form of the enzyme, it is suggested that its wide prevalence in populations of the Far North of America and Asia is associated with adaptation to an Arctic diet deficient in sucrose.

Exceptional Chemotherapy Response in Metastatic Colorectal Cancer Associated With Hyper-Indel-Hypermutated Cancer Genome and Comutation of POLD1 and MLH1.

A73-year-old woman with metastatic colon cancer experienced a complete response to chemotherapy with dose-intensified irinotecan that has been durable for 5 years. We sequenced her tumor and germ line DNA and looked for similar patterns in publicly available genomic data from patients with colorectal cancer. Tumor DNA was obtained from a biopsy before therapy, and germ line DNA was obtained from blood. Tumor and germline DNA were sequenced using a commercial panel with approximately 250 genes. Whole-genome amplification and exome sequencing were performed for POLE and POLD1. A POLD1 mutation was confirmed by Sanger sequencing. The somatic mutation and clinical annotation data files from the colon (n = 461) and rectal (n = 171) adenocarcinoma data sets were downloaded from The Cancer Genome Atlas data portal and analyzed for patterns of mutations and clinical outcomes in patients with POLE- and/or POLD1-mutated tumors. The pattern of alterations included APC biallelic inactivation and microsatellite instability high (MSI-H) phenotype, with somatic inactivation of MLH1 and hypermutation (estimated mutation rate > 200 per megabase). The extremely high mutation rate led us to investigate additional mechanisms for hypermutation, including loss of function of POLE. POLE was unaltered, but a related gene not typically associated with somatic mutation in colon cancer, POLD1, had a somatic mutation c.2171G>A[p.Gly724Glu]. Additionally, we noted that the high mutation rate was largely composed of dinucleotide deletions. A similar pattern of hypermutation (dinucleotide deletions, POLD1 mutations, MSI-H) was found in tumors from The Cancer Genome Atlas. POLD1 mutation with associated MSI-H and hyper-indel-hypermutated cancer genome characterizes a previously unrecognized variant of colon cancer that was found in this patient with an exceptional response to chemotherapy.

Homozygous Deletion Mutation of the FERMT1 Gene in a Chinese Patient with Kindler Syndrome.

Dear Editor: Kindler syndrome (KS, OMIM173650) is a rare autosomal recessive genodermatosis characterized by trauma-induced skin blistering, cutaneous atrophy, progressive poikiloderma and photosensitivity1. KS is caused by mutation in the FERMT1 gene1. This gene encodes kindlin-1, which regulates not only keratinocyte adhesion through β1-class integrins but also proliferation and differentiation of cutaneous stem cells by promoting αvβ6 integrin–mediated transforming growth factor-β activation and inhibiting Wnt ligand expression2. A 12-year-old Chinese boy presented with recurrent blistering after mild trauma from the age of 1 year. There is no family history. Physical examination revealed generalized skin atrophy with some scattered resolving blisters on the extremities and poikiloderma on the face and neck (Fig. 1). He has also suffered from pruritus and photosensitivity. A skin biopsy from non-blistered area on his leg revealed hyperkeratosis and epidermal atrophy (Fig. 2A). Immunofluorescence (IF) mapping of a blister showed that keratins 5 and 14 were localized at the roof of the blister while laminin 5 and type IV collagen were localized at the base (Fig. 2B, C). Electron microscopic findings of non-blistered skin revealed widening of the lamina lucida and reduplication of the lamina densa (Fig. 2D). DNA extracted from the patient's blood was processed for direct nucleotide sequencing and we detected a homozygous dinucleotide deletion mutation at c.994_995delCA in exon 8. The mutation causes frameshift and a premature stop codon (Fig. 2E). Based on these results, we diagnosed the patient with KS. Fig. 1 Clinical features of the patient. (A, B) Poikiloderma on the face and neck. (C) Atrophic change on the dorsal aspect of hand. (D) Skin atrophy with resolving blisters on the leg. Fig. 2 (A) Histological examination. Hyperkeratosis and epidermal atrophy (H&E, ×400). (B, C) Immunofluorescence mapping shows separation between keratin 5 (KRT5) and type IV collagen (COL4). KRT5 is localized at the roof of the blister (B) while ... KS usually manifests at birth with trauma-induced skin blistering that is more prominent on the extremities and tends to regress with age3. However, as patients age, skin atrophy localized to the dorsal aspects of the hands and feet become generalized and progressive skin poikiloderma manifests. Most patients also show photosensitivity3. Although our patient had no history of mucosal lesions, mucosal involvement is common1. Histopathology and IF mapping of a blister are not informative for diagnosis because the plane of cleavage can be variable at the level of the basement membrane zone1. Transmission electron microscopy of the non-blistered skin shows extensive reduplication of the lamina densa which is a characteristic diagnostic feature of KS4. The diagnostic golden standard is sequencing of the FERMT1 gene1. To date, more than 60 FERMT1 gene mutations and 150 KS patients have been reported and one Chinese and 4 Japanese patients have been reported in Eastern Asia5 (https://grenada.lumc.nl/LOVD2/mendelian_genes/home.php?select_db=FERMT1). Most mutations cause premature termination of translation, therefore loss of kindlin-1 function and most patients are related with consanguineous marriages3. Recently, Youssefian et al.4 first reported the c.994_995delCA mutation in a 19-year-old Iranian female patient. Interestingly, that patient had no mucosal involvement except dental plaques. This may imply an association between the mutation and mucosal-sparing features. Has et al.3 suggested genotype-phenotype correlations in KS because some patients with a missense mutation or in-frame deletion demonstrate a mild phenotype. However, only two cases including ours have been reported to date and our patient might be too young to have developed mucosal lesions. Further studies are needed to clarify this association.

Codon expansion and systematic transcriptional deletions produce tetra-, pentacoded mitochondrial peptides

Genes include occasionally isolated codons with a fourth (and fifth) silent nucleotide(s). Assuming tetracodons, translated hypothetical peptides align with regular GenBank proteins; predicted tetracodons coevolve with predicted tRNAs with expanded anticodons in each mammal, Drosophila and Lepidosauria mitogenomes, GC contents and with lepidosaurian body temperatures, suggesting that expanded codons are an adaptation of translation to high temperature. Hypothetically, continuous stretches of tetra- and pentacodons code for peptides. Both systematic nucleotide deletions during transcription, and translation by tRNAs with expanded anticodons could produce these peptides. Reanalyses of human nanoLc mass spectrometry peptidome data detect numerous tetra- and pentapeptides translated from the human mitogenome. These map preferentially on (BLAST-detected) human RNAs matching the human mitogenome, assuming systematic mono- and dinucleotide deletions after each third nucleotide (delRNAs). Translation by expanded anticodons is incompatible with silent nucleotides in the midst rather than at codon 3′ extremity. More than 1/3 of detected tetra- and pentapeptides assume silent positions at codon extremity, suggesting that both mechanisms, regular translation of delRNAs and translation of regular RNAs by expanded anticodons, produce this peptide subgroup. Results show that systematically deleting polymerization occurs, and confirm serial translation of expanded codons. Non-canonical transcriptions and translations considerably expand the coding potential of DNA and RNA sequences.

Insulin Growth Factor 1 Receptor Expression Is Associated with NOTCH1 Mutation, Trisomy 12 and Aggressive Clinical Course in Chronic Lymphocytic Leukemia

Insulin growth factor 1 receptor (IGF1R) is emerging as an important gene in many solid and hematological cancers and its over expression has been reported to be associated with aggressive disease and pharmacologic resistance. Specifically, the IGF1R-IGF1-2 interaction was recently described to be involved in the constitutive activation of many important cell signaling such as NOTCH1 and PI3K/Akt pathways that play a key role in many solid and hematological cancers. In this study we performed a clinical and biological investigations about the role of IGF1R expression in a large and representative prospective series of chronic lymphocytic leukemia (CLL) in Binet A clinical stage enrolled in observation O-CLL1 protocol (clinicaltrial.gov identifier NCT00917540). Total RNA extraction, preparation of DNA single-stranded sense target, and hybridization to gene expression profiling arrays were carried out according to manufacturer’s protocols in 217 CLL patients enrolled in the multicentre O-CLL1 protocol. Gene expression data has been deposited in the National Centre for Biotechnology Information’s Gene Expression Omnibus database http://www.ncbi.nlm.mih.gov/geo and are accessible through series accession number GSE51529. High IGF1R expression was significantly associated with IGHV unmutated (IGHV-UM) status (p<0.0001), high ZAP-70 and CD38 expression (p<0.0001) and unfavorable cytogenetic deletion [i.e. del(11)(q23) and del(17)(p13)], particularly with del(11)(q23) (p=0.03). Cases with del(13)(q14) as single lesion were characterized by the lowest IGF1R gene levels. On the contrary, among the most common cytogenetic aberrations, trisomy 12 showed stronger IGF1R expression compared with the other patients (p<0.0001) and this association was independent from IGHV mutational status. Patients with stereotyped HCDR3 sequences showed a greater IGF1R expression compared to not stereotyped HCDR3 (p=0.001) even if this can be related to the high frequency of IGHV-UM among stereotyped HCDR patients. Interestingly, subset #4 patients, who are known to exhibit an indolent clinical course and distinct biological profile, were also characterized by lower IGF1R expression compared to other M-IGHV and UM-IGHV patients. NOTCH1 c.7541_7542delCT mutation was investigated by next generation sequencing Roche 454 technology in 199 (92%) patients (Lionetti et al, BJH 2014). Globally, median depth of coverage was 1510x, ranging from 605 to 2842. Mutant allele frequency estimated by NGS ranged from 0.02% to 75% of total reads per sample. The presence of NOTCH1 mutation was confirmed by ASO-PCR and Sanger sequencing in all patients with allele burden higher > 0.7% (31; 15.5%) and 7% (19; 9.5%) respectively. We considered as mutated only the 31 patients in whom the presence of the dinucleotide deletion was confirmed by ARMS-PCR. Patients carrying NOTCH1 mutation were characterized by a greater IGF1R expression compared with wild type cases (p=0.002). In addition high IGF1R expression was not significantly different comparing patients with low and high NOTCH1 mutation burden. In order to avoid the bias represented by the strong association between NOTCH1 and trisomy 12, we compared the IGF1R expression between NOTCH1 mutated and wild type cases excluding trisomy 12, and confirmed the previous association (p=0.004). IGF1R expression represented a strong clinical prognostic factor in our CLL cohort: by Kaplan-Maier analysis we observed a significant time to first treatment stratification in all CLLs, in all IGHV-UM and in all IGHV-M patients (p<0.0001). Furthermore, IGF1R retained its significance in multivariate analysis with most important clinical and molecular prognostic factors (CD38 expression, unfavorable FISH and IGHV mutational status). Overall, our study shows the importance of IGF1R expression in CLL and its strong association with specific clinical and biological features, confirming the interest for the study of this gene as a potential prognostic factor and its possible role as a therapeutic target in a specific group of CLL patients carrying trisomy 12 and NOTCH1 mutations. DisclosuresNo relevant conflicts of interest to declare.