Multi-step Mutations Research Articles

Mutational analysis of 23 autosomal short tandem repeats based on trio paternity testing in the Korean population

This study aimed to estimate A-STR mutation rates in 2,317 Korean parent–child trios by examining 20 Combined DNA Index System (CODIS) core loci (D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, CSF1PO, FGA, TH01, TPOX, vWA, D1S1656, D2S441, D2S1338, D10S1248, D12S391, D19S433, and D22S1045) and three non-CODIS loci (Penta E, Penta D, and SE33). Locus-specific mutation rate estimates varied from 0.00 to 8.63 × 10-3 per generation, with an average mutation rate of 1.62 × 10-3 (95 % CI, 1.39–1.88 × 10-3). We also combined data from previous studies to obtain comprehensive genetic values for the Korean population, and the average mutation rate was 1.59 × 10-3 (95 % CI, 1.38–1.82 × 10-3). Single-step mutations (95.69 %) and double-step mutations (3.35 %) were observed in the mutation pattern analysis, and cases expected to have multi-step mutations (0.96 %) were also observed. Large-sized alleles exhibited more loss mutations than gain mutations, and paternal mutations (62.68 %) were more frequently observed than maternal mutations (19.62 %). The calculated values and features of the 23 A-STRs explored in this study are expected to play a crucial role in establishing criteria for forensic genetic interpretation.

Considerations on the application of a mutation model for Y-STR interpretation

If Y-STR profiling is to be more effective in criminal casework, the methods used to evaluate evidential weight require improvement. Many forensic scientists assign an evidential weight by estimating the number of times a Y-STR profile obtained from a questioned sample has been observed in YHRD datasets. More sophisticated models have been suggested but not yet implemented into routine casework, e.g. Andersen & Balding [1]. Mutation is inherent to STR meiosis (or inheritance) and is encountered in practice. We evaluated a mutation model that can be incorporated into a method for assigning evidential weight to Y-STR profiles, an essential part of bringing any method into practice. Since an important part of implementation to casework is communication, the article is written in an accessible format for practitioners as well as statisticians.The mutation component within the MUTEA model by Willems et al. [2] incorporates the potential for multistep mutations and a tendency for alleles to revert towards a central length, reflecting observed mutation data, e.g. [3]. We have estimated the parameters in this model and in a simplified symmetric version of this model, using sequence data from father/son pairs [4] and deep-rooted pedigrees [5]. Both datasets contain multistep mutations, which may have an effect on models based on simulations [1].We introduce Beta-Binomial and Beta-Geometric conjugate analyses for estimating rate and step parameters for the mutation models presented here, which require only summations and multiplications. We proved mathematically that the parameters can be estimated independently. We show the importance of reporting the variability of the parameters and not only a point estimate. The parameters can be easily incorporated into statistical models, and updated sequentially as more data becomes available. We recommend fuller publication of data to enable the development and evaluation of a wider range of mutation models.

Genetic inconsistency at the D6S1043 locus caused by microdeletion at 6q15.

In the practice of parentage testing, short tandem repeat (STR) genetic inconsistencies occasionally occur and are usually treated as genetic mutations. However, they arise for various reasons. To elucidate the reasons for their occurrence, this study investigates a typical trio. For the D6S1043 locus, the genotype of the biological mother comprised the heterozygous alleles "7,20"; that of the child, allele 20; and that of the alleged father, a heterozygous allele "11,13," revealing a 7-step mutation. Different kits were first used to verify the data. The locus map, primers, and core sequences were then analyzed. Ultimately, the STR and single nucleotide polymorphisms of 6q were tested to determine the microdeletion range. The results revealed that this was indeed a true trio, and the underlying cause of the genetic inconsistency at this locus was a microdeletion of approximately 0.74-1.78 Mb in 6q15. Overall, genetic inconsistencies detected during practical work, and particularly rare multi-step mutations, cannot be directly identified as STR mutations. Different tools should be used to examine the causes of genetic inconsistencies from various perspectives and improve the effectiveness of genetic evidence.

The sequence of the repetitive motif influences the frequency of multistep mutations in Short Tandem Repeats

Microsatellites, or Short Tandem Repeats (STRs), are subject to frequent length mutations that involve the loss or gain of an integer number of repeats. This work aimed to investigate the correlation between STRs’ specific repetitive motif composition and mutational dynamics, specifically the occurrence of single- or multistep mutations. Allelic transmission data, comprising 323,818 allele transfers and 1,297 mutations, were gathered for 35 Y-chromosomal STRs with simple structure. Six structure groups were established: ATT, CTT, TCTA/GATA, GAAA/CTTT, CTTTT, and AGAGAT, according to the repetitive motif present in the DNA leading strand of the markers. Results show that the occurrence of multistep mutations varies significantly among groups of markers defined by the repetitive motif. The group of markers with the highest frequency of multistep mutations was the one with repetitive motif CTTTT (25% of the detected mutations) and the lowest frequency corresponding to the group with repetitive motifs TCTA/GATA (0.93%). Statistically significant differences (α = 0.05) were found between groups with repetitive motifs with different lengths, as is the case of TCTA/GATA and ATT (p = 0.0168), CTT (p < 0.0001) and CTTTT (p < 0.0001), as well as between GAAA/CTTT and CTTTT (p = 0.0102). The same occurred between the two tetrameric groups GAAA/CTTT and TCTA/GATA (p < 0.0001) – the first showing 5.7 times more multistep mutations than the second. When considering the number of repeats of the mutated paternal alleles, statistically significant differences were found for alleles with 10 or 12 repeats, between GATA and ATT structure groups. These results, which demonstrate the heterogeneity of mutational dynamics across repeat motifs, have implications in the fields of population genetics, epidemiology, or phylogeography, and whenever STR mutation models are used in evolutionary studies in general.

Verification of a loss of heterozygosity at the D8S1179 locus in a paternity case by the MiSeq FGx system.

Capillary electrophoresis is widely used to study short tandem repeats (STRs) in forensic genetics. However, next-generation sequencing platforms have become a new strategy for forensic DNA typing. In this study, we report a false four-step STR mutation between an alleged father (AF) and child in a paternity case. A total of 23 autosomal STR loci were evaluated using the Huaxia™ Platinum and Goldeneye™ 20A kits, revealing a single mismatch in D8S1179 between the AF (10/10) and the male child (14/14). Additional Y-STR typing of the AF and child was performed, and the results were consistent with those based on 27 Y-STR loci. To further confirm the experimental results, we sequenced the individuals using the MiSeq FGx system and detected 10/15 unbalanced alleles in the D8S1179 locus of the AF and 14/15 unbalanced alleles in the D8S1179 locus of the child. Sanger sequencing revealed that both the AF and child had the C→G point mutation in the primer binding region of D8S1179 resulting in allelic dropout. Therefore, the verification of STR typing by different sequencing systems is helpful for the interpretation of results in cases of multistep STR mutations.

How frequently are Autosomal and X-STRs multistep mutations perceived as single-step?

Short tandem repeats (STRs) incur in length mutations that involve the loss or gain of repeats. STR mutation rates are usually estimated considering the rates of observed Mendelian incompatibilities in one generation familial configurations. When considering multistep mutations, for the autosomal and X-chromosomal modes of genetic transmission, underestimations are inevitable when using this approach (MIA), due to the occurrence of mutational events deceptively perceived as involving fewer steps. The rate of this occurrence depends on the mode of genetic transmission considered, the parental origin of the mutation, the type of familial configuration considered, and the genotypic background of the progenitor(s). MIA biases were weighted and compared for the diploid and haplodiploid modes of transmission, using familial genotypic configurations (parent(s)-child duos and trios) generated resorting to Python™ and real population databases from Norway, Somalia, and Spain for 10 Aut-STRs and Argentina, Eastern Asia, and Northern Europe for 12 X-STRs. One two- or one three-step mutation was simulated in each of the 1,000,000 familial configurations. The frequency with which mutations could be interpreted as involving fewer steps, when the most parsimonious reasoning is employed, was computed. Results showed that the magnitude and type of biases depend on the type of familial data and the genetic mode of transmission considered, being higher in duos than in trios, both in autosomes and the X chromosome. Indeed, whether X- or Aut-STRs are analyzed, trios generally provide better estimates and should be favored over duos. The pooling of the two types of data is not advised. The greater the number of steps involved in the mutation, the worst the estimates obtained. In X-chromosomal analyzes, trios with a paternal mutation presented the best estimates and mother-daughter duos the worst; mother-son duos showed similar estimates to trios when a maternal mutation was considered.

Cytotoxic, genotoxic, and toxicogenomic effects of heated tobacco products and cigarette smoke in human primary keratinocytes.

INTRODUCTIONHeated tobacco products (HTPs) appear to be less harmful to health than conventional cigarettes (CCs). However, limited analytical data are available to support this claim. This study aimed to compare the cytotoxic, genotoxic, and toxicogenomic effects of HTPs and CCs in carcinogenesis via multistep gene mutations in the oral mucosal cells.METHODSCigarette smoke extract (CSE) was obtained from HTPs and CCs. Primary human oral keratinocytes (HOKs) were treated with 5% and 20% CSE from HTPs and CCs. Cell survival rate assays were performed after 6, 12, and 24 h. After 6 h, DNA double-strand breaks (DSBs) were evaluated using anti-γH2AX antibodies with immunohistochemistry. mRNAs expressions of mediator of DNA damage checkpoint 1 (MDC1) and ataxia telangiectasia and Rad3-related protein (ATR), were analyzed. Expressions of miR-22 and miR-185 were analyzed because miR-22 targets MDC1 and miR-185, ATR.RESULTSThe HOKs had equivalent survival rates after exposure to the same concentrations of CSE from CCs and HTPs. HTPs increased foci formation of γH2AX in HOKs, as did CCs (without CSE vs 20% HTP, p<0.05; without CSE vs 20% CC, p<0.05). Expressions of MDC1 and ATR decreased in cells exposed to CSE from CCs and HTPs (MDC1: without CSE vs 20% HTP, p<0.05; without CSE vs 20% CC, p<0.05; ATR: without CSE vs 20% HTP, p<0.05; without CSE vs 20% CC, p<0.05). Expressions of miR-22 and miR-185 were not significantly increased when exposed to CSE from CCs or HTPs.CONCLUSIONSHTPs and CCs had similar cytotoxic effects. HTPs are genotoxic, can cause DSBs, and have toxicogenomic damage because they inhibit the MDC1 and ATR-CHK1 DNA repair pathways in the oral mucosa. The miRNA-mRNA axis was not related to these inhibitions.

Diagnostic and prognostic significance of miRNA-511 and miRNA-16 expressions in adult patients with B-acute lymphoblastic leukemia

Background Acute lymphoblastic leukemia (ALL) is a neoplastic disease that results from multistep somatic mutations in a single lymphoid progenitor cell. MicroRNAs (miRNAs) are critical regulators of gene expression, tumor suppression, and oncogenesis. Aim To evaluate miRNA-511 and miRNA-16 expression in Egyptian adult patients with B-ALL. Patients and methods A total of 37 newly diagnosed adult patients with B-ALL admitted to Alexandria Main University Hospital in 2019 were included. Complete blood count, bone marrow aspiration, immunophenotyping, BCR-ABL testing, karyotyping, miRNA extraction using miRNeasy Mini followed by cDNA synthesis AQ6 (RQ-PCR combines cDNA synthesis from RNA templates using miScript II RT kit), and finally real-time PCR for miRNA-511 and miRNA-16 expression were among the investigations that were conducted. Results Mean age of patients with ALL was 30.65 ± 10.39 years, with male to female ratio of 1.4 : 1. Cytogenetic findings showed that only three patients had favorable risk, and the rest were either intermediate risk (19) or high risk (15). Among the high-risk group, there were 11 patients with Philadelphia chromosome (BCR-ABL 190) positive. Regarding the expression of miRNAs, most patients showed overexpression of both miRNA-16 and miRNA-511. MiRNA-511 was overexpressed in 81.1% (30) patients; among these patients, 43.3% (13) had adverse cytogenetic findings. MiRNA-16 was overexpressed in 70.3% (26) of patients, and half of them (13) had adverse cytogenetic findings. receiver operating characteristic curves showed diagnostic significance in B-ALL for miRNA-16, with sensitivity of 75.7% and specificity of 80%, and for miRNA-511, sensitivity was 89.2% and specificity was 90% (P&lt;0.05). Conclusion MiRNA-16 and miRNA-511 were significantly overexpressed in adult patients with B-ALL. They have a role in diagnosis but a weak role in patient prognosis.

ALL-443: Diagnostic and Prognostic Significance of miRNA-511 and miRNA-16 Expression in Adult B-Acute Lymphoblastic Leukemia Patients

Acute lymphoblastic leukemia (ALL) is a neoplastic disease that results from multistep somatic mutations in a single lymphoid progenitor cell. MicroRNAs (miRNAs) are critical regulators of gene expression, tumor suppression, and oncogenesis. <h3>Aim:</h3> To evaluate miRNA-511 and miRNA-16 expression in Egyptian adult B-ALL. <h3>Subjects and Methods:</h3> 37 newly diagnosed adult B-ALL patients admitted to Alexandria Main University Hospital in 2019. Investigations included CBC, bone marrow aspiration, immunophenotyping, BCR-ABL testing, karyotyping, miRNA extraction using miRNeasy Mini Kit, qRT-PCR combined with cDNA synthesis from RNA templates using miScript II RT Kit, followed by real-time PCR for miR-511 and miR-16 expression analysis. <h3>Results:</h3> Mean age of ALL patients was 30.65 ± 10.39 years, with a male to female ratio of 1.4:1. Cytogenetic findings showed only 3 patients had favorable risk, and the rest were either intermediate risk (19) or high risk (15). Among the high-risk group, 11 patients were Philadelphia (BCR-ABL 190)-positive. Regarding the expression of the miRNAs, most patients showed the overexpression of both miR-16 and miR-511. MiR-511 was overexpressed in 81.1% (30) of patients; among these patients, 43.3% (13) had adverse cytogenetic findings. MiR-16 was overexpressed in 70.3% (26) of patients; half of them (13) had adverse cytogenetic findings. ROC curves showed the diagnostic significance in B-ALL of miR-16, with a sensitivity of 75.7% and a specificity of 80%; for miR-511, the sensitivity was 89.2%, and specificity was 90%, (p<0.05). Among the studied group, 9 (24%) patients developed COVID-19 viral infection, and of those, 3 (33%) patients died, comprising 21% of all deaths. The three patients were either refractory or relapsed and treated mostly as palliative cases. <h3>Conclusion:</h3> MiR-16 and miR-511 were significantly overexpressed in adult B-ALL. They have a role in diagnosis but a weak role in patients' prognosis.

Chronological genome and single-cell transcriptome integration characterizes the evolutionary process of adult T cell leukemia-lymphoma

Subclonal genetic heterogeneity and their diverse gene expression impose serious problems in understanding the behavior of cancers and contemplating therapeutic strategies. Here we develop and utilize a capture-based sequencing panel, which covers host hotspot genes and the full-length genome of human T-cell leukemia virus type-1 (HTLV-1), to investigate the clonal architecture of adult T-cell leukemia-lymphoma (ATL). For chronologically collected specimens from patients with ATL or pre-onset individuals, we integrate deep DNA sequencing and single-cell RNA sequencing to detect the somatic mutations and virus directly and characterize the transcriptional readouts in respective subclones. Characteristic genomic and transcriptomic patterns are associated with subclonal expansion and switches during the clinical timeline. Multistep mutations in the T-cell receptor (TCR), STAT3, and NOTCH pathways establish clone-specific transcriptomic abnormalities and further accelerate their proliferative potential to develop highly malignant clones, leading to disease onset and progression. Early detection and characterization of newly expanded subclones through the integrative analytical platform will be valuable for the development of an in-depth understanding of this disease.

Cell-Free DNA Genotyping Analysis in Diffuse Large B-Cell Lymphoma; The Correlation between TP53 Gene Mutation and Cereblon Gene Mutation

Introduction: Recently cell-free DNA (cfDNA) genotyping analysis has been utilized as a non-invasive procedure for detecting tumor specific genes or clarifying gene mutations instead of tumor sample biopsies. In diffuse large B-cell lymphoma (DLBCL), cfDNA analysis has been proceeding and the relationships between somatic gene mutation and the disease status have been considered. We retrospectively analyzed somatic gene mutations in DLBCL and examined the correlation between mutant genes and clinical outcomes by using serum cfDNA samples. Patients and Methods: 50 patients newly diagnosed with DLBCL (including 4 patients mixed with follicular components) in our institute between March 2016 and March 2017 were enrolled in this study. All cases were sub-divided into germinal center B-cell (GCB) or non-germinal center B-cell (NGC) through immuno-staining, as CD10, bcl-6, and MUM-1 as Hans algorithm with biopsied tumor specimen. The stage at diagnosis and evaluation of treatment effects were assessed by PET-CT scan and bone marrow aspiration. All patients were treated by R-CHOP-like regimens with or without radiation. The serum samples from the patients were obtained before treatment and the cfDNA was extracted from the serum using a Maxwell RSC cfDNA Plasma kit. Using genomic DNA derived from cfDNA, multiplex polymerase chain reaction (PCR) was performed, and a sequence library was then constructed with an Ion Custom Amplicon panel. The panel for the sequence library was designed using an Ion AmpliSeq DesignerTM. 121 targeted genes were selected. The genomes were sequenced using the Ion ProtonTM System. We compared the validation of the sequence results dependent on the characteristics and prognoses of the patients. Connections between each gene mutation and clinical features were assessed using Fisher's exact test. Mann-Whitney U test was used for evaluating factors associated with the number of gene mutations. Survivals were estimated by the Kaplan-Meier method and differences were compared using the log-rank test. This protocol was approved by the institutional review board and the Genomic Review Board of the Japanese Foundation for Cancer Research. Results: Median age was 65.4 years old (range 33-83), 27 patients were male and 23 were female. The stage of Ⅲ or Ⅳ were 17 (34%), IPI high or high intermediate were 17 (34%), and 20 (40%) had LDH&gt;ULN. 27 patients (54%) were GCB and 23 (46%) were NGC. With a median follow up was 26.6 months (range 6-35). Factors affecting both shorter overall survival (OS) and progression-free survival (PFS) were LDH&gt;ULN (P=0.037 and P&lt;0.001), NGC (P=0.012 and P=0.002), IPI≧3 (P=0.023 and P&lt;0.001) in univariate analysis. According to the cfDNA gene mutation analysis, the mutations of HDAC4 (88%), KDM3B (86%), HDAC6 (86%), KDM1A (84%), CREBBP (80%), JMJD1C (78%), and EGR1 (62%) were frequently detected in all DLBCL patients. CREBBP, PCLO, and KDM5A tended to be relatively abundant in NGC (P=0.084, 0.087, and 0.085, respectively). No other mutations had deviation in either GCB or NGC and correlated with other prognostic factors. 7 patients had TP53 mutation, 6 were TP53 p.N178H and 1 was TP53 p.P58L. All cases with TP53 p.N178H mutation were accompanied by more than 27 types of other gene mutations. This was significantly abundant compared to patients without TP53 p.N178H (mean of 38 types vs 16 types, P&lt;0.001). Patients with 15 or more types of gene mutations tended toward poor PFS (2-year PFS of 89.5% vs 77.2%, P=0.092). Additionally, 7 patients had cereblon (CRBN) p.F101S mutation and 5 of 7 were concurrent with TP53 p.N178H mutation. Discussion and Conclusion: This study showed serum cfDNA could be used as an alternative resource for analysis by tumor specimen. Our results indicated that TP53 mutation occurred in latter period undergoing multistep genetic variation and it showed the tendency that multistep mutations were related to poor prognosis. Interestingly, relative rate in TP53 mutation accompanied by CRBN mutation was high. In some reports, it was described that deletion p53 caused drug resistance including immunomodulator which targets CRBN in multiple myeloma cases. Zijun Y et al mentioned CRBN expression correlated with favorable prognosis in DLBCL with wild type TP53. Further studies are warranted to confirm the relationships between TP53 and CRBN mutations and its prognosis of DLBCL. Disclosures Mishima: Chugai-Roche Pharmaceuticals Co.,Ltd.: Consultancy. Yokoyama:Chugai-Roche Pharmaceuticals Co.,Ltd.: Consultancy. Nishimura:Celgene K.K.: Honoraria; Chugai-Roche Pharmaceuticals Co.,Ltd.: Consultancy. Hatake:Takeda Pharmaceutical Co.,Ltd.: Honoraria; Celgene K.K.: Research Funding; Janssen Pharmaceutical K.K.: Research Funding. Terui:Bristol-Myers Squibb, Celgene, Janssen, Takeda, MSD, Eisai, Ono, and Chugai-Roche Pharmaceuticals Co.,Ltd.: Honoraria; Bristol-Myers Squibb K.K.: Research Funding.

Estimating Y-Str Mutation Rates and Tmrca Through Deep-Rooting Italian Pedigrees

In the population genomics era, the study of Y-chromosome variability is still of the greatest interest for several fields ranging from molecular anthropology to forensics and genetic genealogy. In particular, mutation rates of Y-chromosomal Short Tandem Repeats markers (Y-STRs) are key parameters for different interdisciplinary applications. Among them, testing the patrilineal relatedness between individuals and calculating their Time of Most Recent Common Ancestors (TMRCAs) are of the utmost importance. To provide new valuable estimates and to address these issues, we typed 47 Y-STRs (comprising Yfiler, PowerPlex23 and YfilerPlus loci, the recently defined Rapidly Mutating [RM] panel and 11 additional markers often used in genetic genealogical applications) in 135 individuals belonging to 66 deep-rooting paternal genealogies from Northern Italy. Our results confirmed that the genealogy approach is an effective way to obtain reliable Y-STR mutation rate estimates even with a limited number of samples. Moreover, they showed that the impact of multi-step mutations and backmutations is negligible within the temporal scale usually adopted by forensic and genetic genealogy analyses. We then detected a significant association between the number of mutations within genealogies and observed TMRCAs. Therefore, we compared observed and expected TMRCAs by implementing a Bayesian procedure originally designed by Walsh (2001) and showed that the method yields a good performance (up to 96.72%), especially when using the Infinite Alleles Model (IAM).

Application of synthetic lethality in the precise oncology

Cancer is a leading cause of death in both developed and developing countries. Most cancers occur as a result of somatically acquired alternations in the genomes of normal cells. The somatic alterations presented in a cancer genome can reach about ten thousands. However, a few such alterations that are able to drive initiation and progression of cancer are defined as “driver” mutations, while all other mutations are assumed to be randomly acquired and termed as “passenger” mutations. Driver mutations confer growth advantage on the cells carrying them and have been positively selected during the evolution of the cancer. Accumulating driver mutations in a cell genome likely result in multi-step mutations of encoded proteins or non-coding transcripts, which change their biological functions of the proteins and transcripts. Driver mutations potentially have been classified into two categories: “oncogenes” that are likely resulted from gain-of-function mutations and “tumor suppressor genes” that may be due to loss-of-function mutations. Together these two types of mutations drive cancer initiation and development. Targeting driver gene mutations have been successfully applied to the treatment of various cancers in humans. In this review, we introduce the concept of driver and passenger mutations, and theory of synthetic lethality (SL). Furthermore, we address the application of SL by targeting synthetic lethal partner of these “driver” genes and the clinical trial that can be designed based on the theory of SL. Synthetic lethal interactions arise when the perturbation of more than one genes simultaneously results in the loss of viability, whereas a deficiency in only one of these genes does not. SL might be extended to synthetic sickness, which is referred to the sickness but not lethality in cells resulting from mutations of those genes, and synthetic dosage lethality by targeting its partner to kill cells with overexpression of one gene. This theory has been applied to genetic studies to determine functional interactions among different genes for decades and has been exploited for the development of new genotype-selective anticancer agents in engineered human tumor cells. In precision oncology, the theory of SL provides greater promise in specifically targeting cancer cells with genetic mutations but not in normal cells. For example, targeting both of the breast-cancer susceptibility genes 1 and 2( BRCA1 and BRCA2 ) deficiencies dramatically sensitized cells to poly(ADP-ribose) polymerase (PARP) inhibitors due to associated defective homologous recombination, which leads to dysfunction of DNA repair in cancer cells. Moreover, oncogenic drivers such as RAS, P53 are not “druggable”, but recently these oncogenic drivers have been studied in successfully screening for their synthetic lethal partners or agents. If a synthetic lethal agent can be combined with conventionally chemotherapy, this will probably benefit cancer patients. Therefore, the application of the SL in cancer researches and clinical trials might represent one of the most important technological breakthrough in the treatment of cancer in the era of precision oncology.

Multistep microsatellite mutation at D18S51 locus in a parentage testing case

Mutations of short tandem repeats (STRs) loci might cause allelic mismatchs between the child and the parents, which entangled the forensic inference in paternity testing. Most of the reported microsatellite mutations are confined to single-step mutations, and multistep mutations were rarely reported and only account for a very limited number of STR mutation events in previously studied cases. Here we reported a paternity case with a mismatch in locus D18S51. The genotypes of the alleged father, the mother and the child in D18S51 locus were 14/23, 15/16, and 15/20, respectively. Examination of 38 autosomal STR loci revealed no mismatches, and the paternity index is above 1.58E+15. These results suggested that the alleged father is the biological father of the child that a rare three-steps mutation had occurred in the paternal allele of D18S51.

Contraction of fully expanded FMR1 alleles to the normal range: predisposing haplotype or rare events?

Fragile X syndrome (FXS), the most common cause of inherited intellectual disability, is due to the expansion over 200 CGGs and methylation of this polymorphic region, in the 5'-UTR (untranslated region) of FMR1 (Xq27.3). We have identified four FXS mosaic males: M1-(CGG)35/(CGG)>200; M2-(CGG)26/(CGG)>200; M3-(CGG)39/(CGG)>200; and M4-(CGG)18/(CGG)125/(CGG)>200. After genotyping their respective mothers, we suggested that normal alleles of these patients resulted from post-zygotic contractions of full expansions. The detection of these four rare independent cases led us to hypothesize the existence of a large-contraction predisposing haplotype in our population. Next, we questioned whether other normal pure CGGs would have arisen through similar contractions from fully expanded alleles. To address these questions, we identified stable single-nucleotide polymorphism (SNP) lineages and related short tandem repeat (STR) haplotypes (DXS998-DXS548-FRAXAC1-FRAXAC2) of the four mosaics, 123 unrelated FXS patients and 212 controls. An extended flanking haplotype (34-44-38-336) shared by mosaics from lineage A suggested a risk lineage-specific haplotype more prone to large contractions. Other normal pure FMR1 alleles from this SNP background also shared phylogenetically close STR haplotypes, although a single (CGG)exp>(CGG)24 contraction or the loss of AGG interruptions may explain their origin. In both scenarios, multistep FMR1 mutations involving the gain or loss of several CGGs seem to underlie the evolution of the repeat.

Theory and statistics of mutation rates: A mathematical framework reformulation for forensic applications

Mutation is a fundamental topic in forensic genetics since the calculation of kinship likelihoods depends on estimates of germinal mutation rates. Germinal mutation rates have been computed by simply proportioning the number of true parent(s)-child genotypic configurations inconsistent with Mendelian segregation. For STRs, current technology is based on PCR fragment size determination and germinal mutations are detected when parent-child do not share alleles’ length. For technical reasons, it is uncommon to obtain the sequence composition of STR alleles, and the identification of the allele that mutated is based on assumptions. It has been assumed that one-step are much more common than multi-step mutations. Whenever a genotypic parent-child configuration is compatible with Mendelian rules by a single-step mutation, this is the one assumed, despite other possible mutational events. Multi-step events are therefore evoked exclusively when a single-step cannot reconcile the observation with the true kinship, leading to an overestimation of single step mutation rates. For any mode of transmission other than purely haploid it is theoretically impossible to identify both the allele at “origin” (in parents) and at “destination” (in offspring). Therefore, a more sophisticated statistical framework than the simple proportioning is then required to properly evaluate mutation rates. Moreover, the possibility of the occurrence of silent alleles should be simultaneously modeled since it has to be considered whenever an apparently homozygous individual is involved.

Early Bone Marrow Response in Pediatric Acute Lymphoblstic Leukemia with Unfavourable and Favourable Presenting Features

Acute lymphoblastic leukemia (ALL) is a neoplastic disease that results from multistep somatic mutations and remains the most common malignancy in children. This study was conducted on 30 pediatric patients diagnosed as ALL and early bone marrow response on day 7 and day 14 was correlated in patients with or without risk factors. The prognostic factors found to have significance were initial total leukocyte (TLC) count < 50000, immunophenotype and early clearance of peripheral blast cells at day 7 with P value of < 0.05. The three risk group patient for leukemic induction failure (LIF) can be considered as high TLC, T cell immunophenotype and patients with persistence of circulating blast cells at day 7.

Tandem-Repeat Patterns and Mutation Rates in Microsatellites of the Nematode Model OrganismPristionchus pacificus

Modern evolutionary biology requires integrative approaches that combine life history, population structure, ecology, and development. The nematode Pristionchus pacificus has been established as a model system in which these aspects can be studied in one organism. P. pacificus has well-developed genetic, genomic, and transgenic tools and its ecologic association with scarab beetles is well described. A recent study provided first mutation rate estimates based on mitochondrial genome sequencing and mutation accumulation line experiments that help resolve rather ancient evolutionary branches. Here, we analyzed the tandem-repeat pattern and studied spontaneous mutation rates for microsatellite markers by using the previously generated mutation accumulation lines. We found that 0.59%–3.83% of the genome is composed of short tandem repeats. We developed 41 microsatellite markers, randomly chosen throughout the genome and analyzed them in 82 mutation accumulation lines after 142 generations. A total of 31 mutations were identified in these lines. There was a strong correlation between allele size and mutation rate in P. pacificus, similar to Caenorhabditis elegans. In contrast to C. elegans, however, there is no evidence for a bias toward multistep mutations. The mutation spectrum of microsatellite loci in P. pacificus shows more insertions than deletions, indicating a tendency toward lengthening, a process that might have contributed to the increase in genome size. The mutation rates obtained for individual microsatellite markers provide guidelines for divergence time estimates that can be applied in P. pacificus next-generation sequencing approaches of wild isolates.

Reliability of genetic bottleneck tests for detecting recent population declines

The identification of population bottlenecks is critical in conservation because populations that have experienced significant reductions in abundance are subject to a variety of genetic and demographic processes that can hasten extinction. Genetic bottleneck tests constitute an appealing and popular approach for determining if a population decline has occurred because they only require sampling at a single point in time, yet reflect demographic history over multiple generations. However, a review of the published literature indicates that, as typically applied, microsatellite-based bottleneck tests often do not detect bottlenecks in vertebrate populations known to have experienced declines. This observation was supported by simulations that revealed that bottleneck tests can have limited statistical power to detect bottlenecks largely as a result of limited sample sizes typically used in published studies. Moreover, commonly assumed values for mutation model parameters do not appear to encompass variation in microsatellite evolution observed in vertebrates and, on average, the proportion of multi-step mutations is underestimated by a factor of approximately two. As a result, bottleneck tests can have a higher probability of 'detecting' bottlenecks in stable populations than expected based on the nominal significance level. We provide recommendations that could add rigor to inferences drawn from future bottleneck tests and highlight new directions for the characterization of demographic history.

Structure-based characterization of canine–human chimeric uricases and its evolutionary implications

Uricase was lost in hominoids during primate evolution, but the inactivation mechanism remains controversial. To investigate the inactivation process of hominoid uricase, chimeric constructions between canine and human uricase were employed to screen the target regions that may contain labile or inactivated mutations in deduced human uricase. Four chimeric uricases were constructed and showed different enzymatic characteristics. Homology modeling, rational site-directed mutagenesis and DNA alignment were used to analyze the changes. Arg119 is conserved in functional mammalian uricases and its side-chains are crucial in maintaining the stability of the β-barrel core. A single CGT (Arg) to CAT (His) mutation at codon 119 that is shared by the human and great ape clade greatly reduces this stability and could cause the loss of uricase activity. We speculate that this missense mutation occurred first and inactivated the uricase protein in humans and great apes and that later the known nonsense mutation at codon 33 occurred and silenced the uricase gene. A single GTC (Val) to GCC (Ala) mutation at codon 296 in canine uricase is regarded as deleterious structural mutation, but such kinds of deleterious mutations have been widely accumulated in extant mammalian uricases. We speculate that a reduction in uricase activity has been an evolutionary tendency in mammals. Moreover, from structure-activity analysis of helix 2 in ancestral primate uricase, we suggest that before the inactivation of hominoid uricase, deleterious structural evolutionary changes had occurred in ancestral primates. The loss of hominoid uricase should be caused by progressive multistep mutations rather than a single mutation event.