Mutation rate analysis at 19 autosomal microsatellites

Previous studies have demonstrated that a large sample size is needed to reliably estimate population‐ and locus‐specific microsatellite mutation rates. Therefore, we conducted a long‐term collaboration study and performed a comprehensive analysis on the mutation characteristics of 19 autosomal short tandem repeat (STR) loci. The STR loci located on 15 of 22 autosomal chromosomes were analyzed in a total of 21 106 samples (11 468 parent–child meioses) in a Chinese population. This provided 217 892 allele transfers at 19 STR loci. An overall mutation rate of 1.20 × 10^?3 (95% CI, 1.06–1.36 × 10^?3) was observed in the populations across 18 of 19 STR loci, except for the TH01 locus with no mutation found. Most STR mutations (97.7%) were single‐step mutations, and only a few mutations (2.30%) comprised two and multiple steps. Interestingly, approximately 93% of mutation events occur in the male germline. The mutation ratios increased with the paternal age at child birth (r = 0.99, p<0.05), but not maternal age. Last, with the combination analysis of the data from the southern Chinese population, we drew a picture of 19 STR mutations in China. In conclusion, the data from this study will provide useful information in parentage testing, kinship analysis, and population genetics.     

Characterization of a highly variable short tandem repeat polymorphism at the D2S1242 locus

We report the evaluation of short tandem repeat (STR) locus D2S1242 (GDB ID G00-309-429) for forensic purposes, investigated by polymerase chain reaction (PCR) amplification and both native and denaturating polyacrylamide gel electrophoresis in 147 unrelated Austrians. No deviations from Hardy-Weinberg expectations were observed. The mean exclusion chance (MEC) was 0.669, the discriminating power (DP) was 0.947, and the observed heterozygosity rate was 0.856. An allelic ladder consisting of eight sequenced alleles (141-167 and 175 bp) was constructed. Sequence analysis revealed that the locus comprised two repeat motifs varying in number between alleles GAAA and GAAG. According to the number of tetranucleotide repeats the smallest allele was designated as 10 and the largest allele as 18.     

Rapid sizing of microsatellite alleles by gel electrophoresis on microfabricated channels: application to the D19S394 tetranucleotide repeat for cosegregation study of familial hypercholesterolemia.

This study evaluated the applicability of microchip electrophoresis to the sizing of microsatellites suitable to genetic, clinical and forensic applications. The evaluation was performed with the D19S394 tetranucleotide (AAAG) repeat characterized by a wide variation in the repeat number (1-17) and a short recombination distance from the low-density lipoprotein (LDL)-receptor gene that makes it suitable to cosegregation analysis of familial hypercholesterolemia (FH). The study was performed with 70 carriers of two LDL-receptor mutations common in northern Italy (i.e., the 4 bp insertion in exon 10 known as FH-Savona and the D200G missense mutation in the exon 4, known as FH-Padova 1) and 100 healthy controls. The polymerase chain reaction (PCR) amplification products prepared with a cosolvent PCR protocol and an antibody-protected polymerase were directly analyzed with an apparatus for high-voltage capillary electrophoresis on microchips and laser-induced fluorescence detection equipped with chips for the analysis of 25-500 bp dsDNA fragments. The test could not be extended to dinucleotide repeats due to the resolution characteristics of the available microchip. This novel approach was able to distinguish 17 microsatellite alleles varying from 0 to 17 repeats. Many of these alleles were quite rare, but the seven more abundant accounted for over the 70% of allele distribution in control population. The standard deviation in the sizing of the most abundant alleles ranged from +0.60 to +/- 0.75 bp. This indicated that the size attribution to a conventional allele using the +/- 1 bp range around it allowed a confidence limit above the 80 %. The sizing of D19S394 obtained this way allowed the cosegregation analysis with both the FH mutations tested. Therefore, this innovative approach to microsatellite sizing was much simpler, but equally effective as traditional capillary electrophoresis, at least with tetranucleotide repeats.     

De novo mutations in sporadic deletional Duchenne muscular dystrophy (DMD) cases

Dinucleotide repeat polymorphism based genetic analysis is a powerful approach to gain insight into rare genetic events like germline mosaicism and de novo mutations. The loss of heterozygosity of polymorphic dinucleotide loci at "deletional hotspot" of dystrophin gene can provide direct evidence of carrier status in female relatives of affected DMD patients with overlapped exonic deletions. We have used 4 STR loci of the central deletional hotspot of the dystrophin gene for genetic analysis in sporadic unrelated DMD families. Twenty-nine mothers of sporadic deletional cases were analysed and their carrier status was determined. Eighteen of them showed heterozygosity in the deleted loci suggesting the occurrence of de novo mutations. In 9 cases, the carrier status was indeterminate while 2 showed germline mosaicism. Our observations reiterated the importance of STR analysis in determining the status of mothers of sporadic deletional DMD cases in order to provide proper genetic counselling.     

Development of multiplex PCR system with 15 X-STR loci and genetic analysis in three nationality populations from China

The aim of this study is to develop a new multiplex PCR system that simultaneously amplifies the 15 X-chromosome short tandem repeats (X-STRs) loci in the same PCR reaction, and to obtain the 15 X-STR loci database in three nationality populations from China. This multiplex system includes DXS7133, DXS6801, DXS981, DXS6809, DXS7424, DXS6789, DXS9898, DXS7132, GATA165B12, DXS101, DXS10075, DXS6800, GATA31E08, DXS10074, and DXS10079, which were successfully analyzed on 1251 DNA samples (670 males and 581 females) from Guangdong Han population, Xinjiang Uigur and Kazakh. The allele frequencies and mutation rates of the 15 loci were investigated, and the allele frequency distribution among different populations was compared. A total of 6-17 alleles for each locus were observed and altogether 170 alleles for all the selected loci were found. Thirteen cases with mutation of the above loci were detected in 11,850 meioses. Pairwise comparisons of the allele frequencies distribution showed significant differences in most loci among different populations. The results indicate that this multiplex system may provide high polymorphism information for kinship testing and relationship investigations, and it is necessary to gain allele frequency and mutation rate of different population for forensic application.     

Imperfect units of an extended microsatellite structure involving single nucleotide changes

Short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) are widely used as markers in human genome studies. We have characterized a highly polymorphic STR locus (D20S85) with (AAAG)n repeats, by a combination of direct DNA sequencing and single-strand conformation polymorphism (SSCP) analysis. Eight STR alleles were first identified on denaturing gels, and SSCP gels were then used to demonstrate the existence of previously indistinguishable multiple alleles at the locus on the basis of variable allelic flanking sequences. This was confirmed by direct sequencing of the alleles. Four transitions, two G to A and two A to G in the 5'-flanking region of the locus at positions 14, 22, 24, and 26 effectively subdivided the STR alleles into two groups, with frequencies of 0.431 and 0.569, respectively. The mutational processes that generated the polymorphisms involved both simple changes in the number of AAAG repeats and single nucleotide mutations in the region flanking the repeat. The findings have potential application in the avoidance of false linkage and association. A composite locus of this nature, with separate STR and SNP evolutionary histories and resulting from different mutational processes, also could have wide application in studies of selection, drift, migration and inbreeding.     

Sequencing and four-state minisatellite variant repeat mapping of the D1S7 locus (MS1) by fluorescence detection

Minisatellite variant repeat mapping by polymerase chain reaction (MVR-PCR) reveals an enormous degree of variation in the human minisatellite regions. The original approach involved the use of 32P-labelled probes to detect the MVR-PCR products generated. To date, the loci mapped include D1S8, D7S21 and D16S309. However, the most polymorphic locus, D1S7 (MS1), which has been used in forensic analysis, has presented technical difficulties, initially due to its short 9 bp repeats that are much shorter than any conventional primer sequences. This was overcome by using the method of "wrapping around" primers employing inosine at the redundancy position. The difficulty of cloning highly repetitive DNA was overcome by utilising specialised competent SURE cells. We report the cloning and sequencing of selected short MS1 alleles to determine the variety of repeat types. This survey revealed nine types, four of which represented greater than 80% of the sequenced repeats. The reported MVR-PCR system maps the MS1 locus for these four common repeat types by fluorescence detection.     

Amino acid-catalyzed dynamic kinetic asymmetric transformations (DYKAT): one-step de novo synthesis of polyketide sugars from racemic β-hydroxy aldehydes

The ability of amino acids to catalyze dynamic kinetic asymmetric transformation (DYKAT) with excellent stereoselectivity is presented. The novel DYKAT process was applied in the proline-catalyzed one-step de novo synthesis of triketide- and deoxysugars, which were formed with excellent diastereoselectivity and up to 99% ee. The de novo synthesis is accomplished via an enzyme-like DYKAT process, which includes continuous amino acid-mediated racemization of the acceptor β-hydroxy aldehyde in combination with amino acid-catalyzed direct selective aldol addition.     

Developmental validation of the Yfiler Platinum PCR Amplification Kit for forensic genetic caseworks and databases

Y chromosome kits are successfully applied in cases where human biological material exists. With the development of genotyping ability, more Y chromosomal markers are needed for finer identification of male individuals and lineages. In this study, a developmental validation of a newly emerged Y chromosome kit that combines two different kinds of markers: 38 Y-STRs and 3 Y-indels are conducted. The results show that this kit has high sensitivity when there is a small amount of DNA (125 pg), more than one male (minor:major = 1:7), or a mixture of males and females (male:female = 125pg:1875pg), inhibited substances (800 μM hematin and more than 1600 ng/μL humic acid). The kit exhibits high precision level with a standard deviation of allele size no more than 0.14 nt. Locus DYS481 shows the largest stutter rate, with three stutters per true allele. Population samples are well identified (MP of 0.001106), and mutations can be observed in father–son pairs (46 mutations in 70 pairs, 10 in locus DYS627). Out of all the population samples, 13.2% belong to haplogroup M117-O2a2b1a1, with their ethnic group being Han Chinese. The results show that this kit can improve the performance of identifying male individuals, obtaining more unique haplotypes (increasing from 894 to 918 of 1000 male samples) and higher discrimination capacity (increasing from 0.942 to 0.955) in this study compared to previous widely used Yfiler Plus kit. Besides, it gives information about their paternal lineages in forensic genetic casework and genealogical database construction.     

Haplotype studies support slippage as the mechanism of germline mutations in short tandem repeats

Germline mutations of human short tandem repeat (STR) loci are expansions or contractions of repeat arrays which are not well understood in terms of the mechanism(s) underlying such mutations. Although polymerase slippage is generally accepted as a mechanism capable to explain most features of such mutations, it is still possible that unequal crossing over plays some role in those events, as most studies in humans could not exclude unequal crossing over (UCO). Crossing over can be studied by analyzing haplotypes using flanking markers. To check for UCO in mutations, we have analyzed 150 paternity cases for which more than the usual trio (mother, child, and father) were available for testing by analyzing 16 STR loci. In a total of 4900 parent-child allele transfers four mutations were observed at different loci (D8S1179, D18S51, D21S11, and SE33/ACTBP2). To identify the mutated allele and to check for UCO, we typed at least four informative loci flanking the mutated locus and used the pedigree data to establish haplotypes. By doing so we were able to exclude UCO in each case. Moreover, we were able to identify the mutations as one-repeat contractions/expansions. Our data thus support slippage as the mechanism of germline mutations in STRs.     

Methodological factors influencing measurement and processing of plasma reelin in humans

Background

Reelin, intensively studied as an extracellular protein that regulates brain development, is also expressed in a variety of tissues and a circulating pool of reelin exists in adult mammals. Here we describe the methodological and biological foundation for carrying out and interpreting clinical studies of plasma reelin.

Results

Reelin in human plasma was sensitive to proteolysis, freeze-thawing and heating during long-term storage, sample preparation and electrophoresis. Reelin in plasma was a dimer under denaturing conditions. Boiling of samples resulted in laddering, suggesting that each of the 8 repeats expressed in reelin contains a heat-labile covalent bond susceptible to breakage. Urinary-type and tissue-type plasminogen activator converted reelin to a discrete 310 kDa fragment co-migrating with the major immunoreactive reelin fragment seen in plasma and also detected in brain. (In contrast, plasmin produced a spectrum of smaller unstable reelin fragments.) We examined archival plasma of 10 pairs of age-matched male individuals differing in repeat length of a CGG repeat polymorphism of the 5'-untranslated region of the reelin gene (both alleles < 11 repeats vs. one allele having >11 repeats). Reelin 310 kDa band content was lower in subjects having the long repeats in all 10 pairs, by 25% on average (p < 0.001). In contrast, no difference was noted for amyloid precursor protein.

Conclusions

Our studies indicate the need for caution in measuring reelin in archival blood samples, and suggest that assays of plasma reelin should take into account three dimensions that might vary independently: a) the total amount of reelin protein; b) the relative amounts of reelin vs. its proteolytic processing products; and c) the aggregation state of the native protein. Reelin-plasminogen activator interactions may affect their roles in synaptic plasticity. Our results also suggest that the human CGG repeat polymorphism affects reelin gene expression, and may affect susceptibility to human disease.

     

Discrimination of A1555G and C1494T point mutations in the mitochondrial 12S rRNA gene by on/off switch

The objective of this study was to apply the “on/off” switch consisting of 3′ phosphorothioate-modified allele specific primers and exo⁺ polymerase in single base discrimination of A1555G and C1494T mutations in the highly conserved sites of the mitochondrial 12S rRNA. The two point mutations are the hotspot mutations associated with either aminoglycoside antibiotics induced deafness or inherited nonsyndromic hearing loss. The PCR products of mitochondrial DNA (mtDNA) 12S rRNA gene were inserted into the pMD19-T vector for transformation into Escherichia coli JM109 competent cells for preparing wild-type pMD19-T/mt vector. Inverse PCR was carried out for mtDNA 12S rRNA gene C1494T and A1555G mutagenesis and DpnI endonuclease degradating methylated pMD19-T/mt vector existing in the inverse PCR products was carried out to construct the mutation-type pMD19-T/mtM vector. These constructed vectors were confirmed by DNA sequencing. Allelic specific primers targeting wild-type and mutation-type templates were designed with 3′ terminal phosphorothioate modification. Two-directional primer extension was performed using Pfu polymerases. Amplified by exo⁺ polymerase, allelic specific primers perfectly matching wild-type allele were extended while no products were produced from primers targeting point-mutated deafness-related allele. Similarly, allelic specific primers perfectly matching point-mutated deafness-related mutation-type allele were extended and no products were yielded from primers targeting wild-type allele. No specific product was observed in the primer extension reaction mediated by on/off switch in screening the mtDNA 12S rRNA gene harboring either C1494T or A1555G mutation in 40 healthy volunteers tested. These data suggest that the “off switch” mediated by exo⁺ polymerase is highly reliable in the diagnosis of monogenic diseases and the novel “on/off” switch has enormous applications in systematic and extended screening of the12S rRNA gene A1555G and C1494T mutations. The established assay can be widely used not only for hearing loss patients but also for normal subjects before the use of aminoglycoside antibiotics.     

Mass spectrometric characterization of human skin elastin peptides produced by proteolytic digestion with pepsin and thermitase

This study investigated peptides resulting from the digestion of human skin elastin with pepsin and thermitase. Characterization of the peptides was performed using two complementary mass spectrometric techniques; LC/ESI-ion trap and nano-ESI-qTOF MS. 155 different peptides were identified using a combined database based and de novo sequencing approach resulting in a total sequence coverage of 65.4% calculated on the basis of the precursor tropoelastin (accession number A32707). A potential hydroxylation was found in 29% of the recovered prolines. Furthermore, the absence of amino acids expressed by exon 26A could be confirmed. However, contrary to earlier studies, amino acids expressed by exon 22 seem to exist.     

Analysis of five rare alleles at the STR loci D1S1656, D12S391, D13S317, Penta D,and D2S441

Short tandem repeat (STR) automatic typing technology is extensively used in forensic laboratories with commercial kits, in rare cases genotyping misinterpretations or mislabeling may occur due to unexpected rare alleles. This study refers to the investigation of several rare alleles observed from routine cases. Besides cross-kit verification with Goldeneye 25A (Beijing PeopleSpot Inc, China) and Huaxia platinum (Thermo Fisher Scientific, USA) kits, the next-generation sequencing technology by MiSeq FGx System (Illumina, USA) was applied to further validation. To solve the inconsistent outcomes reached by the above mentioned approaches at D2S441 locus, single gene amplification, gene cloning, and genetic sequencing was also performed. As a result, five rare alleles were detected. Two novel alleles of allele 3 at the D13S317 locus and allele 5 at the D2S441 locus were found; three previously reported alleles of allele 9 at D1S1656 locus, allele 19 at Penta D locus, and allele 28 at D12S391 locus in STRBase were initially supplemented with sequence information. We, therefore, propose that such uncommon observations with rare events should be carefully investigated and interpreted.     

Genotyping short tandem repeats using flow injection and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

Characterizing polymerase chain reaction (PCR) amplicons has been accomplished for the first time using flow injection analysis coupled to electrospray ionization mass spectrometry (ESI-MS). The PCR amplicons were amplified at the human tyrosine hydroxylase short tandem repeat locus from an individual homozygotic for the 9.3 allele. One product was amplified using Pfu polymerase and yielded a blunt-ended amplicon of 82 base-pairs (bp) in length. The second PCR product was amplified using Taq polymerase that resulted in an amplicon with cohesive termini of 82 bp plus either mono- or diadenylation. The two PCR amplicons were alternatively injected using a 0.5-microL loop at 2 microM for the Pfu amplicon and 1 microM for the Taq amplicon with a flow rate of 200 nL/min during data acquisition. Both PCR amplicons were accurately identified using mass measurements illustrating the compatibility of ESI-MS for genotyping short tandem repeat sequences and the potential for high-throughput genotyping of large PCR amplicons.     

Fifty-nine bp repeat polymorphism in the uncommon intron 36 of the human mucin gene MUC5B

A variable number of tandem repeat (VNTR) polymorphism within the intron 36 of the human mucin gene MUC5B, which is mapped to chromosome 11 band p15.5, have been identified using Southern blotting experiments. This polymorphism can be easily assayed by polymerase chain reaction (PCR) to detect linkage of inherited disorder. Five alleles were observed in 86 unrelated individuals due to 3-8 direct perfect repeats of 59 bp. This repeat has the particularity to begin at the end of the preceding exon. Southern blot experiments revealed the locus specificity of the repeat. The sequence of the repeat unit does not match the consensus sequence of Chi-related minisatellites.     

Human FEN-1 can process the 5'-flap DNA of CTG/CAG triplet repeat derived from human genetic diseases by length and sequence dependent manner

Trinucleotide repeat (TNR) instability can cause a variety of human genetic diseases including myotonic dystrophy and Huntington's disease. Recent genetic data show that instability of the CAG/CTG repeat DNA is dependent on its length and replication origin. In yeast, the RAD27 (human FEN-1 homologue) null mutant has a high expansion frequency at the TNR loci. We demonstrate here that FEN-1 processes the 5'-flap DNA of CTG/CAG repeats, which is dependent on the length in vitro. FEN-1 protein can cleave the 5'-flap DNA containing triplet repeating sequence up to 21 repeats, but the activity decreases with increasing size of flap above 11 repeats. In addition, FEN-1 processing of 5'-flap DNA depends on sequence, which play a role in the replication origin-dependent TNR instability. Interestingly, FEN-1 can cleave the 5'-flap DNA of CTG repeats better than CAG repeats possibly through the flap-structure. Our biochemical data of FEN-1's activity with triplet repeat DNA clearly shows length dependence, and aids our understanding on the mechanism of TNR instability.     

High-throughput genotyping of repeat polymorphism in the regulatory region of serotonin transporter gene by gel microchip electrophoresis.

Large-scale genotyping of the repeat polymorphism in the regulatory region of the serotonin transporter gene (5-HTTLPR) was attempted by polymerase chain reaction (PCR) amplification followed by gel microchip electrophoresis analysis. The multilane (96) format of the gel microchip system allowed parallel separation of a large number of samples. The separation and visualization of the PCR amplicons from either the 5-HTTLPR short allele (number of repeats are 14) or the 5-HTTLPR long form (16 repeats) was completed in a few minutes. Genotyping of healthy Caucasian individuals showed that the short allele had a somewhat lower frequency (0.42) than the long form (0.58), and the genotype frequencies fulfilled the criteria of the Hardy-Weinberg equilibrium (chi = 0.012, p = 0.994). Based on these results, gel microchip electrophoresis system proved to be a powerful tool for high throughput genotyping of repeat polymorphism.     

Automated Antibody De Novo Sequencing and Its Utility in Biopharmaceutical Discovery

Applications of antibody de novo sequencing in the biopharmaceutical industry range from the discovery of new antibody drug candidates to identifying reagents for research and determining the primary structure of innovator products for biosimilar development. When murine, phage display, or patient-derived monoclonal antibodies against a target of interest are available, but the cDNA or the original cell line is not, de novo protein sequencing is required to humanize and recombinantly express these antibodies, followed by in vitro and in vivo testing for functional validation. Availability of fully automated software tools for monoclonal antibody de novo sequencing enables efficient and routine analysis. Here, we present a novel method to automatically de novo sequence antibodies using mass spectrometry and the Supernovo software. The robustness of the algorithm is demonstrated through a series of stress tests.

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