Two rare autosomal recessive neurological disorders identified by combined genetic approaches in a single consanguineous family with multiple offspring.

INTRODUCTION: Neurodevelopmental disorders (NDDs) refer to a broad range of diseases including developmental delay, intellectual disability, epilepsy, autism spectrum disorders, and attention-deficit/hyperactivity disorder caused by dysfunctions in tightly controlled brain development. The genetic backgrounds of NDDs are quite heterogeneous; to date, recessive or dominant variations in numerous genes have been implicated. Herein, we present a large consanguineous family from Turkiye, who has been suffering from NDDs with two distinct clinical presentations. METHODS AND RESULTS: Combined in-depth genetic approaches led us to identify a homozygous frameshift variant in NALCN related to NDD and expansion of dodecamer repeat in CSTB related to Unverricht-Lundborg disease (ULD). Additionally, we sought to functionally analyze the NALCN variant in terms of mRNA expression level and current alteration. We have both detected a decrease in the level of premature stop codon-bearing mRNA possibly through nonsense-mediated mRNA decay mechanism and also an increased current in patch-clamp recordings for the expressed truncated protein. CONCLUSION: In conclusion, increased consanguinity may lead to the revealing of distinct rare neurogenetic diseases in a single family. Exome sequencing is generally considered the first-tier diagnostic test in individuals with NDD. Yet we underline the fact that customized approaches other than exome sequencing may be used as in the case of ULD to aid diagnosis and better genetic counseling.

The Need for Establishing a Universal CTG Sizing Method in Myotonic Dystrophy Type 1.

The number of cytosine-thymine-guanine (CTG) repeats ('CTG expansion size') in the 3'untranslated region (UTR) region of the dystrophia myotonica-protein kinase (DMPK) gene is a hallmark of myotonic dystrophy type 1 (DM1), which has been related to age of disease onset and clinical severity. However, accurate determination of CTG expansion size is challenging due to its characteristic instability. We compared five different approaches (heat pulse extension polymerase chain reaction [PCR], long PCR-Southern blot [with three different primers sets-1, 2 and 3] and small pool [SP]-PCR) to estimate CTG expansion size in the progenitor allele as well as the most abundant CTG expansion size, in 15 patients with DM1. Our results indicated variability between the methods (although we found no overall differences between long PCR 1 and 2 and SP-PCR, respectively). While keeping in mind the limited sample size of our patient cohort, SP-PCR appeared as the most suitable technique, with an inverse significant correlation found between CTG expansion size of the progenitor allele, as determined by this method, and age of disease onset (r = -0.734, p = 0.016). Yet, in light of the variability of the results obtained with the different methods, we propose that an international agreement is needed to determine which is the most suitable method for assessing CTG expansion size in DM1.

Mitochondrial DNA damage in the hair bulb: can it be used as a noninvasive biomarker of local exposure to low LET ionizing radiation?

Purpose: Our aim was to evaluate whether mitochondrial DNA (mtDNA) damage in hair bulbs could be a suitable biomarker for the detection of local exposure to ionizing radiation.Materials and methods: Mouse hair was collected 4 and 24 hours, 3 and 10 days after single whole-body exposure to 0, 0.1, and 2 Gy radiation. Pubic hair (treated area) and scalp hair (control area) were collected from 13 prostate cancer patients before and after fractioned radiotherapy with an average total dose of 2.7 Gy to follicles after five fractions. Unspecified lesion frequency of mtDNA was analyzed with long PCR, large mtDNA deletion levels were tested with real-time PCR.Results: Unspecified lesion frequency of mtDNA significantly increased in mouse hair 24 hours after irradiation with 2 Gy, but variance among samples was high. No increase in lesion frequency could be detected after 0.1 Gy irradiation. In prostate cancer patients, there was no significant change in either the unspecified lesion frequency or in the proportion of 4934-bp deleted mtDNA in pubic hair after radiotherapy. The proportions of murine 3860-bp common deletion, human 4977-bp common deletion and 7455-bp deleted mtDNA were too low to be analyzed reliably.Conclusions: Our results suggest that the unspecified lesion frequency and proportion of large deletions of mtDNA in hair bulbs are not suitable biomarkers of exposure to ionizing radiation.

Full-Length Open Reading Frame Amplification of Hepatitis C Virus.

The purpose of this method is to amplify the full coding sequence of hepatitis C virus (HCV) by a single round reverse transcriptase-polymerase chain reaction (RT-PCR) approach. Our method relies on a highly robust and sensitive RNA extraction procedure and cutting-edge RT-PCR enzymes, all of which have been rigorously tested and optimized. This will not only allow for robust amplification of the entire open reading frame (ORF) of HCV for sequencing by Sanger or next-generation sequencing (NGS), but can also be used for cloning of the ORF of uncharacterized samples and for linkage analysis of mutations on individual genomes spanning the entire ORF. The method has been validated on a variety of samples, including sera from HCV patients and cell-culture supernatants.

Deep sequencing reveals the mitochondrial DNA variation landscapes of breast-to-brain metastasis blood samples.

Breast-to-brain metastasis (BBM) often represents a terminal event, due to the inability of many systemic treatments to cross the blood-brain barrier (BBB), rendering the brain a sanctuary site for tumour cells. Identifying genetic variations that can predict the patients who will develop BBM would allow targeting of adjuvant treatments to reduce risk while disease bulk is minimal. Germ-line genetic variations may contribute to whether a BBM forms by influencing the primary tumour subtype that presents, or by influencing the host response to the tumour or treatment regimen, or by facilitating transition of tumour cells across the BBB and establish a viable brain metastasis. The role of mitochondrial DNA (mtDNA) variants specifically in BBM is underexplored. Consequently, using a sensitive deep sequencing approach, we characterized the mtDNA variation landscapes of blood samples derived from 13 females who were diagnosed with early-onset breast cancer and later went on to develop BBM. We also predicted the potential pathogenic significance of variations identified in all mtDNA-encoded oxidative phosphorylation (OXPHOS) proteins using 3D protein structural mapping and analysis, to identify variations worthy of follow-up. From the 70 variations found in protein coding regions, we reveal novel links between three specific mtDNA variations and altered OXPHOS structure and function in 23% of the BBM samples. Further studies are required to confirm the origin of mtDNA variations, and whether they correlate with (1) the predicted alterations in mitochondrial function and (2) increased risk of developing breast-to-brain metastasis using a much larger cohort of samples.

Complete mitochondrial genome and taxonomic revision of Cardiodactylus muiri Otte, 2007 (Gryllidae: Eneopterinae: Lebinthini).

In the present study, we report the high-coverage complete mitochondrial genome (mitogenome) of the cricket Cardiodactylus muiri Otte, 2007. The mitogenome was sequenced using a long-PCR approach on an Ion Torrent Personal Genome Machine (PGM) for next generation sequencing technology. The total length of the amplified mitogenome is 16,328 bp, representing 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and one noncoding region (D-loop region). The new sets of long-PCR primers reported here are invaluable resources for future comparative evolutionary genomic studies in Orthopteran insects. The new mitogenome sequence is compared with published cricket mitogenomes. In the taxonomic part, we present new records for the species and describe life-history traits, habitat and male calling song of the species; based on observation of new material, the species Cardiodactylus buru Gorochov & Robillard, 2014 is synonymized under C. muiri.

Comparison of Two Different PCR-based Methods for Detection of GAA Expansions in Frataxin Gene.

BACKGROUND: Expansion of GAA trinucleotide repeats is the molecular basis of Friedreich's ataxia (FRDA). Precise detection of the GAA expansion repeat in frataxin gene has always been a challenge. Different molecular methods have been suggested for detection of GAA expansion, including; short-PCR, long-PCR, Triplet repeat primed-PCR (TP-PCR) and southern blotting. The aim of study was to evaluate two PCR-based methods, TP-PCR and long-PCR, and to explore the use of TP-PCR accompanying with long-PCR for accurate genotyping of FRDA patients. METHODS: Blood samples were collected from six Iranian patients suspected to FRDA, who referred to the Department of Medical Genetics at Tehran University of Medical Sciences during the year 2014. For one of these patients' four asymptomatic members of the family were also recruited for the analysis. DNA extraction was performed by two different methods. TP-PCR and long-PCR were carried out in all samples. The type of this study is assessment / investigation of methods. RESULTS: Using a combination of the above methods, the genotypes of all samples were confirmed as five homozygous mutants (expanded GAA repeats), two heterozygous and three homozygous normal (normal repeat size). The results obtained by TP-PCR are consistent with long-PCR results. CONCLUSION: The presence or absence of expanded alleles can be identified correctly by TP-PCR. Performing long-PCR and Fluorescent-long-PCR enables accurate genotyping in all samples. This approach is highly reliable. It could be successfully used for detection of GAA expansion repeats.

Mitochondrial genomes of Heterakis gallinae and Heterakis beramporia support that they belong to the infraorder Ascaridomorpha.

Heterakis gallinae and Heterakis beramporia are the most prevalent nematode infecting native chicken breed, causing major economic losses. In the present study, the complete mitochondrial genomes (mt) of H. gallinae and H. beramporia were amplified by long-PCR and then sequenced. The complete mt genomes of H. gallinae and H. beramporia were 13,973bp and 14,012bp in size, respectively. Both mt genomes contain 12 protein-coding genes, 22 transfer RNA genes and 2 ribosomal RNA genes. All genes are transcribed in the same direction and the gene arrangement is identical to Ascaridia spp. Phylogenetic analysis based on the 12 protein-coding genes revealed that the family Heterakidae (represented by H. gallinae and H. beramporia) was more closely related to the infraorder Ascaridomorpha than it was to the infraorder Oxyuridomorpha. The present study determined the complete mt genome sequences for two Heterakis species, providing useful markers for studying the systematics, population genetics, and molecular epidemiology of these Heterakis parasites.

Mitogenomic sequence and phylogenetic placement of the Hortle's whipray Himantura hortlei (Elasmobranchii: Dasyatidae).

Chondrichthyans are a class of fishes threatened with habitat destruction and fishing pressures. The current study presents the complete mitochondrial genome sequence (17,688 bp) of the vulnerable Hortle's whipray, Himantura hortlei. The mitochondrial genome arrangement is consistent with that seen in most vertebrates, containing 13 protein-coding, 22 tRNA, 2 rRNA genes, and 1 control region. Phylogenetic analyses were performed based on mitogenome and ND2 sequences. Under our taxon sampling scheme, Himantura hortlei was found to be most closely related to H. fai.

The first complete mitochondrial genome of stag beetle from China, Prosopocoilus gracilis (Coleoptera, Lucanidae).

The complete mitochondrial genome (mitogenome) of Prosopocoilus gracilis (Coleoptera: Lucanidae) that is endemic to Southern China is determined. The circular genome is 736 bp in length and comprises 13 protein-coding genes, 22 tRNA, 2 rRNA genes and a control region. Gene order is identical to that of the putative ancestral arrangement of insects. The nucleotide composition of heavy strand is A (36.6%), C (22.6%), T (29.5%) and G (11.3%). All protein-coding genes start with a typical ATN codon except for the gene COI that uses AAC as the start codon. tRNA-Ser (AGN) uses the anticodon UCU instead of the commonly used GCU. Both maximum likelihood and Bayesian analyses support the monophyly of Lucanidae and the sister relationship of Nigidionus and the remaining sampled genera. Two species of Prosopocoilus were not recovered as a monophyletic group.

The complete mitochondrial genome of the endangered spotback skate, Atlantoraja castelnaui.

Chondrichthyes are a highly threatened class of organisms, largely due to overfishing and other human activities. The present study describes the complete mitochondrial genome (16,750 bp) of the endangered spotback skate, Atlantoraja castelnaui. The mitogenome is arranged in a typical vertebrate fashion, containing 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and 1 control region.

A description of the mitogenome of the Endangered Taiwanese angelshark, Squatina formosa.

Squatinid sharks are among the most threatened of cartilaginous fishes. Here we describe the complete mitochondrial genome sequence (16,690 bp) of the Endangered Taiwanese angelshark, Squatina formosa. It has 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 1 control region in the typical vertebrate arrangement.

Long-PCR based next generation sequencing of the whole mitochondrial genome of the peacock skate Pavoraja nitida (Elasmobranchii: Arhynchobatidae).

We determined the complete mitochondrial genome sequence (16,760 bp) of the peacock skate Pavoraja nitida using a long-PCR based next generation sequencing method. It has 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 1 control region in the typical vertebrate arrangement. Primers, protocols, and procedures used to obtain this mitogenome are provided. We anticipate that this approach will facilitate rapid collection of mitogenome sequences for studies on phylogenetic relationships, population genetics, and conservation of cartilaginous fishes.

The complete mitochondrial genome sequence of Nemateleotris decora (gobiiformes, gobiidae).

We determined the mitochondrial genome (mitogenome) sequence of Nemateleotris decora by using a long polymerase chain reaction (PCR) method and next-generation sequence (NGS) technology. The total length of N. decora mitogenome is 16 502 bp, consisting of 13 protein-coding genes, 22 transfer RNAs, two ribosomal RNAs genes, and a non-coding control region. The overall base composition of N. decora is 25.22% for A, 25.90% for T, 30.69% for G, and 18.19% for C. Our results showed the complete mitogenome is a good marker for the phylogenetic study.

Complete mitogenome of three flyingfishes Cheilopogon unicolor, Cheilopogon arcticeps and Cheilopogon atrisignis (Teleostei: Exocoetidae).

The complete mitogenomes of Cheilopogon unicolor, C. arcticeps and C. atrisignis were determined by the next-generation sequencing (NGS) method. The assembled mitogenome of C. unicolor, C. arcticeps and C. atrisignis consist of 16 529 bp, 16 530 bp and 16 530 bp, respectively. Three mitogenomes contain the typical gene complement including 13 protein-coding genes, 22 transfer RNAs, two ribosomal RNA genes and a non-coding D-loop. The length of D-loop is 870 bp (C. unicolor and C. arcticeps) and 869 bp (C. atrisignis), located between tRNA-Pro and tRNA-Phe. Phylogenetic analysis indicates that Cheilopogon is not monophyly. The mitogenomes of C. unicolor, C. atrisignis and C. arcticeps may provide useful information for phylogentic and population genetic analysis for flyingfishes.

A long PCR-based approach for DNA enrichment prior to next-generation sequencing for systematic studies.

PREMISE OF THE STUDY: We present an alternative approach for molecular systematic studies that combines long PCR and next-generation sequencing. Our approach can be used to generate templates from any DNA source for next-generation sequencing. Here we test our approach by amplifying complete chloroplast genomes, and we present a set of 58 potentially universal primers for angiosperms to do so. Additionally, this approach is likely to be particularly useful for nuclear and mitochondrial regions. • METHODS AND RESULTS: Chloroplast genomes of 30 species across angiosperms were amplified to test our approach. Amplification success varied depending on whether PCR conditions were optimized for a given taxon. To further test our approach, some amplicons were sequenced on an Illumina HiSeq 2000. • CONCLUSIONS: Although here we tested this approach by sequencing plastomes, long PCR amplicons could be generated using DNA from any genome, expanding the possibilities of this approach for molecular systematic studies.

Prevalence of aac(6')-Ie-aph(2″)-Ia resistance gene and its linkage to Tn5281 in Enterococcus faecalis and Enterococcus faecium isolates from Tabriz hospitals.

BACKGROUND AND OBJECTIVE: High-level gentamicin resistance (HLGR: MIC ≥ 500 µg/ml) in Enterococci is mediated by aminoglycoside modifying enzymes which is mainly encoded by aac(6')-Ie-aph(2″)-Ia gene. The aim of this study was to evaluate the frequency of aac(6')-Ie-aph(2″)-Ia gene in clinical isolates of Enterococcus facium and Enterococcus faecalis collected from hospitals in northwest of Iran. MATERIALS AND METHODS: In the present study a total of 111 enterococcus isolates were collected from 4 hospitals during a two year period (July 2009-August 2011). Bacterial identification and species determination were carried out by standard biochemical tests. Antimicrobial susceptibility was evaluated by Kirby Bauer disc diffusion method. MICs were determined by agar dilution method. The frequency of aac(6')-Ie-aph(2″)-Ia gene in the isolates was determined by PCR. The carriage of resistance gene on Tn5281 transposon was identified by long PCR and dot-blot hybridization methods. RESULTS: Antibiotic susceptibility tests revealed that the highest resistance was against streptomycin (74.77%) and erythromycin (67.58%) whereas the highest susceptibility was observed to vancomycin (81.1%). 36 isolates (32.43%) were identified as HLGR, 34(94.44%) of them had resistant gene in their genome. Long PCR studies revealed that 88% of HLGR clinical isolates harboured Tn5281. The aac(6')-Ie-aph(2″)-Ia resistance gene was present on Tn5281 transposon in all 32 isolates according to dot blot hybridization test. CONCLUSION: The results of this study indicated that aac(6')-Ie-aph(2″)-Ia resistance gene is highly prevalent in gentamicin resistant isolates. Carriage of aac(6')-Ie-aph(2″)-Ia resistance gene on Tn5281 transposable element suggests possible contribution of this transposone on dissemination of resistance gene among enterococcus isolates.