Orofacial Muscle Weakening in Facioscapulohumeral Muscular Dystrophy (FSHD) Patients

Background: Facioscapulohumeral muscular dystrophy is the third most commonly found type of muscular dystrophy. The aim of this study was to correlate the D4Z4 repeat array fragment size to the orofacial muscle weakening exhibited in a group of patients with a genetically supported diagnosis of FSHD. Methods: Molecular genetic analysis was performed for 52 patients (27 female and 25 male) from a group that consisted of 36 patients with autosomal dominant pedigrees and 16 patients with either sporadic or unknown family status. The patients were tested with the southern blotting technique, using EcoRI/Avrll double digestion, and fragments were detected by a p13E-11 telomeric probe. Spearman’s correlation was used to compare the fragment size with the degree of muscle weakening found in the forehead, periocular and perioral muscles. Results: A positive non-significant correlation between the DNA fragment size and severity of muscle weakness was found for the forehead (r = 0.27; p = 0187), the periocular (r = 0.24; p = 0.232) and the left and right perioral (r = 0.29; p = 0.122), (r = 0.32; p = 0.085) muscles. Conclusions: Although FSHD patients exhibited a decrease in muscular activity related to the forehead, perioral, and periocular muscles the genotype–phenotype associations confirmed a weak to moderate non-significant correlation between repeat size and the severity of muscle weakness. Orofacial muscle weakening and its association with a D4Z4 contraction alone may not have the significance to serve as a prognostic biomarker, due to the weak to moderate association. Further studies with larger sample sizes are needed to determine the degree of genetic involvement in the facial growth in FSHD patients.

Molecular Genetic Analysis with Microsatellite-like Loci Reveals Specific Dairy-Associated and Environmental Populations of the Yeast Geotrichum candidum

Geotrichum candidum is an environmental yeast, also found as part of the cheese surface microbiota, where it is important in the ripening of many traditional cheeses, such as Camembert. We have previously developed a Multi Locus Sequence Typing (MLST) scheme, which differentiated five clades, of which one contained only environmental isolates, two were composed almost entirely of dairy isolates, and two others contained a mixture of dairy, environmental, and miscellaneous food isolates. In order to provide a simple method to uniquely type G. candidum strains, and in addition to permit investigation of the population structure at a fine level, we describe here a molecular analysis using a set of twelve highly discriminating microsatellite-like markers. The present study consolidates the previously suggested division between dairy and environmental strains, and in addition distinguishes a specifically European group of environmental strains. This analysis permitted the discrimination of 72 genotypes from the collection of 80 isolates, while retaining the underlying meaningful phylogenetic relation between groups of strains.

Karyotype and plasmotype characteristics in wheat-rye hybrids with the different structural organization of the nuclear genome

Aim. Genome structure analysis and plasmotype identification in wheat-rye hybrids of various types (triti- cale, secalotriticum) and ploidy level. Mcth0ds. Cytological and molecular-genetic analysis. Rcsults. The karyotype and plasmotype analysis was carried out in 11 stable lines of secondary recombinant hexaploid triticale with the introgression of D-genome chromosomes of the wheat (A/B/DRR, 2n = 6x = 42), 14 stable and highly productive secalotriticum lines of F6–16 generations (Secalotriticum, S/RRAABB, 2n = 6x = 42), 9 stable lines of tetraploid triticale (A/BRR, 2n = 4x = 28). By means of differential chromosome staining, the chromosomal composition of the experimental material was characterized and the intergenomic substitution and translocation of chromosomes were detected. The PCR-RFLP analysis of the 18S/5S mitochondrial (mt) repeat and the ndhH-region of chloroplast DNA showed that these organ- elle DNA regions are in the homoplasmic state and belong to rye-type cytoplasm in secalotriticum lines and wheat-type cytoplasm in tetraploid and secondary recombinant hexaploid triticale lines. C0nclusi0ns. Cytological and molecular genetic analysis revealed significant genetic diversity of the created gene pool of wheat-rye hybrids by nuclear-cytoplasmic structure. The synthesized linear material of wheat-rye hybrids may be used in cytogenetic research and practical breeding.

Genetic Features of Triticale–Wheat Hybrids with Vaviloid-Type Spike Branching

Vaviloid spike branching, also called sham ramification, is a typical trait of Triticum vavilovii Jakubz. and is characterized by a lengthening of the spikelet axis. In this article, we present the results of a study of three triticale–wheat hybrid lines with differences in terms of the manifestation of the vaviloid spike branching. Lines were obtained by crossing triticale with hexaploid wheat, T. aestivum var. velutinum. The parental triticale is a hybrid of synthetic wheat (T. durum × Ae. tauschii var. meyrei) with rye, S. cereale ssp. segetale. Line 857 has a karyotype corresponding to hexaploid wheat and has a spike morphology closest to normal, whereas Lines 808/1 and 844/4 are characterized by the greatest manifestation of vaviloid spike branching. In Lines 808/1 and 844/4, we found the substitution 2RL(2DL). The karyotypes of the latter lines differ in that a pair of telocentric chromosomes 2DS is detected in Line 808/1, and these telocentrics are fused into one unpaired chromosome in Line 844/4. Using molecular genetic analysis, we found a deletion of the wheat domestication gene Q located on 5AL in the three studied hybrid lines. The deletion is local since an analysis of the adjacent gene B1 showed the presence of this gene. We assume that the manifestation of vaviloid spike branching in two lines (808/1 and 844/4) is associated with a disturbance in the joint action of genes Q and AP2L2-2D, which is another important gene that determines spike morphology and is located on 2DL.

Introduction to Molecular Diagnostics of Insects

Insects play an important role in biocenoses due to their abundance and wide (cosmopolitan) distribution. Many insects are crop pests. An effective pest control could be realized in case of proper species identification, which is usually managed by morphological analysis. Molecular methods allow to deep study of many issues of insect biology. In particular, traditional approach can not ordinary identify a species at all stages of their life cycle, whereas molecular methods can it. This review covers a wide range of issues related to the molecular genetic analysis of insects. In the first section we consider the methods of fixation and storage of insect specimens, as well as their impact on DNA quality. Further, we provide general information on population study design. Various schemes of DNA extraction, examples of both express techniques and more thorough protocols for DNA extraction and their purification are provided. In addition, methods of DNA isolation that allow to preserve a specimen integrity for further morphological studies are considered. The methods of DNA quality control are described in detail, that is important for PCR analysis. The last section provides various methods of PCR analysis, that we exemplify by studies aimed to elucidate both fundamental issues and practical problems.

First Results from the Prospective German Registry for Childhood Glaucoma: Phenotype–Genotype Association

Childhood glaucoma is a heterogeneous disease and can be associated with various genetic alterations. The aim of this study was to report first results of the phenotype–genotype relationship in a German childhood glaucoma cohort. Forty-nine eyes of 29 children diagnosed with childhood glaucoma were prospectively included in the registry. Besides medical history, non-genetic risk factor anamnesis and examination results, genetic examination report was obtained (23 cases). DNA from peripheral blood or buccal swab was used for molecular genetic analysis using a specific glaucoma gene panel. Primary endpoint was the distribution of causative genetic mutations and associated disorders. Median age was 1.8 (IQR 0.6; 3.8) years, 64% participants were female. Secondary childhood glaucoma (55%) was more common than primary childhood glaucoma (41%). In 14%, parental consanguinity was indicated. A mutation was found in all these cases, which makes consanguinity an important risk factor for genetic causes in childhood glaucoma. CYP1B1 (30%) and TEK (10%) mutations were found in primary childhood glaucoma patients. In secondary childhood glaucoma cases, alterations in CYP1B1 (25%), SOX11 (13%), FOXC1 (13%), GJA8 (13%) and LTBP2 (13%) were detected. Congenital cataract was associated with variants in FYCO1 and CRYBB3 (25% each), and one case of primary megalocornea with a CHRDL1 aberration. Novel variants of causative genetic mutations were found. Distribution of childhood glaucoma types and causative genes was comparable to previous investigated cohorts. This is the first prospective study using standardized forms to determine phenotypes and non-genetic factors in childhood glaucoma with the aim to evaluate their association with genotypes in childhood glaucoma.

Constitutional chromothripsis of the APC locus as a cause of genetic predisposition to colon cancer

PurposeApproximately 20% of patients with clinical familial adenomatous polyposis (FAP) remain unsolved after molecular genetic analysis of the APC and other polyposis genes, suggesting additional pathomechanisms.MethodsWe applied multidimensional genomic analysis employing chromosomal microarray profiling, optical mapping, long-read genome and RNA sequencing combined with FISH and standard PCR of genomic and complementary DNA to decode a patient with an attenuated FAP that had remained unsolved by Sanger sequencing and multigene panel next-generation sequencing for years.ResultsWe identified a complex 3.9 Mb rearrangement involving 14 fragments from chromosome 5q22.1q22.3 of which three were lost, 1 reinserted into chromosome 5 and 10 inserted into chromosome 10q21.3 in a seemingly random order and orientation thus fulfilling the major criteria of chromothripsis. The rearrangement separates APC promoter 1B from the coding ORF (open reading frame) thus leading to allele-specific downregulation of APC mRNA. The rearrangement also involves three additional genes implicated in the APC–Axin–GSK3B–β-catenin signalling pathway.ConclusionsBased on comprehensive genomic analysis, we propose that constitutional chromothripsis dampening APC expression, possibly modified by additional APC–Axin–GSK3B–β-catenin pathway disruptions, underlies the patient’s clinical phenotype. The combinatorial approach we deployed provides a powerful tool set for deciphering unsolved familial polyposis and potentially other tumour syndromes and monogenic diseases.

A Novel Mutation Diagnosing in Allan–Herndon–Dudley's Syndrome

Allan–Herndon–Dudley's syndrome (AHDS) is a rare X-linked recessive disease that causes abnormal serum thyroid function tests, severe hypotonia, intellectual disability, and motor deficit due to a mutation in the monocarboxylate transporter 8, which is a thyroid hormone transporter. A 6-month-old male patient presented to our outpatient clinic with a serious hypotonia complaint. With a preliminary diagnosis of AHDS, a molecular genetic examination was performed. The molecular genetic analysis detected a new previously unidentified variant in the SLC16A2 gene. This case has been presented to report the AHDS, which is a rare cause of hypotonia in patients presenting/consulting with severe hypotonia, global developmental delay, and abnormal thyroid function test results. Besides, a novel pathogenic mutation in the SLC16A2 gene has been described in the present article.

Pathogenic Aspects of Inherited Platelet Disorders

Inherited platelet disorders (IPDs) constitute a large heterogeneous group of rare bleeding disorders. These are classified into: (1) quantitative defects, (2) qualitative disorders, or (3) altered platelet production rate disorders or increased platelet turnover. Classically, IPD diagnostic is based on clinical phenotype characterization, comprehensive laboratory analyses (platelet function analysis), and, in former times, candidate gene sequencing. Today, molecular genetic analysis is performed using next-generation sequencing, mostly by targeting enrichment of a gene panel or by whole-exome sequencing. Still, the biochemical and molecular genetic characterization of patients with congenital thrombocytopathias/thrombocytopenia is essential, since postoperative or posttraumatic bleeding often occurs due to undiagnosed platelet defects. Depending upon the kind of surgery or trauma, this bleeding may be life-threatening, e.g., after tonsillectomy or in brain surgery. Undiagnosed platelet defects may lead to additional surgery, hysterectomy, pulmonary bleeding, and even resuscitation. In addition, these increased bleeding symptoms can lead to wound healing problems. Only specialized laboratories can perform the special platelet function analyses (aggregometry, flow cytometry, or immunofluorescent microscopy of the platelets); therefore, many IPDs are still undetected.