Variants of the HNF4A and HNF1A genes in patients with impaired glucose metabolism and dyslipidemia

Maturity onset diabetes of the young is a dominantly inherited form of monogenic diabetes, diagnosed mainly before the age of 35 years. Mutations in the HNF1A and HNF4A genes are associated with diabetes mellitus of the HNF1A-MODY and HNF4A-MODY subtypes, respectively. These two forms of MODY are characterized by dyslipidemia in addition to impaired glucose metabolism due to the altered function HNF1A and HNF4A proteins. The aim of this study was a genetic analysis of young patients with the MODY phenotype and dyslipidemia with a burdened family history. Material and methods. The probands underwent targeted DNA sequencing using the Illumina MiSeq NGS System. The target panel included the coding regions and splicing sites of MODY-associated genes: HNF4A, GCK, HNF1A, PDX1, HNF1B, NEUROD1, KLF11, CEL, PAX4, INS, BLK, KCNJ11, ABCC8, and APPL1. Results. A heterozygous single nucleotide deletion NM_000457.4: c.153del (3’rule) was found in proband P1 in the HNF4A gene. In proband P2, single nucleotide deletion NM_000545.8: c.335del (3 ‘rule) in the HNF1A gene was detected in a heterozygous state. Both variants are located in the coding parts of the genes, led to a shift in the reading frame and have not been described in the literature and databases earlier. Conclusions. Taking into account the phenotypic features of probands, we assume that the variants NM_000545.8: c.335del (rule 3) in the HNF1A gene and NM_000457.4: c.153del (rule 3) of the HNF4A gene are associated with different MODY subtypes in these individuals. After verification of MODY-HNF1A and MODY-HNF4A diagnosis, it is necessary to monitor the lipid profile parameters (total cholesterol, low and high density lipoprotein cholesterol, triglycerides) and prescribe appropriate drug therapy.

Characterization of the Leucistic Texas Rat Snake Pantherophis obsoletus

Albinism and leucism are phenotypes resulting from impaired melanin pigmentation in the skin and skin appendages. However, melanin pigmentation of eyes remains unaffected in leucism. Here, using transmission electron microscopy, we show that the leucistic morph of the Texas rat snake (Pantherophis obsoletus lindheimeri) lacks both melanophores and xanthophores in its skin and exhibits a uniform ivory white color generated by iridophores and collagen fibers. In addition, we sequenced the full genome of a leucistic individual and obtained a highly-contiguous near-chromosome quality assembly of 1.69 Gb with an N50 of 14.5 Mb and an L50 of 29 sequences. Using a candidate-gene approach, we then identify in the leucistic genome a single-nucleotide deletion that generates a frameshift and a premature termination codon in the melanocyte inducing transcription factor (MITF) gene. This mutation shortens the translated protein from 574 to 286 amino acids, removing the helix-loop-helix DNA-binding domain that is highly conserved among vertebrates. Genotyping leucistic animals of independent lineages showed that not all leucistic individuals carry this single-nucleotide deletion. Subsequent gene expression analyses reveal that all leucistic individuals that we analyzed exhibit a significantly decreased expression of MITF. We thus suggest that mutations affecting the regulation and, in some cases, the coding sequence of MITF, the former probably predating the latter, could be associated with the leucistic phenotype in Texas rat snakes. MITF is involved in the development and survival of melanophores in vertebrates. In zebrafish, a classical model species for pigmentation that undergoes metamorphosis, larvae and adults of homozygous mitfa mutants lack melanophores, show an excess of iridophores and exhibit reduced yellow pigmentation. On the contrary, in the leucistic Texas rat snake, a non-metamorphic species, only iridophores persist. Our results suggest that fate determination of neural-crest derived melanophores and xanthophores, but not of iridophores, could require the expression of MITF during snake embryonic development.

Identification of species in the genus Nitraria L. (Nitrariaceae) based on nucleotide variability of nuclear ribosomal DNA

Intragenomic polymorphism of ITS1 and ITS2 of nuclear ribosomal DNA sequences was analysed in 33 samples belonging to the Nitraria species N. schoberi, N. sibirica, and N. komarovii. The nucleotide variability of the ITS region was detected in the Nitraria species as single-nucleotide substitutions (mainly transitions) and single-nucleotide deletion. Information about the nucleotide variability of fragments is given for the first time by us. The ITS1-5.8S-ITS2 region contained 17 phylogenetically informative single-nucleotide polymorphisms. Eleven single-nucleotide substitutions (transitions, C/T) were detected in ITS1. The ITS2 spacer contained 273–274 bp and was more conservative. A total of 5 phylogenetically informative single-nucleotide polymorphisms (4 transitions: C/T, G/A, one transversion: G/C), one single-nucleotide deletion (T/–) were detected in ITS2. The average GC content was 61.5 %. The GC content was lower in N. sibirica (59.2 %) than in N. schoberi and N. komarovii (62.7 %). It has been shown that the shorter ITS2 is a suitable molecular marker separating these species, due to the low interspecific variability and simultaneous available intraspecific variability. Phylogenetic ML and BI trees constructed separately for the ITS1 and ITS2 spacers, as well as separately for the full-size ITS region and the ITS2 spacer, were congruent. The results obtained on the intraspecific differentiation of N. sibirica revealed two main ribotypes among the samples of this species: the main Siberian sibirica-ribotype and the main Kazakh sibiricaribotype. Geographical features of the distribution of N. sibirica ribotypes, as well as the presence of significant differences between the main Siberian and Kazakh sibirica-ribotypes (3 single-nucleotide substitutions) indicated significant inter-population differences and taxonomic heterogeneity of N. sibirica. Most likely, the processes of homogenization of nuclear ribosomal DNA of N. sibirica samples, the origin of which is associated with hybridization and speciation, are currently continuing.

Using the ITS2 transcribed spacer of nuclear ribosomal DNA to identify species of the genus Nitraria

Intragenomic polymorphism of ITS2 of nuclear ribosomal DNA sequences was analysed in 30 samples belonging to Nitraria schoberi, N. sibirica. The nucleotide variability of the ITS2 region was detected in the studied Nitraria species as single-nucleotide substitutions (mainly transitions) and single-nucleotide deletion. Five ribotypes of Nitraria were identified in Russia, Kazakhstan and Tajikistan. These ribotypes formed two haplogroups that belong to the species N. schoberi and N. sibirica, respectively. The high importance of the discriminatory role of the ITS2 spacer in identifying species of the genus Nitraria is confirmed.