2 research outputs found
MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION OF MICRODELETION SYNDROMS
Introduction: Microdeletion syndromes are an extensive group of diseases affecting various organs
and systems. These diseases are caused by deletions of small sections of chromosomes. Previously,
most microdeletion syndromes were described as pathologies of unknown origin and were not even
associated with “chromosomal breakdowns”, since it was not possible to conduct subtle and accurate
genetic diagnostics. Microdeletion and microduplication syndromes are defined as a group of clinically
recognisable disorders characterised by a small (< 5 Mb) deletion or duplication of a chromosomal segment
spanning multiple disease genes. Clinically well described syndromes, for which the involvement
of multiple disease genes has been established or is strongly suspected, such as: 1p36 deletion syndrome,
Wolf-Hirschhorn syndrome, Cri-du-Chat syndrome, Sotos syndrome, Saethre-Chotzen syndrome,
Williams-Beuren syndrome, Williams-Beuren duplication syndrome, Langer-Giedion syndrome, WAGR
syndrome, Prader-Willi syndrome, Angelman syndrome, Rubinstein-Taybi syndrome, Miller-Dieker syndrome,
Lissencephaly-1, Smith-Magenis syndrome, Potocki-Lupski syndrome, Alagille syndrome, Di-
George syndrome, 22q11.2microduplication syndrome, Phelan-McDermid syndrome.
Methods: MLPA (Multiplex Ligation-dependent Probe Amplification) is the go-to technique for studying
gene copy number variations (CNVs) associated with disease. The power of the technique lies in its
versatility: MLPA can be used to detect copy number changes in anything from complete chromosomes
to single exons. This SALSA MLPA probemix for detection microdeletion syndromes contains 52MLPA
probes with amplification products between 130 and 483 nucleotides (nt). The probes detect sequences
involved in a distinct subset of microdeletion and microduplication disorders.
Results: At the clinical diagnostic laboratory, examined 57 children with suspected microdeletion
syndrome. Among them, a heterozygous deletion was found of SNRPN-u5, SNRPN-CpGisl, UBE3A-10,
UBE3A-1genes that corresponds to Prader-Willi/Angelman syndrome, heterozygous deletion ( DQ=0.5)
CLTCL1-3, CDC45-1, GNB1L-8, DGCR8-2, ZNF74-2, MED15-10, SNAP29-3 genes (Di Georgia syndrome) and
ELN-4, ELN-6, ELN-27 heterozygous deletion of Williams-Beuren syndrome.
Conclusion: This diagnostic method allows you to detect microdeletions and microduplications, which
are often not noticeable with standard cytogenetic analysis
Mutation Analysis of the NRLP3 Gene in Children With Cryopyrin-Associated Fever
Introduction: CAPS is a group of severe multi-system, auto-inflammatory diseases with an autosomal
dominant type of inheritance associated with mutations in the cryopyrin NLRP3 gene located on the first
chromosome (1q44). CAPS-syndrome is represented by three phenotypes: familial cold urticarial, Muckle
– Wells syndrome, and CINCA/NOMID syndrome. Today, it is quite a lot known about CAPS-syndrome,
but due to the nonspecific, chronic, remitting course of the disease, diagnosis and, consequently, treatment
of the patient is hindered. The diagnosis is based on the results of molecular genetic approach.
Methods: The DNAs were extracted from the whole blood of patients with suspected periodic fever
(according to the manufacturer’s instructions). The presence or absence of mutations of the NLRP3 gene
were determined by PCR using specific primers followed by purification of amplicons and Sanger sequencing.
Results of Sanger sequencing were analyzed using Seq 6, Sequencher 2.0 software.
Results: The molecular characteristic of the NLRP3 gene includes 10 exons, 32.952base pairs. As of
today, according to the register and database https://infevers.umai-montpellier.fr/web/search.php, 204
mutations of the NLRP3 gene are known, but only 19 mutations are considered pathogenic. Taking into
account that the majority of pathogenic mutations are located in exon 3 of the NLRP3 gene we designed
primers of the 3 exon. After optimizing the primers of 3 exons, we examined 2patients by Sanger sequencing.
Analysis of the electrophoregrams using the Mutation Testing resource revealed a heterozygous
carrier of c. 732G>A (p.Ala 244=; rs3806268) as prediction polymorphism. The frequency of this
polymorphism is high in different populations; the frequency of alleles is about 0.555. The results of
genetic testing allow us to consider the detected variant as the cause of cryopyrin-related syndrome only
if there is sufficient clinical and anamnestic data to confirm the diagnosis.
Conclusion: Thus, this result does not exclude the presence of mutations in other exons, it is necessary
to conduct next generation sequencing of WES. Designing primers were optimized and can use to confirm
diagnosis in molecular genetic tools