@2024 Afarand., IRAN

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ISSN: 2251-8215    Sarem Journal of Reproductive Medicine 2018;2(1):13-18

Background
‎... [1-4]. Congenital nystagmus is a relatively common ocular disease, the most common heritable ‎pattern of which is dominant in X [5, 6].‎
Previous Studies
‎... [6-10]. Tarpey et al. in the study of 16 families with congenital X-linked idiopathic nystagmus ‎‎(XLICN) and the sequencing of all exons of the genes between the two markers DXS1047 and DXS1041 ‎showed that in 15 families of 16 families, the FRMD7 gene which is located in the Xq26 -q27 region, is ‎the carrier of the mutation [11]. ... [12-17]. ‎
Aim(s)
The purpose of this study was to evaluate the mutations of FRMD7 gene in congenital nystagmus ‎associated with chromosome X in an Iranian family population.‎
Research type

Research society, place and time

Sampling method and number
A 15-year-old boy belonging to a family from West Azerbaijan Province came to the Department of ‎Genetics at Sarem Hospital, and it was found that he has a congenital idiopathic nystagmus disease. He ‎had acute vision and no other clinical findings. The second boy suffered from this disease, was from an ‎apparently non-familial marriage to asymptomatic parents. The family tree illustrates the history of ‎disorder in the five generations of the family (Fig. 1). There were also 11 other people with normal ‎mental state and non-motor movements and oculogyric movements of the eye and reduced visual ‎acuity without any neurological and morphological disorders in the genealogy.‎The gene's infiltration rate in women was 30%. Therefore, it was decided that, in the first step, the ‎FRMD7 gene d be examined by indirect linkage and direct sequencing. This large family had about 50 ‎members, of whom 12 had this disease. ‎ Used devices & materials ‎ DNA extraction: 5 cc of blood was collected from the family members and stored in an EDTA tube. ‎DNA extraction was carried out using columnar method and by qiagen kit using saturated salt method. At first, 2 ml of blood was injected into a 15 ml falcone and centrifuged with 10 ml of cold water to a ‎volume of 12 ml and centrifuged for 15 minutes at 6000 rpm at 8°C. Then, 10 ml of the surface liquid ‎was discarded and pipetted with Pasteur pipette, and again, 10 ml of cold water was added to the ‎sample and shaken well to dissolve all the deposition in water. Then, it was centrifuged at 8 ° C for 15 ‎minutes at 6000 rpm. The second phase was repeated twice and was performed with the same ‎centrifuge program. In the next step, the precipitate was well pumped and 10 ml of solution A was ‎poured onto it to reach 12 ml volume. Samples were then centrifuged at 6000 rpm at 8°C. surface liquid ‎was discarded and white sediment was remained, and 1.5 ml of B solution was added to each sample. ‎Then, 30 μl SDS 10% and 25 μL proteinase K were added to each sample at 25 mg / ml, and then the ‎samples were shaken well in a banner machine at 65 ° C for 30 minutes. Then they were added 500 ‎micro litre NaCl 5 M and it was placed in a refrigerator at 4 ° C for 20 minutes. At this stage, the ‎samples were centrifuged at 4 C° with 4000rpm, with a slight deposition of white and a nonsense ‎liquid.‎ A new 15-millimeter Falcon was prepared for the previous falcon. Then, 2 ml of 96% cold alcohol was ‎poured into the flask containing the samples and the liquid surface was transferred to another clean 15 ‎ml tube. Then the falcons were shaken a few times to observe the DNA bundle at this stage. In a 1.5 mm ‎micro tube, which was previously written on the patient's specification, 1000 ml of 70% alcohol was ‎poured into the microscope and transferred to a microscope of 1.5 micro tube, with a slow-paced ‎pipette. The specimens were then centrifuged for 3 minutes at 13000 rpm, causing clapping to occur as ‎a white sediment in the micro tube at the bottom. At this stage, to remove the alcohol from the ‎specimen, it was completely covered by the sampler. These samples were placed at room temperature ‎for 15-20 minutes to dry. Finally, 100-200 microliters of distilled water were added to the samples and ‎placed at room temperature for 24 hours and transferred to the freezer a-20 ° C the next day.‎ ‎ DNA quality and quantity analysis: The analysis of UV absorption by nucleotides provides an ‎accurate and simple estimation of the concentration of the acid nucleic in the sample. When the DNA ‎sample is pure and does not contain significant amounts of contamination, such as protein or organic ‎solvents, purines and pyrimidines in acid nuclei exhibit peak absorbance at wavelengths of about 260 ‎nm. To determine the purity of the sample, the OD260 / OD280 ratio must be determined. The ‎presence of impurities or excessive DNA deficiency in the sample prevents the correct analysis. ‎500 micro liters of dipped water was poured into a cuvette and then the spectrophotometer was ‎calibrated at wavelengths of 260 and 280 nm. 5 μl of each DNA sample was diluted with 500 μl volume ‎of two-volume distilled water and was used as a double-distilled water as Blank. OD260 and OD280 ‎values were read from the spectrophotometer and the OD260 / OD280 ratio was calculated. For the ‎pure RNA, the OD260 / OD280 ratio is 2.2-2.3 and for pure DNA, this ratio is close to 1.8. An increase ‎in this ratio usually indicates the presence of an RNA, which can be tested by agarose gel testing. In this ‎case, the RNA is observed as smear and moves ahead of the DNA. The ratio below 1.8 is usually ‎indicative of phenolic or protein contamination. The phenolic or protein contamination is also shown ‎by a ratio of OD230 / OD260 greater than 0.5. When it was assured that the sample contained pure ‎DNA, its DNA concentration was calculated by formula‏.‏ Electrophoresis of the sample was determined on the agarose gel DNA extracted DNA and the DNA ‎value of each sample was calculated by comparing the color intensity of the sample with standard DNA ‎bands and also the presence or absence of RNA was determined by this method. RNA moves in form of ‎smear and moves ahead of DNA.‎ DNA extraction: The blood of patients and subjects with normal phenotype was extracted by Salting out ‎and qiagen kits. To evaluate the quality of the extracted specimens, 3 μl of DNA extracted by saturated ‎salt method was electrophoresed on the agarose gel 1.5% (figure 2).Mutation Analysis: A mutation study was performed using direct sequencing of FMRD7 gene by ‎Sanger method, in which the 12 exons and adjoining intron regions were sequenced after amplification ‎by PCR reaction. The sequencing results determined the nucleotide changes in the target gene. In cases ‎where the sequence change is de novo, markers are very helpful. In other words, in the event of a new ‎mutation, the association of the disease in the family with the FRMD7 gene, along with the absence of ‎the desired mutation in a healthy 100-person group, can be effective in preventing mutation disease. In ‎cases where the mutation is pathogenic, it is also easy to confirm the cause of the disease in the family. ‎Direct sequencing results were investigated on the ABI 3130x. To rule out the possibility that the ‎mutation detected in the FRMD7 gene is a community-specific polymorphism, we examined 100 ‎chromosomes of healthy individuals that were ethnically matched to this family by the method of ‎limiting the examination throughout the fragment of polymorphism (RFLP), that mutated allele was not ‎observed among the control group.‎ ‎ ‎‎
Used devices&materials

Findings by Text
PCR of the FMRD7 gene exons: The quality and size of each single exon of the FRMD7 gene were ‎evaluated on the gel electrophoresis (Fig. 3).‎Direct sequencing: Exon 1, which was heterozygote in female patients with mutation, and the organic ‎synthesis of cytosine in a single allele is converted to thymine (Fig. 4).‎Exon 1 was also found in male patients with mutations (cytosine-thymine conversion) (Fig. 5).‎A review of all coding exons and non-codon-containing intranial regions revealed a nucleotide change ‎in nucleotide # 37 of the FRMD7 gene. This mutation is located at Exon # 1. The bioinformatics of this ‎mutation indicated the pathogenicity of this mutation. This change in the nucleotide has not been ‎reported before (Fig. 6).‎Changing cytosine to thymine in nucleotide number 37 (c.37C → T) resulted in the lack of translation of ‎glutamine amino acids, which resulted in the conversion to the end (Amb) at position 13 (Q13X) and a ‎kind of meaningless change. This change creates a defective protein that quickly disappears. The rest ‎of the FRMD7 encoding regions were not mutated.
Main comparison to the similar studies
‎... [18]. Watkins et al. Have shown that mutation in the CASK gene can lead to congenital nystagmus ‎associated with mental retardation in boys [19]. ... [20, 21]. There is no study in Iranian patients with congenital nystagmus, and we identified this mutation in the ‎family C.37C> T nonsense, which could be considered a pathogenic mutation, which has not yet been ‎reported in the literature. Since we have succeeded in identifying a new mutation in the sample size, we ‎included only one Iranian family; on this basis, the Iranian population might be a rich source to detect ‎more mutations and even genes associated with congenital nystagmus.
Suggestions
More sample studies are recommended in Iranian patients with congenital nystagmus to evaluate the ‎prevalence of this mutation and other mutations in the FRMD7 gene.
Limitations

Conclusions
This study led to the identification of c.37C> T mutations. The mutation has led to a change in the ‎organic synthesis of cytosine into thymine, which results in the lack of translation of glutamine amino ‎acids, which result in defective protein production and it easily disappears.
Acknowledgments

Conflict of interest

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