The Effect of Short-term Mediterranean-DASH Intervention for Neurodegenerative Delay (MIND) Diet on Hunger Hormones, Anthropometric Parameters, and Brain Structures in Middle-aged Overweight and Obese Women: A Randomized Controlled Trial.

Background: The rising prevalence of obesity, as well as its detrimental effects on the brain, has drawn attention to specific dietary patterns. This study aimed to examine the effect of the Mediterranean-DASH Intervention for Neurodegenerative Delay (MIND) pattern on anthropometric parameters, hunger hormones, and brain structures in overweight and obese women. Methods: This randomized trial was conducted in Shiraz between October 2018 and March 2019. We analyzed 37 healthy women with a mean age of 48±5.38 years and a Body Mass Index (BMI) of 32±0.69 Kg/m2. Participants were randomly allocated to a hypocaloric modified MIND diet or a hypocaloric control diet. Differences in anthropometric, laboratory analysis, and brain structure were determined at baseline and three-month follow-up. Data were analyzed using SPSS 22.0. Independent and paired sample t test were used to determine between and within differences. We also used mixed-model ANOVA to compare the mean differences between two-factor groups. Results: A more significant weight reduction (P<0.0001), BMI (P<0.0001), percentage of body fat (P=0.03), waist circumference (P=0.01), and Leptin concentration (P=0.03) were found in the MIND diet group. The results also showed a significant increase in Ghrelin (P=0.002) and GLP-1 (P=0.01) levels in the MIND diet group. The findings revealed no differences in the whole and regional brain structures between the two groups. Conclusion: For the first time, this study showed that the MIND diet intervention could improve the devastating effect of obesity on metabolic profiles and anthropometric parameters. However, we could not find its effect on brain structures.Trial registration number: IRCT20190427043387N1.A preprint of this study was published at https://www.medrxiv.org/content/10.1101/2020.06.28.20142018v1.

Two Novel Mutations in LAMC2 Gene in Iranian Families Affected by Junctional Epidermolysis Bullosa.

Background: Junctional epidermolysis bullosa (JEB) is an autosomal recessive skin disorder with defective adhesion of dermal- epidermal within the lamina lucida region of the basement membrane zone. The main characterization of JEB is blistering and fragile skin and mucous membrane. Laminins are noncollagenous part of basement membrane and classified as a family of extracellular matrix glycoprotein. Laminins contain three chains: Laminin α, Laminin ß and Laminin γ. LAMC2 (laminin subunit gamma 2) gene encodes γ subunit of laminin and its mutation contributes to JEB. Here, we report a disease-causing nonsense mutation and a large deletion mutation in LAMC2 gene in two families affected by JEB. Methods: Whole exome sequencing (WES) was carried out on the mother of patient in family I and the patient himself in family II to detect the underlying mutations. Then, sanger sequencing was performed to confirm the identified mutations. Results: Next generation sequencing (NGS) data analysis of the first family showed a novel, nonsense mutation in LAMC2 gene (LAMC2: NM_005562: exon14:c.C2143T: p.R715X). The heterozygous state of the mutation was confirmed by sanger sequencing in the parents and unaffected brother. In Family II, NGS data had no coverage in the large area of LAMC2 gene. Thus, to confirm the possible deletion sanger sequencing was done and blasting of sequence showed the deleted region of 9.4 kb (exon10-17) in LAMC2 gene. Conclusion: In summary, current study reported a novel disease-causing premature termination codon (PTC) mutation in LAMC2 gene and a large deletion mutation in patients affected by JEB.

Inferior Spear-like Lens Opacity as a Sign of Keratoconus.

PURPOSE: To report 21 cases of typical inferior feather-shape lens opacity associated with keratoconus. METHODS: In this cross-sectional study, we evaluated the association of keratoconus with inferior feather-shape lens opacity in refractive surgery candidates. Visual acuity, demographic, refractive, and topographic characteristics of 26 eyes of 21 patients with inferior feather-shape lens opacity were evaluated in detail. Pedigree analysis was also performed to assess possible inheritance. RESULTS: Overall, 2122 out of 33,368 cases (6.4%) without lens opacity had keratoconus, while 20 out of 21 patients (95.2%) with peculiar lens opacity had definite keratoconus (P < 0.001). Lens opacity was bilateral in 5 cases (24%), and keratoconus was bilateral in all 20 patients with lens opacity. Nine eyes out of thirty-six with a complete data record (25%) had a severe keratoconus and underwent deep lamellar keratoplasty, while 11 (31%) had forme fruste keratoconus. Pedigrees were drawn for eight patients, most families of whom suggested an X-linked recessive inheritance. CONCLUSION: The present study was the first to investigate patients with a peculiar inferior feather-shape lens opacity accompanied by bilateral keratoconus, which was observed in 95% of the patients. This finding should raise awareness as to the possibility of diagnosing keratoconus in the eyes of the patients with these characteristics.

High dietary inflammatory index (DII) scores increase odds of overweight in adults with rs9939609 polymorphism of FTO gene.

BACKGROUND: Obesity/overweight is a public health problem globally. Dietary induced inflammation is among the most critical risk factors modulating overweight/obesity. Some people genetically are at higher risk for obesity/overweight. The first gene contributing to conventional forms of human obesity is the FTO gene. The associations between genes like the FTO, inflammation, and obesity/overweight have been investigated in limited studies. We aimed to investigate the association between the dietary inflammatory index (DII) and odds of obesity/overweight in adults with rs9939609 polymorphism of the FTO gene. METHODS: A valid 168-item semi-quantitative food frequency questionnaire was used to assess dietary intake. To investigate the role of diet in the development of inflammation, we used the DII, which is predictive of serum inflammatory markers' levels. The Inclusion criteria were defined as body mass index (BMI) from 24.9 to 29.9 kg/m2, age from 20 to 45 years, not participating in a weight management program during two past months, and no weight loss greater than 5%. We determined the genotypes of FTO rs9939609 polymorphism via amplification refractory mutation system polymerase chain reaction (ARMS-PCR) in a retrospective chart review. RESULTS: Results obtained from modeling the DII as a continuous variable and odds of obesity/overweight showed a significant association after multivariate adjustment for sex, height, fat mass, systolic blood pressure (SBP), and total energy intake (OR = 2.83 CI = 1.16-6.91). In addition, logistic regression models with the DII as a dichotomous variable adjusting for sex, height, fat mass, SBP, and total energy intake showed subjects with the DII score<0.49 were at 2.5 times higher odds of having overweight compared to subjects with the DII ≥0.49 (ORDII<0.49/≥0.49 = 2.44 CI = 1.12-5.32). CONCLUSION: Dietary induced inflammation significantly is related to odds of overweight in adults with rs9939609 polymorphism of the FTO gene. Suggesting an anti-inflammatory diet containing vitamins and minerals such as vitamin A, thiamine, niacin, and zinc, manganese, and selenium and recommending a reduction in the most inflammatory factors of diet, including saturated and trans fatty acids, could be a new strategy in the treatment and or controlling of obesity/overweight as a public health problem.

Novel pathogenic variants in NLRP7, NLRP5, and PADI6 in patients with recurrent hydatidiform moles and reproductive failure.

Recurrent hydatidiform moles (RHMs) are human pregnancies with abnormal embryonic development and hyperproliferating trophoblast. Biallelic mutations in NLRP7 and KHDC3L, members of the subcortical maternal complex (SCMC), explain the etiology of RHMs in only 60% of patients. Here we report the identification of seven functional variants in a recessive state in three SCMC members, five in NLRP7, one in NLRP5, and one in PADI6. In NLRP5, we report the first patient with RHMs and biallelic mutations. In PADI6, the patient had four molar pregnancies, two of which had fetuses with various abnormalities including placental mesenchymal dysplasia and intra-uterine growth restriction, which are features of Beckwith-Wiedemann syndrome and Silver Russell syndrome, respectively. Our findings corroborate recent studies and highlight the common oocyte origin of all these conditions and the continuous spectrum of abnormalities associated with deficiencies in the SCMC genes.

CRISPR/Cas9 knock-in toward creating a Rett syndrome cell model with a synonymous mutation in the MECP2 gene.

BACKGROUND: Rett syndrome is an X-linked dominant neurodevelopmental disease caused by mutation in the methyl-CpG-binding protein 2 (MECP2) gene. This gene encodes a methylated DNA-binding protein, which acts as a transcriptional regulatory factor. The present study aimed to establish a cell model of Rett syndrome with the MECP2 synonymous mutation c.354G>T (p.Gly118Gly). In addition, the molecular mechanism of pathogenesis of this mutation was also investigated. METHODS: To create a cell line containing the synonymous variant in MECP2 locus, the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated homology-directed repair precise gene editing method was used. In addition, employing the synthesis of cDNA, the effect of this variant on splicing was investigated. RESULTS: Using this model and molecular analysis, we found that the c.354G>T synonymous variant created a novel 5' cryptic splice donor site within the exon 3 of MECP2 gene, which resulted in the deletion of 25 nucleotides at the 3' end of exon 3 and presumably protein truncation. CONCLUSIONS: The results of the present study show that an apparently neutral synonymous polymorphism, which may be commonly classified as non-pathogenic, may indeed lead to the creation of an aberrant splice site, thereby resulting in disease.

The association between FTO rs9939609 gene polymorphism and anthropometric indices in adults.

BACKGROUND: Fat mass and obesity-associated gene (FTO) is the most studied obesity-related gene up to date. We aimed to assess anthropometric indices in carriers of FTO rs9939609 polymorphism with overweight across Iranian population (Shiraz) to find out the associations of this polymorphism with obesity indices. METHODS: This was a cross-sectional study conducted on 198 overweight healthy adults aged 20-45 years old. We assessed the body composition of the participants using bioelectrical impedance analyzer. In addition, we measured the waist circumference (WC) and hip circumference (HC). Waist to hip ratio (WHR) and waist to height ratio (WHtR) were also calculated by equations. The participants' genotype was determined by ARMS-PCR. Also, data analysis was performed using SPSS software version 20 and R software version 3.6.2. RESULTS: The mean values of body mass index (BMI) and age of the participants were 26.93 ± 1.13 kg/m2 and 33.33 ± 6.35 years old, respectively. Homozygous carriers of A-allele had significantly higher values for BMI (0.60 kg/m2, p = 0.026), WHR (0.04 unit, p = 0.003), and WHtR (0.02 unit, p = 0.030) than the homozygous carriers of T-allele. Individuals with AA genotype had greater WC (2.66 cm, p = 0.042, and 4.03 cm, p = 0.002), fat mass (2.24 kg, p = 0.004, and 3.02 kg, p = 0.001), and trunk fat (1.53 kg, p = 0.001, and 2.08 kg, p = 0.001) compared to those with AT and TT genotypes, respectively. Interestingly, after adjustment of the confounders, significant associations were observed among rs9939609 polymorphism and BMI, Wt, WC, trunk fat percentage, WHR, and WHtR. CONCLUSIONS: A-allele of the FTO rs9939609 polymorphism was indicated to be associated with greater general and central obesity in adult population of Shiraz, Iran.

Founder Effect of KHDC3L, p.M1V Mutation, on Iranian Patients with Recurrent Hydatidiform Moles.

BACKGROUND: Recurrent hydatidiform moles (RHMs) are an unusual pregnancy with at least two molar gestations that are associated with abnormal proliferation of trophoblastic tissue and a failure in the embryonic tissues development. Three maternal-effect genes, including NLRP7, KHDC3L, and PADI6 have been identified as the cause of RHMs. The present study aimed to understand the association of a founder mutation with the incidence and prevalence of a disease in different individuals of a population. METHODS: 14 unrelated Iranian patients with recurrent reproductive wastage, including at least two HMs, entered this study. In order to find a possible mutation in KHDC3L, all the 14 samples were Sanger sequenced. For haplotype analysis, three single nucleotide polymorphisms (SNPs) were selected with highest Minor Allele Frequency along KHDC3L. RESULTS: A common KHDC3L mutation with the same haplotype was identified in four out of 14 patients with RHM. Regarding the present study, c.1A>G is the highest reported mutation in KHDC3L so far and is also the first report of the homozygous state that has led to RHM. CONCLUSION: c.1A>G mutation in KHDC3L is the highest reported mutation around the world. Our data also demonstrated the presence of founder effects for this particular mutation in Iranian populations. These data suggest that the high frequency of this mutation is potentially responsible for a higher rate of RHM in Iran.

Two novel mutations in the MECP2 gene in patients with Rett syndrome.

Rett syndrome (RTT) is an X-linked severe neurological disorder. Mutations in Methyl-CpG-Binding Protein2 (MECP2) gene are the main cause of RTT disease. In this study, we report the results of screening the MECP2 gene for mutations in 7 Iranian patients with RTT syndrome. MECP2 sequencing identified two novel mutations in the heterozygous state, a splice mutation, c.354G>T, p.Gly119Gly, resulting in a premature splice-donor site and a 20-bp deletion, c.1167-1186del20 (p.P390Rfs), leading to modifying the c-terminal parts of the protein and it also changes the reading frames of all coding sequence downstream of the mutation. Multiple sequence alignment showed that amino acid changes occurred in the well conserved protein regions across species. Based on the results of this study and literature reviews, about 70% of mutations are found in exon 3 and 4 of the MECP2 gene, and mutations in exon 4 are more common than other exons. Therefore, it is recommended that exon 4 to be a priority for screening the genetic analysis of RTT patients.

Association of FTO rs9939609 polymorphism with serum leptin, insulin, adiponectin, and lipid profile in overweight adults.

FTO gene polymorphisms are associated with obesity and food intake. This study aimed to investigate the association of FTO rs9939609 polymorphism genotypes with serum glucose, lipid profile and serum hormones level. This cross-sectional study was carried out on 196 randomly selected overweight adults. Anthropometric measurements including weight, height, body mass index (BMI), fat mass, and fat-free mass were assessed. Serum TGs, total cholesterol, HDL cholesterol, LDL cholesterol, glucose and insulin levels were measured. The FTO gene was Genotyped for rs9939609 polymorphism. Dietary intake was assessed by avalid 168-item semi-quantitative food frequency questionnaire (FFQ). The homozygotes for the FTO rs9939609 risk allele (A) had higher serum leptin (p = 0.005, F: 5.131) and lower HDL (p = 0.001, F: 7.687) level than TT genotype. The differences between TT and AT genotypes were not significant. The association remained significant for HDL level after adjustments for age and sex, calorie intake, physical activity, and BMI. The association between rs9939609 polymorphism genotypes and leptin was disappeared after adjustments for calorie intake and physical activity. In conclusion, rs9939609 risk allele was associated with higher serum leptin and lower HDL levels in overweight people. Further studies are warranted.

Segmental Duplications as a Complement Strategy to Short Tandem Repeats in the Prenatal Diagnosis of Down Syndrome.

BACKGROUND: Quantitative fluorescence-polymerase chain reaction (QF-PCR) is an inexpensive and accurate method for the prenatal diagnosis of aneuploidies that applies short tandem repeats (STRs) as a chromosome-specific marker. Despite its apparent advantages, QF-PCR is not applicable in all cases due to the presence of uninformative STRs. This study was carried out to investigate the efficiency of a method based on applying segmental duplications (SDs) in conjunction with STRs as an alternative to stand-alone STR-based QF-PCR for the diagnosis of Down syndrome. METHODS: Fifty amniotic fluid samples from pregnant women carrying Down syndrome fetuses, 9 amniotic fluid samples with 1 or without any informative STR marker (inconclusive), and 100 normal samples were selected from Shiraz, Iran, between October 2015 and December 2016. Analysis was done using an in-house STR-SD-based multiplex QF-PCR and the results were compared. Statistical analysis was performed using MedCalc, version 14. RESULTS: All the normal, Down syndrome, and inconclusive samples were accurately identified by the STR-SD-based multiplex QF-PCR, yielding 100% sensitivity and 100% specificity. Karyotype analysis confirmed all the cases with normal or trisomic results. CONCLUSION: The STR-SD-based multiplex QF-PCR correctly identified all the normal and trisomy 21 samples regardless of the absence of informative STR markers. The STR-SD-based multiplex QF-PCR is a feasible and particularly useful assay in populations with a high prevalence of homozygote STR markers.

Further delineation of the phenotype caused by a novel large homozygous deletion of GRID2 gene in an adult patient.

Different mutations in glutamate receptor ionotropic delta 2 (GRID2) gene cause cerebellar ataxia in human. We report the largest homozygous deletion of the GRID2 gene reported to date, most probably causing complete loss of the gene product. Our patient presents mainly early onset cerebellar ataxia, cerebellar atrophy, nystagmus, and developmental delay with the least amount of intellectual disability.

A Novel Mutation in NLRP7 Related to Recurrent Hydatidiform Mole and Reproductive Failure.

BACKGROUND: Hydatidiform mole (HM) is an abnormal human pregnancy with excessive trophoblastic proliferation and abnormal embryonic development, dividing into two complete HM (CHM) and partial HM (PHM) groups. One subcategory of the CHMs is recurrent and familial, which is known as biparental HM (BiHMs) or recurrent HM (RHM). NLRP7, KHDC3L and PADI6 are maternal-effect genes involved in RHMs. NLRP7 is a major gene responsible for RHMs. This study was performed on patients with molar pregnancies and miscarriage. The aim of this study was to genetic screen for mutations in NLRP7 and KHDC3L genes in an affected woman with previous history of 5RHM and the sibling with history of miscarriage. MATERIALS AND METHODS: In this experimental study, DNA was extracted from blood samples. KHDC3L and NLRP7 were polymerase chain reaction (PCR) amplified. The PCR products were purified and Sanger sequenced. RESULTS: In this study, there is no mutation in KHDC3L gene but a novel mutation was identified in the NACHT domain of NLRP7 gene. Patient with five recurrent moles had this mutation in the homozygous state while her sister with one miscarriage and one normal child showed this mutation in the heterozygous state. CONCLUSION: In this study, we identified a new mutation in NLRP7 gene of a patient with recurrent HM. Following egg donation, this patient has a normal boy. The sister of this patient with heterozygous mutation has a spontaneous abortion and one normal child that confirm the impact of a defective allele of NLRP7 on reproductive wastage in a recent finding.

Novel mutation in the MED23 gene for intellectual disability: A case report and literature review.

MED23 deficiency causes the autosomal recessive Intellectual Disability (ID). Here we report an Iranian case with nonsyndromic ID presenting with developmental delay, microcephaly, hypotonia, severe ID, speech delay, and spasticity, who was homozygous for the novel MED23 c.670C>G variant. These results expand the clinical and mutation spectrum of MED23 deficiency.

An immunocompetent patient with a nonsense mutation in NHEJ1 gene.

BACKGROUND: DNA double-strand breaks (DSBs) are among the most deleterious types of DNA damage. DSBs are repaired by homologous recombination or non-homologous end-joining (NHEJ). NHEJ, which is central to the process of V(D)J recombination is the principle pathway for DSB repair in higher eukaryotes. Mutations in NHEJ1 gene have been associated with severe combined immunodeficiency. CASE PRESENTATION: The patient was a 3.5-year-old girl, a product of consanguineous first-degree cousin marriage, who was homozygous for a nonsense mutation in NHEJ1 gene. She had initially presented with failure to thrive, proportional microcephaly as well as autoimmune hemolytic anemia (AIHA), which responded well to treatment with prednisolone. However, the patient was immunocompetent despite having this pathogenic mutation. CONCLUSIONS: Herein, we report on a patient who was clinically immunocompetent despite having a pathogenic mutation in NHEJ1 gene. Our findings provided evidence for the importance of other end-joining auxiliary pathways that would function in maintaining genetic stability. Clinicians should therefore be aware that pathogenic mutations in NHEJ pathway are not necessarily associated with clinical immunodeficiency.

Enhancing Stability of Destabilized Green Fluorescent Protein Using Chimeric mRNA Containing Human Beta-Globin 5' and 3' Untranslated Regions.

BACKGROUND: In spite of recent progress in mRNA technologies and their potential applications for treatment of human diseases, problems such as the transient nature of mRNA limit the stability of gene up-regulation and, thus, potentially reduce mRNA efficiency for gene therapy. Using human ß-globin 5' and 3' untranslated regions (UTRs), this study aimed to develop the different chimeric constructs of mRNAs to increase the stability of destabilized green fluorescent protein (EGFPd2) in HEK 293 cells. METHODS: Purified human ß-globin (HBG) 5'-3'UTRs, and the coding sequence of destabilized green fluorescent protein (EGFPd2) were amplified separately and ligated to each other using SOEing PCR method in a different format. As controls, the original construct of EGFPd2 under the control of T7 promoter was used. Following in vitro transcription, HEK 293 cells were then transfected with several constructs and incubated at 37°C in a CO2 incubator. They were monitored under a fluorescence microscope every four hours for the first 24 hr, then every 12 hr afterwards. The resulting fluorescence was measured as a surrogate for translation efficiency and duration. RESULTS: By monitoring the HEK cells over 48 hr, cells transfected with mRNA with various HBG UTRs showed significantly different fluorescence intensity and stability in comparison with the pEGFPd2 prototype (control transcript) overtime. Overall, the images show that replacement of the 3' UTR end of the prototype vector pGFPd2 with the 3' end of ß-globin mRNA increases the half-life of the chimeric mRNA for more than 32 hr. CONCLUSION: This result indicates that ß-globin 3' UTR would definitely increase the half-life of mRNA and may help to decrease the mRNA therapeutic dosage in the treatment of diseases associated with mRNA therapy.

The First Case of a Small Supernumerary Marker Chromosome 18 in a Klinefelter Fetus: A Case Report.

Small supernumerary marker chromosomes (sSMCs), or markers, are abnormal chromosomal fragments that can be hereditary or de novo. Despite the importance of sSMCs diagnosis, de novo sSMCs are rarely detected during the prenatal diagnosis process. Usually, prenatally diagnosed de novo sSMCs cannot be correlated with a particular phenotype without knowing their chromosomal origin and content; therefore, molecular cytogenetic techniques are applied to achieve this goal. The present study aimed to characterize an sSMC in a case of Klinefelter syndrome using an in-house microsatellite analysis method and fluorescent in situ hybridization (FISH) technique. Amniotic fluid was collected from a pregnant woman who was considered to have risk factors for trisomy higher than the screening cut-off. Karyotype analysis was followed by the amplification of different microsatellite loci and FISH technique. Karyotype analysis identified a fetus with an extra X chromosome and also an sSMC with unknown identity. Further investigation of the parents showed that the sSMC is de novo. Microsatellite amplification by quantitative fluorescent PCR (QF-PCR) and FISH analysis showed that the sSMC is a derivative of chromosome 18. Eventually, the patient decided to terminate the pregnancy. Here, the first case of the coincidence of sSMC 18 in a Klinefelter fetus is reported.

Advanced molecular approaches pave the road to a clear-cut diagnosis of hereditary retinal dystrophies.

Purpose: The aim of this study was to identify the molecular genetic basis of hereditary retinal dystrophies (HRDs) in five unrelated Iranian families. Methods: Whole exome sequencing and Sanger sequencing were performed in all families. Variants were analyzed using various bioinformatics databases and software. Results: Based on the selected strategies, we identified potentially causative variants in five families with HRDs: the novel homozygous deletion mutation c.586_589delTTTG (p.F196Sfs*56) in the TTC8 gene of family A, the novel homozygous missense mutation c.2389T>C (p.S797P) in the CRB1 gene in family B, the novel homozygous frameshift mutation c.2707dupA (p.S903Kfs*66) in the LRP5 gene in family C, the novel homozygous splice mutation c.584-1G>T in the MERTK gene in family D, and the novel homozygous missense mutation c.1819G>C (p.G607R) rs61749412 in the ABCA4 gene of family E. Conclusions: This study highlights the presence of five novel variants associated with retinal dystrophies in selected Iranian families with hereditary blindness.