A Study of Associations Between rs9349379 (PHACTR1), rs2891168 (CDKN2B-AS), rs11838776 (COL4A2) and rs4880 (SOD2) Polymorphic Variants and Coronary Artery Disease in Iranian Population.

Recent genome-wide association studies reported the association of polymorphic alleles of PHACTR1 (rs9349379 (G)), CDDKN2B-AS1 (rs2891168 (G)), COL4A2 (rs11838776 (A)) and SOD2 (rs4880 (T)) with increased risk of coronary artery disease (CAD). The aim of our study was to assess the association of genetic variants with risk of CAD and its severity and in Southeast Iranian population. This study was examined in 250 CAD-suspected patients (mean age 53.49 ± 6.9 years) and 250 healthy individuals (mean age 52.96 ± 5.9 years). The Taqman SNP genotyping assay was used for genotyping of rs9349379 and rs2891168 variants. Tetra-primer Amplified refractory mutation system-PCR (Tetra-primer ARMS-PCR) was employed for rs11838776 and rs4880. Multivariate logistic regression analyses indicated that the G allele of rs9349379 and rs2891168 were associated with increased risk of CAD. The GG homozygous genotype of rs9349379 and rs2891168 had also been associated with risk of CAD. Additionally, the AG genotype of rs2891168 was associated with CAD. The significance of association of rs2891168 (G, GG, AG) increases with severity of CAD; but the rs9349379 (G, GG) have shown reverse association with severity of CAD. The genetic variants of COL4A2 (rs11838776) and SOD2 (rs4880) reflected no association with CAD in Southeast Iranian population. The findings of this study revealed that the PHACTR1 (rs9349379) and CDKN2B-AS1 (rs2891168) genetic variants might serve as genetic risk factor in CAD.

A novel homozygous splice-site mutation in SCARB2 is associated with progressive myoclonic epilepsy with renal failure.

BACKGROUND: Progressive myoclonic epilepsy-4 with or without renal failure (EPM4) is a rare neurological autosomal recessive disorder caused by mutations in SCARB2 gene. In this study, we described clinical features and genetic causes of an Iranian family with two affected individuals whose clinical manifestations closely resembled progressive myoclonus epilepsy. METHODS: Our proband was a 38-year-old male with a history of tremor, generalized seizures, action myoclonus, ataxia, and dysarthria that presumptive diagnosed as progressive myoclonus epilepsy. His older sister has the same symptoms. Whole-exome sequencing of DNA sample from the proband was performed. Candidate variant and cosegregation were confirmed by direct sequencing. Functional prediction of candidate variant was performed using appropriate prediction tools. RESULTS: Genetic analysis identified a homozygous splicing c.423+1 G>A variant in the SCARB2 gene of the proband and his affected sister. Segregation study identified heterozygous state in four unaffected family members (parents and two children). The variant is localized at the first nucleotide of intron 3 and was not detected among in-house healthy controls. This variant was not reported in genetic databases and predicted to potentially alter the 5' donor splice site and disease causing using online prediction tools. It was classified as a likely pathogenic variant according to ACMG standards and guidelines. CONCLUSION: This is the first report that demonstrates c.423+1 G>A variant in the SCARB2 gene segregating with the phenotype of EPM4 in a consanguineous Iranian family.

Haplotype Analysis in Carriers of ß-Globin Gene Mutation Facilitates Genetic Counseling in ß-Thalassemia: A Cross-Sectional Study in Kerman Province, Iran.

BACKGROUND: ß-thalassemia is characterized by reduced synthesis of the hemoglobin beta chain that results in microcytic hypochromic anemia and reduced amounts of hemoglobin A (HbA) on hemoglobin analysis. ß-thalassemias are caused by mutations in the ß-globin gene, inherited in an autosomal recessive manner. Determining molecular defects in couples carrying ß-thalassemia is a prerequisite for prenatal diagnosis of the disease. In this regards, database of ß-globin gene haplotypes facilitates mutation detection of the gene and helps genetic counselors to reach the goals of ß-thalassemia prevention program. METHODS: In this cross-sectional study, 255 couples attended genetic counseling between December 2017 and January 2019 in Afzalipour Hospital, Kerman University of Medical Scinces, Kerman, Iran as suspicious of ß-thalassemia carriers. Furthermore, they were investigated using amplification refractory mutations system-polymerase chain reaction and restriction fragment length polymorphism methods for mutation screening and haplotype analysis of polymorphic sites in ß-globin gene cluster, respectively. RESULTS: We identified 20 different types of ß-globin gene mutation in 449 ß-thalassemia carriers. Analysis of the pattern of Hind III/Gγ, Hinf I/5'ß, Hinc II/3'Ψß, Rsa I/5'ß, AvaII/ß and Hind III/Aγ polymorphic sites in 257 alleles of informative families revealed 17 different haplotypes. Haplotype 1 (77.24%) showed strong linkage with the most common mutation IVSI-5 while haplotype 5 (66.67%) was associated with the second frequent mutation IVSII-1. CONCLUSION: To our knowledge, these ß-globin haplotypes are reported for the first time which are different with those found in other parts of Iran. The current haplotypes pattern data makes the counseling of ß-thalassemia carriers more straightforward and the process of mutation screening faster and more accurate.

Quantification of circulating miR-517c-3p and miR-210-3p levels in preeclampsia.

MicroRNAs (miRNAs, miRs) are small regulatory non-coding RNAs that regulate gene expression by incomplete complementary attachment to the 3'UTR, 5'UTR, ORF and promoter regions of target mRNAs. We compared plasma levels of miR-210-3p and miR-517c-3p as cell-free microRNAs (cfmiRNAs) in preeclamptic (n = 20) and healthy women (n = 20). These miRs are responsible for cell growth and proliferation, placental hypoxia, immune response and apoptosis. We found higher expression levels of miR-210 and miR-517c in preeclamptic cases (+3.34 and +2.27 fold change, respectively). This is the first study that evaluates the plasma levels of miR-517c in preeclamptic cases by real time PCR (RT-PCR) technique. This study can lead to new opportunities for research about the roles of miRNAs in preeclampsia etiology or new biomarkers.

A Novel Splice Site Mutation of the ATM Gene Associated with Ataxia Telangiectasia.

OBJECTIVES: Ataxia telangiectasia (AT) is a rare autosomal recessive disorder caused by mutation in the Ataxia telangiectasia mutated (ATM) gene. This disorder is characterized by progressive cerebellar ataxia, telangiectasia, immunodeficiency and a predisposition to leukemia/lymphoma. In this study, we investigated a family with a new mutation in ATM, confirmed by molecular genetic test. MATERIALS&METHODS: Four members of a family including a symptomatic AT patient, his parents and sibling were examined for ATM gene defects at Kerman University Hospital, Kerman, Iran in 2016. DNA was extracted from peripheral leukocytes and the coding regions and exon-intron boundaries of ATM gene were amplified by next-generation sequencing technique. The identified mutation was tested in all members of the family. RESULTS: Molecular analyses identified a homozygous T to G substitution in c.7308-6 position resulting in a novel acceptor splice site in intron 49 of the ATM gene in the index patient. Parents and sibling of the proband were heterozygous for the same mutation. CONCLUSION: The variant c.7308-6T>G is predicted to be pathogenic due to impaired splice site causing exon skipping. This newly found frameshift mutation cosegregated as an autosomal recessive trait as expected for Ataxia telangiectasia syndrome.

A novel homozygous frameshift mutation in the FUCA1 gene causes both severe and mild fucosidosis.

AIMS: Fucosidosis is a rare autosomal recessive lysosomal storage disorder caused by α-L-fucosidase deficiency as a result of FUCA1 gene mutations. Here, we studied clinical features and the molecular basis of fucosidosis in a family from Iran, including two probands and nine family members. METHODS: DNA sample of two probands were screened for gene defects using a next generation sequencing technique. The sequencing processes were performed on an Illumina Hiseq 4000 platform. Sequence reads were analysed using BWA-GATK. RESULTS: Next generation sequencing revealed a frameshift mutation caused by 2 bp deletion (c.837_838 delTG; p.Cys279) in the FUCA1 gene. The identified mutation was tested in all participants. Homozygous patients had almost all the complications associated with fucosidosis, while heterozygous carriers were unaffected. CONCLUSIONS: The variant c.837_838 delTG; p.Cys279 has not been reported previously and is predicted to be pathogenic due to a premature stop codon.

Effects of α-thalassaemia mutations on the haematological parameters of ß-thalassaemia carriers.

BACKGROUND: Thalassaemia is a haemoglobin disorder caused by a reduction in, or a complete absence of, the production of α- or ß-globin genes. Detection of ß-thalassaemia carriers is the first step in the prenatal diagnosis of the disease and is based primarily on the differences between levels of blood cell indices. Since co-inheritance of ß- and α-thalassaemia mutations modulates the haematological parameters of heterozygote ß-thalassaemia indices, understanding the influence of this interaction is helpful for identification of disease carriers. OBJECTIVE: To determine the effects of α-thalassaemia mutations on the haematological parameters of ß-thalassaemia carriers. METHOD: We used gap-PCR and amplification refractory mutation system techniques to find any α- and/or ß-thalassaemia mutations in 270 subjects who were suspected to be thalassaemia carriers. The mean values of the haematological parameters in α, ß-thalassaemia and ß-thalassaemia carriers were compared. RESULTS: Significant differences in mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and HbA2 were found between the two groups. Patients who were α, ß-thalassaemia carriers had higher mean values of MCV and MCH, whereas HbA2 levels were higher in simple ß-thalassaemia. No marked differences were found in mean cell haemoglobin (Hb) concentration and Hb blood cell indices. The value of MCV, MCH and HbA2 were significantly different between α,ß-thalassaemia and simple ß-thalassaemia in men and women, but the mean values of Hb in the two groups differed markedly only in men. CONCLUSION: We conclude that co-inheritance of α- and ß-thalassaemia mutations may result in misdiagnosis of ß-thalassaemia carriers. Therefore, in genetic counselling of patients with a near-normal range of blood cell indices the possibility that they may carry α, ß-thalassaemia mutations must be considered.

Homozygosity mapping and whole exome sequencing reveal a novel homozygous COL18A1 mutation causing Knobloch syndrome.

The aim of this study was to identify the genetic basis of a chorioretinal dystrophy with high myopia of unknown origin in a child of a consanguineous marriage. The proband and ten family members of Iranian ancestry participated in this study. Linkage analysis was carried out with DNA samples of the proband and her parents by using the Human SNP Array 6.0. Whole exome sequencing (WES) was performed with the patients' DNA. Specific sequence alterations within the homozygous regions identified by whole exome sequencing were verified by Sanger sequencing. Upon genetic analysis, a novel homozygous frameshift mutation was found in exon 42 of the COL18A1 gene in the patient. Both parents were heterozygous for this sequence variation. Mutations in COL18A1 are known to cause Knobloch syndrome (KS). Retrospective analysis of clinical records of the patient revealed surgical removal of a meningocele present at birth. The clinical features shown by our patient were typical of KS with the exception of chorioretinal degeneration which is a rare manifestation. This is the first case of KS reported in a family of Iranian ancestry. We identified a novel disease-causing (deletion) mutation in the COL18A1 gene leading to a frameshift and premature stop codon in the last exon. The mutation was not present in SNP databases and was also not found in 192 control individuals. Its localization within the endostatin domain implicates a functional relevance of endostatin in KS. A combined approach of linkage analysis and WES led to a rapid identification of the disease-causing mutation even though the clinical description was not completely clear at the beginning.

Sengers syndrome: six novel AGK mutations in seven new families and review of the phenotypic and mutational spectrum of 29 patients.

BACKGROUND: Sengers syndrome is an autosomal recessive condition characterized by congenital cataract, hypertrophic cardiomyopathy, skeletal myopathy and lactic acidosis. Mutations in the acylglycerol kinase (AGK) gene have been recently described as the cause of Sengers syndrome in nine families. METHODS: We investigated the clinical and molecular features of Sengers syndrome in seven new families; five families with the severe and two with the milder form. RESULTS: Sequence analysis of AGK revealed compound heterozygous or homozygous predicted loss-of-function mutations in all affected individuals. A total of eight different disease alleles were identified, of which six were novel, homozygous c.523_524delAT (p.Ile175Tyrfs*2), c.424-1G > A (splice site), c.409C > T (p.Arg137*) and c.877 + 3G > T (splice site), and compound heterozygous c.871C > T (p.Gln291*) and c.1035dup (p.Ile346Tyrfs*39). All patients displayed perinatal or early-onset cardiomyopathy and cataract, clinical features pathognomonic for Sengers syndrome. Other common findings included blood lactic acidosis and tachydyspnoea while nystagmus, eosinophilia and cervical meningocele were documented in only either one or two cases. Deficiency of the adenine nucleotide translocator was found in heart and skeletal muscle biopsies from two patients associated with respiratory chain complex I deficiency. In contrast to previous findings, mitochondrial DNA content was normal in both tissues. CONCLUSION: We compare our findings to those in 21 previously reported AGK mutation-positive Sengers patients, confirming that Sengers syndrome is a clinically recognisable disorder of mitochondrial energy metabolism.

Identification of a SLC19A2 nonsense mutation in Persian families with thiamine-responsive megaloblastic anemia.

Thiamine-responsive megaloblastic anemia (TRMA) is an autosomal recessive syndrome characterized by early-onset anemia, diabetes, and hearing loss caused by mutations in the SLC19A2 gene. We studied the genetic cause and clinical features of this condition in patients from the Persian population. A clinical and molecular investigation was performed in four patients from three families and their healthy family members. All had the typical diagnostic criteria. The onset of hearing loss in three patients was at birth and one patient also had a stroke and seizure disorder. Thiamine treatment effectively corrected the anemia in all of our patients but did not prevent hearing loss. Diabetes was improved in one patient who presented at the age of 8months with anemia and diabetes after 2months of starting thiamine. The coding regions of SLC19A2 were sequenced in all patients. The identified mutation was tested in all members of the families. Molecular analyses identified a homozygous nonsense mutation c.697C>T (p.Gln233*) as the cause of the disease in all families. This mutation was previously reported in a Turkish patient with TRMA and is likely to be a founder mutation in the Persian population.

Identification of homozygous WFS1 mutations (p.Asp211Asn, p.Gln486*) causing severe Wolfram syndrome and first report of male fertility.

Wolfram syndrome (WFS) is a neurodegenerative genetic condition characterized by juvenile-onset of diabetes mellitus and optic atrophy. We studied clinical features and the molecular basis of severe WFS (neurodegenerative complications) in two consanguineous families from Iran. A clinical and molecular genetic investigation was performed in the affected and healthy members of two families. The clinical diagnosis of WFS was confirmed by the existence of diabetes mellitus and optic atrophy in the affected patients, who in addition had severe neurodegenerative complications. Sequencing of WFS1 was undertaken in one affected member from each family. Targeted mutations were tested in all members of relevant families. Patients had most of the reported features of WFS. Two affected males in the first family had fathered unaffected children. We identified two homozygous mutations previously reported with apparently milder phenotypes: family 1: c.631G>A (p.Asp211Asn) in exon 5, and family 2: c.1456C>T (p.Gln486*) in exon 8. Heterozygous carriers were unaffected. This is the first report of male Wolfram patients who have successfully fathered children. Surprisingly, they also had almost all the complications associated with WFS. Our report has implications for genetic counseling and family planning advice for other affected families.

Congenital insensitivity to pain with anhidrosis in an Iranian patient.

Congenital insensitivity to pain with anhidrosis is a rare disease of the nervous system which causes one to lose his/her feeling of pain. The disease is subtype four of hereditary sensory and autonomic neuropathy (HSAN IV) that results from NTRK1 gene defect. Direct sequencing was performed to screen NTRK1 for mutations. The result revealed a homozygous deletion of adenine on intron 14 that may cause the disease.

BRCA1 Gene Mutations in Breast Cancer Patients from Kerman Province, Iran.

BACKGROUND: Breast cancer is the most common malignancy in Iranian women. Mutations in BRCA1 gene is one of the important genetic predisposing factors in breast cancer. This gene is a tumor suppressor that plays an important role in regulating the functions of RAD51 protein for strand invasion in homologous recombination repair. METHODS: The BRCA1 gene has amplified in the DNA isolated from breast cancer patients' leukocytes, using Polymerase Chain Reaction technique. The PCR products have sequenced using an automated DNA sequencer and subsequently obtained data have aligned with the human BRCA1 DNA sequences available online. RESULTS: In this study, we have considered nine different mutations on 60 examined chromosomes from 30 patients, living in Kerman province. A deletion of one adenine (c.1017delA) and insertion of one cytosine (c.969InsC) have found as the most frequent (20%) mutation in this survey. A substitution of thymine for adenine (c.999T>A) has detected as the second common BRCA1 gene defect (6.7%). The other mutations have identified as single nucleotide replacement including: c.792A>C, c.825G>C, c.822T>A, c.1068A>G, c.969A>T and c.966T>C. CONCLUSION: The identified BRCA1 mutations were in accordance with the previous reports. To our knowledge, four mutations: (c.969InsC, c.792A>C, c.825G>C, c.822T>A) which have identified in this study, have not been previously reported in the literature. A larger cohort study would help identifying all relevant BRCA1 mutations in this population.

Senior-Loken syndrome secondary to NPHP5/IQCB1 mutation in an Iranian family.

Senior-Loken syndrome (SLS) is a rare autosomal recessive disease characterized by nephronophthisis and early-onset retinal degeneration. We used a large Iranian family with SLS to establish a molecular genetic diagnosis. Following clinical evaluation, we undertook homozygosity mapping in two affected family members and mutational analysis in known SLS genes coinciding with regions of homozygosity. In a region of homozygosity coinciding with a known SLS locus on chromosome 3q21.1, we found a homozygous non-sense mutation R332X in NPHP5/IQCB1. This is the first report of a molecular genetic diagnosis in an Iranian kindred with SLS.

Spectrum of α-globin gene mutations in the Kerman Province of Iran.

Mutation of the α-globin gene may result in α-thalassemia (α-thal) which is characterized by a reduction or complete absence of the gene expression. In this study, 607 individuals with low levels of blood cell indices and normal Hb A(2) were referred to our laboratory for investigation of any α-thal mutations. We used the gap-polymerase chain reaction (gap-PCR) method and an α-globin strip assay kit to detect the mutation. Our results showed that -α(3.7) was the most common mutation (83.8%) in the overall mutated alleles of the α-globin gene. The second and third most frequent α-globin gene defects were codon 19 (α2) and IVS-I, -5 nt/αα (α2), 5.7 and 4.2%, respectively. We found that the spectrum of α-globin gene mutation in Kerman Province was in accordance with what was previously reported in other Iranian provinces where malaria has selected these protective traits.

A family with the 619 bp deletion on the beta-globin gene found in Kerman Province, Iran.

In the present study, we report the first case of a 619 bp deletion on the beta-globin gene, found in a family from Kerman Province, Iran. This mutation is frequent in Pakistan, but it has not been previously reported in any part of Iran. This study revealed that all family members have this beta(0)-thalassemia (beta(0)-thal) mutation.

The ERCC1/XPF endonuclease is required for completion of homologous recombination at DNA replication forks stalled by inter-strand cross-links.

Both the ERCC1-XPF complex and the proteins involved in homoIogous recombination (HR) have critical roles in inter-strand cross-link (ICL) repair. Here, we report that mitomycin C-induced lesions inhibit replication fork elongation. Furthermore, mitomycin C-induced DNA double-strand breaks (DSBs) are the result of the collapse of ICL-stalled replication forks. These are not formed through replication run off, as we show that mitomycin C or cisplatin-induced DNA lesions are not incised by global genome nucleotide excision repair (GGR). We also suggest that ICL-lesion repair is initiated either by replication or transcription, as the GGR does not incise ICL-lesions. Furthermore, we report that RAD51 foci are induced by cisplatin or mitomycin C independently of ERCC1, but that mitomycin C-induced HR measured in a reporter construct is impaired in ERCC1-defective cells. These data suggest that ERCC1-XPF plays a role in completion of HR in ICL repair. We also find no additional sensitivity to cisplatin by siRNA co-depletion of XRCC3 and ERCC1, showing that the two proteins act on the same pathway to promote survival.

The ERCC1/XPF endonuclease is required for efficient single-strand annealing and gene conversion in mammalian cells.

The mammalian ERCC1-XPF endonuclease has a suggested role in the repair of DNA double-strand breaks (DSB) by single-strand annealing (SSA). Here, we investigated the role of ERCC1 in homologous recombination in mammalian cells, and confirm a role of ERCC1 in SSA. Interestingly, we also report an unexpected role for ERCC1 in gene conversion. This provides support that gene conversion in mammalian somatic cells is carried out through synthesis-dependent strand annealing, rather than through a double Holliday Junction mechanism. Moreover, we find low frequencies of SSA and gene conversion in G1-arrested cells, suggesting that SSA is not a frequent DSB repair pathway in G1-arrested mammalian cells, even in the presence of perfect repeats. Furthermore, we find that SSA is not influenced by inhibition of CDK2 (using Roscovitine), ATM (using Caffeine and KU55933), Chk1 (using CEP-3891) or DNA-PK (using NU7026).

Spontaneous homologous recombination is induced by collapsed replication forks that are caused by endogenous DNA single-strand breaks.

Homologous recombination is vital to repair fatal DNA damage during DNA replication. However, very little is known about the substrates or repair pathways for homologous recombination in mammalian cells. Here, we have compared the recombination products produced spontaneously with those produced following induction of DNA double-strand breaks (DSBs) with the I-SceI restriction endonuclease or after stalling or collapsing replication forks following treatment with thymidine or camptothecin, respectively. We show that each lesion produces different spectra of recombinants, suggesting differential use of homologous recombination pathways in repair of these lesions. The spontaneous spectrum most resembled the spectra produced at collapsed replication forks formed when a replication fork runs into camptothecin-stabilized DNA single-strand breaks (SSBs) within the topoisomerase I cleavage complex. We found that camptothecin-induced DSBs and the resulting recombination repair require replication, showing that a collapsed fork is the substrate for camptothecin-induced recombination. An SSB repair-defective cell line, EM9 with an XRCC1 mutation, has an increased number of spontaneous gammaH2Ax and RAD51 foci, suggesting that endogenous SSBs collapse replication forks, triggering recombination repair. Furthermore, we show that gammaH2Ax, DSBs, and RAD51 foci are synergistically induced in EM9 cells with camptothecin, suggesting that lack of SSB repair in EM9 causes more collapsed forks and more recombination repair. Furthermore, our results suggest that two-ended DSBs are rare substrates for spontaneous homologous recombination in a mammalian fibroblast cell line. Interestingly, all spectra showed evidence of multiple homologous recombination events in 8 to 16% of clones. However, there was no increase in homologous recombination genomewide in these clones nor were the events dependent on each other; rather, we suggest that a first homologous recombination event frequently triggers a second event at the same locus in mammalian cells.