Pancreatitis as a Main Consequence of APOC2-Related Hypertriglyceridemia: The Role of Nonsense and Frameshift Variants.

APOC2-related hypertriglyceridemia occurs due to biallelic variants of this gene. Here, genotype-phenotype architecture of all pathogenic APOC2 variants is investigated among heterozygous and homozygous individuals. Clinical heterogeneity of various types of the variants is also described, and pancreatitis in more than half of homozygotes carrying chain-termination variants is highlighted as well. For this study, patients were selected who had a plasma triglyceride level above 250 mg/dL. The coding and intronic regions of the APOC2 gene were amplified using the Sanger sequencing to investigate the presence of variants. The genotypes, lipid profiles, and detailed clinical features were documented for all APOC2-related patients and heterozygous individuals. Pathogenicity of the variants was predicted and categorized using available bioinformatics tools such as MutationTaster and PolyPhen-2 and ACMG criteria. MetaDome and Phyre2 were applied for structural and functional in silico analyses. 40% (12 out of 30) of APOC2 variants were chain-termination (nonsense and frameshift) variants. These types of variants were determined in 60.53% of patients. 55% of these patients showed pancreatitis followed by lipemia retinalis (29%), abdominal pain (24%), hepatosplenomegaly (24%), and xanthomas (18%). The mean age of onset was about 22 years old. In at least 50% of 38 homozygous individuals, the TG level was more than 2000 mg/dL. More than 25% of heterozygous individuals showed at least one symptom. Pancreatitis and a severe form of HTG were found in 5 and 2% of heterozygous individuals, respectively. The main clinical features of APOC2-related hypertriglyceridemia include pancreatitis, lipemia retinalis, abdominal pain, hepatosplenomegaly, and xanthomas. Nonsense and frameshift homozygous variants usually lead to a severe form of hypertriglyceridemia. Pancreatitis is one of the main consequences of these types of mutations; thus, it is important to consider this point when evaluating asymptomatic individuals. Heterozygous individuals may become symptomatic due to the role of unknown modifying agent including environmental genetic factors.

Evaluation of the presence of integrons, sul and smqnr genes and the prevalence of antibiotic resistance in Stenotrophomonas maltophilia clinical isolates.

OBJECTIVES: The objective of this investigation was to examine the mechanisms associated with antibiotic resistance in Stenotrophomonas maltophilia clinical isolates retrieved from hospitalized patients undergoing open heart surgery in a Heart Center located in Tehran, Iran. MATERIALS AND METHODS: This investigation encompassed a cross-sectional study of 60 S. maltophilia isolates, which were procured from diverse clinical specimens. Primary identification of the isolates was conducted through conventional microbiologic methods and subsequently verified by means of PCR primers. The E-test was utilized to establish the minimum inhibitory concentrations (MICs). PCR was then employed to ascertain the antibiotic resistance genes (sul1, sul2, Smqnr and intl1 - intl3). RESULTS: In this study, a total of sixty clinical isolates of S. maltophilia were collected, with the majority of them being obtained from Intensive Care Units (ICU) (n = 54; 90%). The disk diffusion method yielded results indicating that 55% of the isolates were sensitive to minocycline, whereas 30% were intermediate and 15% were found to be resistant. Additionally, the MIC results revealed that the resistant rates of the isolates towards ceftazidime, cotrimoxazole and levofloxacin were 46.7%, 1.7% and 5%, respectively. The PCR amplification of three classes of integrons genes indicated that fifteen (25%) of the isolates carried int1, while no detection for intl2 and intl3 was reported. Furthermore, the prevalence of antibiotic resistance genes (sul1, sul2, and Smqnr) was identified in 15 (25%), 6 (10%), and 28 (46.7%) isolates, respectively. CONCLUSION: The reported increasing rate of antibiotic resistance and mobile genetic elements that could extend the resistance genes to other strains in the hospital, finally it could be an alarming issue for healthcare settings that need special attention to this strain and the epidemiological study on this issue.

Neurofibromatosis-Noonan syndrome and growth deficiency in an Iranian girl due to a pathogenic variant in NF1 gene.

BACKGROUND: Mutations in NF1 gene could cause allelic disorders with clinical spectrum of Neurofibromatosis type 1 to Noonan syndrome. Here, a 7-year-old Iranian girl is described with Neurofibromatosis-Noonan syndrome due to a pathogenic variant in NF1 gene. METHODS: Clinical evaluations were performed along with genetic testing using whole exome sequencing (WES). The variant analysis including pathogenicity prediction was also done using bioinformatics tools. RESULTS: The chief compliant of the patient was short stature and lack of proper weight gain. Other symptoms were developmental delay, learning disability, inadequate speech skill, broad forehead, hypertelorism, and epicanthal folds, low set ears and webbed neck. A small deletion, c.4375-4377delGAA, was found in NF1 gene using WES. This variant was classified as pathogenic according to ACMG. CONCLUSIONS: NF1 variants may show variable phenotypes among the patients; identifying such variants is helpful in therapeutic management of the disease. WES is considered as an appropriate test to diagnose Neurofibromatosis-Noonan syndrome.

Clinical and Molecular Findings of Autosomal Recessive Spastic Ataxia of Charlevoix Saguenay: an Iranian Case Series Expanding the Genetic and Neuroimaging Spectra.

Autosomal recessive spastic ataxia of Charlevoix Saguenay (ARSACS) is now increasingly identified from all countries over the world, possibly rendering it one of the most common autosomal recessive ataxias. Here, we selected patients harboring SACS variants, the causative gene for ARSACS, in a large cohort of 137 patients with early-onset ataxia recruited from May 2019 to May 2021 and were referred to the ataxia clinic. Genetic studies were performed for 111 out of 137 patients (81%) which led to a diagnostic rate of 72.9% (81 out of 111 cases). Ten patients with the molecular diagnosis of ARSACS were identified. We investigated the phenotypic and imaging spectra of all confirmed patients with ARSACS. We also estimated the frequency of ARSACS in this cohort and described their clinical and genetic findings including seven novel variants as well as novel neuroimaging findings. While the classic clinical triad of ARSACS is progressive cerebellar ataxia, spasticity, and sensorimotor polyneuropathy, it is not a constant feature in all patients. Sensorimotor axonal-demyelinating neuropathy was detected in all of our patients, but spasticity and extensor plantar reflex were absent in 50% (5/10). In all patients, brain magnetic resonance imaging (MRI) showed symmetric linear hypointensities in the pons (pontine stripes) and anterior superior cerebellar atrophy as well as a hyperintense rim around the thalami (thalamic rim). Although infratentorial arachnoid cyst has been reported in ARSACS earlier, we report anterior temporal arachnoid cyst in two patients for the first time, indicating that arachnoid cyst may be an associated imaging feature of ARSACS. We also extended molecular spectrum of ARSACS by presenting 8 pathogenic and one variant of unknown significance (VUS) sequence variants, which 7 of them have not been reported previously. MetaDome server confirmed that the identified VUS variant was in the intolerant regions of sacsin protein encoded by SACS.

Genetic testing of leukodystrophies unraveling extensive heterogeneity in a large cohort and report of five common diseases and 38 novel variants.

This study evaluates the genetic spectrum of leukodystrophies and leukoencephalopathies in Iran. 152 children, aged from 1 day to 15 years, were genetically tested for leukodystrophies and leukoencephalopathies based on clinical and neuroradiological findings from 2016 to 2019. Patients with a suggestive specific leukodystrophy, e. g. metachromatic leukodystrophy, Canavan disease, Tay-Sachs disease were tested for mutations in single genes (108; 71%) while patients with less suggestive findings were evaluated by NGS. 108 of 152(71%) had MRI patterns and clinical findings suggestive of a known leukodystrophy. In total, 114(75%) affected individuals had (likely) pathogenic variants which included 38 novel variants. 35 different types of leukodystrophies and genetic leukoencephalopathies were identified. The more common identified disorders included metachromatic leukodystrophy (19 of 152; 13%), Canavan disease (12; 8%), Tay-Sachs disease (11; 7%), megalencephalic leukodystrophy with subcortical cysts (7; 5%), X-linked adrenoleukodystrophy (8; 5%), Pelizaeus-Merzbacher-like disease type 1 (8; 5%), Sandhoff disease (6; 4%), Krabbe disease (5; 3%), and vanishing white matter disease (4; 3%). Whole exome sequencing (WES) revealed 90% leukodystrophies and genetic leukoencephalopathies. The total diagnosis rate was 75%. This unique study presents a national genetic data of leukodystrophies; it may provide clues to the genetic pool of neighboring countries. Patients with clinical and neuroradiological evidence of a genetic leukoencephalopathy should undergo a genetic analysis to reach a definitive diagnosis. This will allow a diagnosis at earlier stages of the disease, reduce the burden of uncertainty and costs, and will provide the basis for genetic counseling and family planning.

GATA4 screening in Iranian patients of various ethnicities affected with congenital heart disease: Co-occurrence of a novel de novo translocation (5;7) and a likely pathogenic heterozygous GATA4 mutation in a family with autosomal dominant congenital heart disease.

BACKGROUND: Congenital heart disease (CHD) is the most common birth defect and a major health problem around the world. However, its exact etiology has remained unclear. Among various genetic contributing factors, GATA4 transcription factor plays a significant role in the CHD pathogenesis. In this study, GATA4 coding sequence was screened in Iranian patients of various ethnicities. METHODS: Sixty six individuals with familial CHD referred to our center were recruited in this study. After receiving written informed consent from each individual or their parents, chromosomal analyses and GATA4 variant screening were performed. Pathogenicity of the suspected variants was evaluated using available online software tools: CADD, Mutation Taster, SIFT, and PolyPhen-2. RESULTS: A total of twelve GATA4 variants were detected including five intronic, 2 exonic and 3 polymorphisms as well as 2 missense mutations, the c.1220C>A and c.1309G>A. Unlike the c.1220C>A, the likely pathogenic heterozygous c.1309G>A has not been previously associated with any phenotype. Here, we not only report, for the first time, a c.1309G>A-related CHD, but also report a novel de novo balanced translocation, 46,XY,t(5;7)(qter13;qter11), in the same patient which may have influenced the disease severity. CONCLUSION: From screening GATA4 sequence in 66 Iranian patients of various ethnicities, we conclude that cytogenetic analysis and PCR-direct sequencing of different candidate genes may not be the best approach for genetic diagnosis in CHD. Applying novel approaches such as next-generation sequencing (NGS) may provide a better understating of genetic contributing factors in CHD patients for whom conventional methods could not reveal any genetic causative factor.

Mosaic trisomy 22 in a 4-year-old boy with congenital heart disease and general hypotrophy: A case report.

BACKGROUND: Trisomy 22 mosaicism is a rare autosomal anomaly with survival compatibility. Recognition of the complete trisomy 22 which is incompatible with life from the mosaic form is critical for genetic counseling. Affected mosaic cases have prevalent clinical presentations such as webbed neck, developmental delay, abnormal ears, cardiac disorders, and microcephaly. Phenotype of these patients is milder than full chromosomal aneuploidy, and the severity of the phenotype depends on the count of trisomic cells. We describe a 4-year-old boy with mosaic trisomy 22 from healthy parents and no family history of any genetic disorders in the pedigree. METHOD AND RESULTS: The patient had determined dysmorphic clinical features including facial asymmetry, cleft palate, gastroenteritis, hydronephrosis, developmental delay, genital anomalies, dysplastic toenails, flattened nasal bridge, congenital heart defect, hearing loss, cryptorchidism, and hypotonic muscle. He is the first reported with hypothyroidism and larynx wall thickness in worldwide and the first with atrial septal defect (ASD) from Iran. Chromosomal analyses using G-banding indicated a de novo Mos 47,XY,+22(6)/46,XY(44) karyotype with no other chromosomal structural changes. CONCLUSIONS: Our observations confirm the importance of cytogenetic analyses for determining the cause of congenital anomalies and provide a useful genetic counseling. In addition, due to the fact that some of mosaic trisomy 22 features are unavoidable such as CHD and general hypotrophy, we suggest including echocardiography test for early diagnosis during the clinical assessment.

Autosomal Recessive Nonsyndromic Arrhythmogenic Right Ventricular Cardiomyopathy without Cutaneous Involvements: A Novel Mutation.

The arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is a genetic disease frequently associated with desmosomal mutations, mainly attributed to dominant mutations in the Plakophilin-2 (PKP2) gene. Naxos and Carvajal are the syndromic forms of ARVD/C due to recessive mutations. Herein, we report an autosomal recessive form of nonsyndromic ARVD/C caused by a mutation in the PKP2 gene. After examination and implementation of diagnostic modalities, the definite diagnosis of ARVD/C was confirmed by detection of ventricular tachycardia with a left bundle branch configuration and a superior axis, T-wave inversion in right precordial leads (i.e., V1-V3) in a 12-lead electrocardiogram, and a right ventricle outflow tract dilatation. Neither cutaneous involvement nor other abnormalities were observed. Genetic testing was performed during which an intronic mutation of c.2577+1G>T in the PKP2 gene was observed homozygously. The c.2577+1G>T disrupts PKP2 mRNA splicing and causes a nonsyndromic form of ARVD/C.

MYO15A splicing mutations in hearing loss: A review literature and report of a novel mutation.

Sensorineural hearing loss (SNHL) is the most prevalent genetic sensory defect in humans, affecting about 1 in 1000 newborns around the world. Non-syndromic SNHL accounts for nearly 70% of hereditary hearing loss and 80% of SNHL cases show an autosomal recessive mode of inheritance (ARNSHL). In the present study, we applied targeted-exome sequencing to a family with a single proband affected by congenital sensorineural hearing loss. 127 known genes were sequenced to find the causative mutation. One novel homozygous donor splice site mutation, c.4596 + 1G > A (IVS12 + 1G > A) was found in MYO15A gene. Analysis of this mutation within the family showed that the mutation segregates with hearing loss. New DNA sequencing technologies could lead to identification of the disease causing variants especially in highly heterogeneous disorders such as hearing loss.

Construction, expression, and activity of a novel immunotoxin comprising a humanized antiepidermal growth factor receptor scFv and modified Pseudomonas aeruginosa exotoxin A.

Overexpression of epidermal growth factor receptor (EGFR) plays a significant role in the development and metastasis of many solid tumors. Strategies based on anti-EGFR immunotoxins have shown promising results in several studies, but immunogenicity of antibody and toxin moieties is a limitation of this type of therapeutics. In the present study, a novel humanized anti-EGFR immunotoxin (huscFv-PE25) was developed by genetic fusing of a humanized anti-EGFR single-chain variable fragment (huscFv) with a modified Pseudomonas aeruginosa exotoxin A (PE25KDEL). The reactivity and toxicity of this immunotoxin with tumor cells were assessed by dot-blot, enzyme-linked immunosorbent assay, and MTT procedures. Results of enzyme-linked immunosorbent assay and dot-blot assay indicated that the immunotoxin recognizes and efficiently binds to EGFR-overexpressing tumor cells. MTT assay showed a specific growth-inhibitory effect of huscFv-PE25 on EGFR-overexpressing A431 cells, without any inhibitory effect on EGFR-negative cells. In conclusion, the results of this study indicated that huscFv-PE25 can recognize and exert an inhibitory effect on EGFR-overexpressing cancer cells, despite its smaller size and lower immunogenicity. This may provide a basis for the development of novel clinical therapeutic agents against EGFR-overexpressing tumor cells.

Design, expression and evaluation of a novel humanized single chain antibody against epidermal growth factor receptor (EGFR).

Various strategies have been attempted for targeting of epidermal growth factor receptor (EGFR), as an essential biomarker in a variety of cancers. Several anti-EGFR antibodies including cetuximab are used in clinics for treatment of EGFR-overexpressing colorectal and head and neck cancers but the efficiency of these antibodies is threatened by their large size and chimeric nature. Humanized single chains antibodies (huscFv) are smaller generation of antibodies with lower immunogenicity may overcome these limitations. This article reports production and evaluation of a novel humanized anti-EGFR scFv. The CDRs of cetuximab heavy and light chains were grafted onto human antibody frameworks as framework donors. To maintain the antigen binding affinity of murine antibody, the murine vernier zone residues were retained in framework regions of huscFv. Additionally, two point mutations in CDR-L1 and CDR-L3 and three point mutations in CDR-H2 and CDR-H3 loops of the humanized scFv (huscFv) were introduced to increase affinity of the huscFv to EGFR. Analysis of results demonstrated that the humanness degree of resultant huscFv was increased as 19%. HuscFv was expressed in BL21 (DE3) and affinity purified via Ni-NTA column. The reactivity of huscFv with EGFR was evaluated by ELISA and dot blot techniques. Analysis by ELISA and dot blot showed that the huscFv was able to recognize and react with EGFR. Toxicity analysis by MTT assay indicated an inhibitory effect on growth of EGFR-overexpressing A431 cells. In conclusion, the huscFv produced in this study revealed decreased immunogenicity while retained growth inhibitory effect on EGFR-overexpressing tumor cells.

GJB2 mutations in deaf population of Ilam (Western Iran): a different pattern of mutation distribution.

Hearing loss is the most common sensory defect caused by heterogeneous factors. Up to now, more than 60 mutations in genes have been documented for nonsyndromic hearing loss. Hence, finding the causal gene in affected families could be a laborious and time-consuming process. GJB2 mutations, here, were investigated among deaf subjects of Ilam for the first time. In this study, we studied 62 unrelated patients with non-syndromic autosomal recessive deafness from 62 families. The most common mutation of GJB2, 35delG was checked, followed by direct sequencing of the GJB2 gene for determination of other mutations. In silico analyses were also performed using available software. In nine families, mutations in the connexin 26 gene were observed. In the studied population, R32H was the most common mutation. 35delG, W24X, and R127H were other mutations found in this study. In silico analyses showed pathogenicity of 35delG, R32H, and W24X but not R127H. Low frequency of GJB2 mutations in this population is probably indicative of the fact that other genes may be involved in nonsyndromic hearing loss in Ilam populations. In the other hand, the vicinity of Ilam and Iraq suggests that GJB2 mutations have likely a low frequency in this population.

A case-control study on the association of common variants of CAPN10 gene and the risk of type 2 diabetes in an Iranian population.

BACKGROUND: Calpain-10 is a ubiquitously expressed protease that serves as an intracellular calcium-dependent cysteine protease and is regarded to be one of the candidate genes for type 2 diabetes mellitus (T2DM). We aimed to identify the association of the common variants of this gene and the risk of T2DM in the Kurdish ethnic group of Iran. METHODS: Study groups included 173 T2DM and 173 normoglycemic subjects. Genotyping was determined by PCR-RFLP. Genotypic and allelic frequencies were then evaluated. Data was analyzed using SPSS software. RESULTS: The allelic frequency of the A-allele of SNP-43 variant was significantly different (p = 0.01) between case and control groups (18% vs. 11%). The genotype frequencies for SNP-43 did not show any significant difference between case and control individuals. However, the dominant model of SNP-43 was found to be significantly associated with T2DM (OR = 1.75, 95% CI = 1.06 - 2.89, p < 0.029). The distribution and allele frequency of other SNPs (SNP-19 and -63) did not show any significant difference between the study groups. For SNP-43, fasting serum insulin (p = 0.043) and HOMA-IR (p = 0.026) were higher in the control subjects with the GA+AA genotype when compared with the GG genotype. Among the T2DM subjects, there was no significant difference in any of the clinical or biochemical parameters between the GG and GA+AA genotypes of SNP-43. Normoglycemic subjects carrying the 2R/3R+3R/3R genotypes of SNP-19 had significantly lower HDL-C (p = 0.034) as compared with those with the 2R/2R genotype. In T2DM subjects, no significant difference was found in any of the clinical or biochemical parameters between 2R/2R and 2R/3R+3R/3R genotypes. T2DM subjects carrying the CT+TT genotypes of SNP-63 variation had significantly higher LDL-C (p = 0.015) as compared with those with the CC genotype. In normoglycemic subjects, no significant difference was found in any of the clinical or biochemical parameters between CC and CT+TT genotypes. CONCLUSIONS: Our findings revealed that there is an association between the SNP-43, but not SNP-19 and -63, and T2DM in the Kurdish ethnic group of West Iran.