Exome sequencing utility in defining the genetic landscape of hearing loss and novel-gene discovery in Iran.

Hearing loss (HL) is one of the most common sensory defects affecting more than 466 million individuals worldwide. It is clinically and genetically heterogeneous with over 120 genes causing non-syndromic HL identified to date. Here, we performed exome sequencing (ES) on a cohort of Iranian families with no disease-causing variants in known deafness-associated genes after screening with a targeted gene panel. We identified likely causal variants in 20 out of 71 families screened. Fifteen families segregated variants in known deafness-associated genes. Eight families segregated variants in novel candidate genes for HL: DBH, TOP3A, COX18, USP31, TCF19, SCP2, TENM1, and CARMIL1. In the three of these families, intrafamilial locus heterogeneity was observed with variants in both known and novel candidate genes. In aggregate, we were able to identify the underlying genetic cause of HL in nearly 30% of our study cohort using ES. This study corroborates the observation that high-throughput DNA sequencing in populations with high rates of consanguineous marriages represents a more appropriate strategy to elucidate the genetic etiology of heterogeneous conditions such as HL.

GJB2 mutations in Iranian Azeri population with autosomal recessive nonsyndromic hearing loss (ARNSHL): First report of c.238 C>A mutation in Iran.

OBJECTIVE: Autosomal-recessive nonsyndromic hearing loss (ARNSHL) is a heterogeneous genetic disorder. Mutations in the gap junction protein beta 2 (GJB2) gene, encoding connexin 26, are a significant cause of ARNSHL in different ethnic groups. This study aimed to identify the frequency and type of GJB2 mutations in the Iranian Azeri population. METHODS: Fifty unrelated families presenting ARNSHL in Ardabil Province, the northwest of Iran, were studied to determine the frequency and type of GJB2 mutations leading to ARNSHL. ARMS-PCR screened all DNA samples to detect c.35delG; p. Gly12Val mutation. In addition, normal samples for c.35delG; p. Gly12Val were analyzed by direct sequencing for other GJB2 mutations. RESULT: Of the fifty families, 13 (26%) showed a GJB2 gene mutation, with c.35delG; p. Gly12Val mutation was the most prevalent one that occurred in eight (61.5%) out of the 13 families. Of the families, two were homozygous for c.358-360delGAC; p. Glu120del mutation, and one was homozygous for c.290dupA; p. Tyr97Ter and c.299-300delAT; p. His100Arg mutations. Also, we detected a novel mutation, c.238C>A; p. Gln80lys, in one of the families. CONCLUSION: Our findings are comparable to previous studies, indicating c.35d3lG; p. Gly12Val mutation in the GJB2 gene is the most common cause of GJB2-related hearing loss in the Iranian Azeri population. Furthermore, our study highlights the significance of ARNSHL screening programs of live births based on local population data in Iran.

Genetics of intellectual disability in consanguineous families.

Autosomal recessive (AR) gene defects are the leading genetic cause of intellectual disability (ID) in countries with frequent parental consanguinity, which account for about 1/7th of the world population. Yet, compared to autosomal dominant de novo mutations, which are the predominant cause of ID in Western countries, the identification of AR-ID genes has lagged behind. Here, we report on whole exome and whole genome sequencing in 404 consanguineous predominantly Iranian families with two or more affected offspring. In 219 of these, we found likely causative variants, involving 77 known and 77 novel AR-ID (candidate) genes, 21 X-linked genes, as well as 9 genes previously implicated in diseases other than ID. This study, the largest of its kind published to date, illustrates that high-throughput DNA sequencing in consanguineous families is a superior strategy for elucidating the thousands of hitherto unknown gene defects underlying AR-ID, and it sheds light on their prevalence.

Iranome: A catalog of genomic variations in the Iranian population.

Considering the application of human genome variation databases in precision medicine, population-specific genome projects are continuously being developed. However, the Middle Eastern population is underrepresented in current databases. Accordingly, we established Iranome database (www.iranome.com) by performing whole exome sequencing on 800 individuals from eight major Iranian ethnic groups representing the second largest population of Middle East. We identified 1,575,702 variants of which 308,311 were novel (19.6%). Also, by presenting higher frequency for 37,384 novel or known rare variants, Iranome database can improve the power of molecular diagnosis. Moreover, attainable clinical information makes this database a good resource for classifying pathogenicity of rare variants. Principal components analysis indicated that, apart from Iranian-Baluchs, Iranian-Turkmen, and Iranian-Persian Gulf Islanders, who form their own clusters, rest of the population were genetically linked, forming a super-population. Furthermore, only 0.6% of novel variants showed counterparts in "Greater Middle East Variome Project", emphasizing the value of Iranome at national level by releasing a comprehensive catalog of Iranian genomic variations and also filling another gap in the catalog of human genome variations at international level. We introduce Iranome as a resource which may also be applicable in other countries located in neighboring regions historically called Greater Iran (Persia).

A novel variant in ASNS gene responsible for syndromic intellectual disability and microcephaly: Case report and literature review.

BACKGROUND: The ASNS (ASNS, MIM 108370) gene variations are responsible for asparagine synthetase deficiency (ASNSD, MIM 615574), a very rare autosomal recessive disease characterized by cerebral anomalies. These patients have congenital microcephaly, progressive encephalopathy, severe intellectual disability, and intractable seizures. METHOD: Clinical characteristics of the patient were collected. Exome sequencing was used for the identification of variants. Sanger sequencing was used to confirm the variant in the target region. The structure of the protein was checked using the DynaMut2 web server. RESULTS: The proband is an 11-year-old Iranian-Azeri girl with primary microcephaly and severe intellectual disability in a family with a consanguineous marriage. Symptoms emerged around the 10-20th days of life, when refractory epileptic gaze and unilateral tonic-clonic seizures initiated without any provoking factor such as fever. A brain MRI revealed no abnormalities except for brain atrophy. The karyotype was normal. Using exome sequencing, we identified a novel homozygous variant of thymine to adenine (NM_001673.5:c.538T>A) in the ASNS gene. Both parents had a heterozygous variant in this location. Subsequently, Sanger sequencing confirmed this variant. We also reviewed the clinical manifestations and MRI findings of the previously reported patients. CONCLUSION: In the present study, a novel homozygous variant was recognized in the ASNS gene in an Iranian-Azeri girl manifesting typical ASNSD symptoms, particularly intellectual disability and microcephaly. This study expands the mutation spectrum of ASNSD and reviews previously reported patients.

De novo KAT6B mutation causes Say-Barber-Biesecker-Young-Simpson variant of Ohdo syndrome in an Iranian boy: a case report.

BACKGROUND: Say-Barber-Biesecker-Young-Simpson (SBBYS) (OMIM #603736, Ohdo syndrome variant) is a rare type of severe blepharophimosis intellectual disability syndrome, which is generally characterized by a global developmental delay, distinctive facial features, and intellectual disability with multiple congenital anomalies, including skeletal involvement, missing, or underdeveloped kneecaps, and genital anomalies, in affected males. It has been shown that mutations in the KAT6B gene, which is a lysine acetyltransferase-encoding gene, have been associated with SBBYS syndrome. All the known variants are dominant de novo mutations that result in protein truncation. CASE PRESENTATION: A 14-year-old Iranian Azeri boy with an intellectual disability, distinct dysmorphic facial features such as open-mouth expression, sparse medial eyebrows, widely spaced upward-slanted eyes, epicanthal folds, broad nasal bridge, low-set ears, anteverted ears, short philtrum, hypertelorism, microphthalmia is presented in this case study. Cryptorchidism was reported. Neurologically, the patient presented with poor eye contact, hypotonia, and speech difficulties. In the skeletal X-ray, underdeveloped kneecaps with some new features were observed. CONCLUSION: We present the first case of SBBYS syndrome in association with some new anomaly features in the Iranian population. Based on this diagnosis, we could provide the patient with a suitable plan of management as well as appropriate genetic counseling for his family.

Immunogenic Potential of the Mediterranean Fever Gene in Patients with Coronavirus Disease: A Cross-Sectional Study.

Background: In December 2019, an outbreak of pneumonia caused by the novel coronavirus disease 2019 (COVID-19) became a pandemic and caused a global health crisis. This study evaluates the immunogenic potential of the Mediterranean fever (MEFV) gene in patients with COVID-19. Methods: A cross-sectional study was conducted from March to April 2020 in various COVID-19 referral centers in Ardabil, Iran. Blood samples of 50 hospitalized patients with confirmed COVID-19 were evaluated for MEFV gene mutation using the amplification refractory mutation system polymerase chain reaction (ARMS-PCR) and Sanger sequencing. Statistical analysis was performed using SPSS software, version 22.0. Results: Mutations of the MEFV gene were found in 6 (12%) of the patients. All mutations were heterozygous, and no homozygous or compound heterozygous forms were detected. The total mutant allele frequency was 6% and the carrier rate was 12%. The most common allele of the MEFV variant was E148Q, detected in 3 (6%) patients. No mutant variant of the MEFV gene was detected in deceased patients. None of the mutation carriers had familial Mediterranean fever (FMF) symptoms or a family history of FMF. Conclusion: MEFV gene mutations may have immunogenic potential in patients with COVID-19. A preprint version of this article has already been published at https://www.researchsquare.com/article/rs-69373/latest.pdf.

A novel compound heterozygous of ß-thalassemia with HbG-Coushatta: case report of Iran.

A 30-year-old male couple from Ardabil city, Iran, were admitted for premarital screening. An abnormal band in HbS/D regions with high levels of HbF and HbA 2 led us to suspect the possibility of a compound heterozygous state of ß-thalassemia in our affected proband. Therefore, beta globin chain sequencing of proband discovered a heterozygote combination of the Hb G-Coushatta [b22 (B4) Glu>Ala, HBB: c.68A>C) with HBB: IVS-II-1 (G>A) mutation as a compound heterozygote.

Infectious rate of ocular discharges in patients with approved COVID-19 respiratory infection.

Background: Since the relation between ocular manifestation and coronavirus disease 2019 (COVID-19) has been not elucidated well, thus this study was performed to assess the transmission to patients and healthcare providers via ocular secretions, and to decide if there is a link between ocular symptoms and COVID-19. Methods: A total of 101 patients who have been diagnosed with COVID-19 based on the clinical symptoms, radiological studies and confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR), and hospitalized in Corona ward of Imam Khomeini Hospital in Ardabil from April 15, 2020 to September 15, 2020. Ocular examination was done and patients' demographical data and ocular symptoms were recorded. Also, ocular secretion specimens were obtained to evaluate the COVID-19 RT-PCR test to detect the virus in ocular secretion specimen. Results: In this study, we observed that 15.8% of patients had positive RT-PCR test for COVID-19 in their ocular secretion specimens. In addition, we showed that there is no difference between male and female as well as age in patients with positive RT-PCR and negative tests obtained from ocular secretion. In addition, we observed that 27 patients (26.7%) were symptomatic patients for ocular manifestations including conjunctivitis, epiphora, injection, discharge, etc., but only 2 patients (1.98%) had follicular conjunctivitis during examination. Besides, nonsymptomatic patients (for ocular manifestations) had significant lower rate of positive RT-PCR test from their ocular secretion. Conclusion: This study showed that ocular secretion might be a source of COVID-19 infection even in nonsymptomatic patients. Thus, self-protection from ocular secretion should be observed by ophthalmologists from all patients (even nonsymptomatic patients).

Phenotype and genotype spectrum of variants in guanine nucleotide exchange factor genes in a broad cohort of Iranian patients.

BACKGROUND: Guanine nucleotide exchange factors (GEFs) play pivotal roles in neuronal cell functions by exchanging GDP to GTP nucleotide and activation of GTPases. We aimed to determine the genotype and phenotype spectrum of GEF mutations by collecting data from a large Iranian cohort with intellectual disability (ID) and/or developmental delay (DD). METHODS: We collected data from nine families with 20 patients extracted from Iranian cohort of 640 families with ID and/or DD. Next-generation sequencing (NGS) was used to identify the causing variants in recruited families. We also compared our clinical and molecular findings with previously reported patients carrying mutations in these GEF genes in the literature published until mid-2021. RESULTS: We identified disease-causing variants in eight GEF genes including ALS2, IQSEC2, MADD, RAB3GAP1, RAB3GAP2, TRIO, ITSN1, and DENND2A. The major clinical manifestations in 203 previously reported cases along with our 20 patients with disease causing variants in eight GEF genes were as follow; speech disorder (85.2%), ID (81.6%), DD (81.1%), inability to walk (71.3%), facial dysmorphisms features (52.4%), abnormalities in skull morphology (55.6%), hypotonia and muscle weakness (47%), and brain MRI abnormalities (43.4%). CONCLUSION: Our study provides new insights into the genotype and phenotype spectrum of mutations in GEF genes.

Azelaic acid stimulates catalase activation and promotes hair growth through upregulation of Gli1 and Gli2 mRNA and Shh protein.

OBJECTIVE: Although azelaic acid is effective for treatment of acne and rosacea, the biological activity of azelaic acid and the effect of its combination therapy with minoxidil were not elucidated with regard to hair growth. MATERIALS AND METHODS: In this study, mouse vibrissae follicles were dissected on day 10 after depilation. Then, the bulb and bulge cells of the hair follicle were treated with minoxidil and azelaic acid for 10 days to evaluate Sonic hedgehog (Shh) protein expression. Moreover, bulge and bulb cells of the hair follicles were cultivated and the expression of Gli1, Gli2, and Axin2 mRNA levels was evaluated using real-time polymerase chain reaction (PCR) analysis. We further investigated the protective effects of azelaic acid against ultraviolet B (UVB) irradiation in cultured bulb and bulge cells by determining catalase activity. An irradiation dose of 20 mJ/cm2 UVB for 4 sec was chosen. RESULTS: The results showed that catalase activity significantly (p<0.05) increased in the bulge cells after exposure to 2.5 mM and 25 mM azelaic acid. Meanwhile, treatment of the bulb cells with azelaic acid (2.5 and 25 mM) did not cause significant changes in catalase activity. We also found that azelaic acid (25 mM) alone upregulated Gli1 and Gli2 expression in the bulge cells and 100 µ minoxidil caused Gli1 and Axin2 overexpression in the bulb region of the hair follicle. Moreover, minoxidil (100 µM) alone and in combination with azelaic acid (25 mM) led to Shh protein overexpression in the hair follicles in vitro and in organ culture. CONCLUSION: Our results indicated a potential role for azelaic acid in the protection of bulge cells from UVB damage and its combination with minoxidil may activate hair growth through overexpression of Shh protein.

Identification of a founder mutation for Pendred syndrome in families from northwest Iran.

OBJECTIVE: Mutations in the SLC26A4 gene cause both Pendred syndrome and autosomal recessive nonsyndromic hearing loss (ARNSHL) at the DFNB4 locus. The SLC26A4 mutations vary among different communities. Previous studies have shown that mutations in the SLC26A4 gene are responsible for the more common syndromic hereditary hearing loss in Iran. This study assesses the possibility of a founder mutation for Pendred syndrome in northwest Iran. MATERIALS AND METHODS: In this study, we performed comprehensive clinical and genetic evaluations in two unrelated families from northwest Iran with nine members affected by hearing loss (HL). After testing short tandem repeat (STR) markers to confirm linkage to the SLC26A4 locus, we screened the SLC26A4 gene by Sanger sequencing of all 21 exons, exon-intron boundaries and the promoter region for any causative mutation. We identified the same causative mutation in these two families as we had detected earlier in two other Azeri families from northwest Iran. To investigate the possibility of a founder effect in these four families, we conducted haplotype analysis, and 14 single nucleotide polymorphisms (SNPs) throughout the SLC26A4 gene were genotyped. RESULTS: Patients in the two families showed the phenotype of Pendred syndrome. A known frameshift mutation (c.965insA, p.N322Fs7X) in exon 8 was identified in the two families, which was the same mutation that we detected previously in two other Azeri families. The results of haplotype analysis showed that all 15 patients from four families shared the founder mutation. Common haplotypes were not observed in noncarrier members. CONCLUSIONS: Based on the results of our two studies, the c.965insA mutation has only been described in Iranian families from northwest Iran, so there is evidence for a founder mutation originating in this part of Iran.

Spectrum of GJB2 (Cx26) gene mutations in Iranian Azeri patients with nonsyndromic autosomal recessive hearing loss.

OBJECTIVE: Hereditary hearing impairment is a genetically heterogeneous disorder. In spite of this, mutations in the GJB2 gene, encoding connexin 26 (Cx26), are a major cause of nonsyndromic recessive hearing loss in many countries and are largely dependent on ethnic groups. The purpose of our study was to characterize the type and prevalence of GJB2 mutations among Azeri population of Iran. METHODS: Fifty families presenting autosomal recessive nonsyndromic hearing loss from Ardabil province of Iran were studied for mutations in GJB2 gene. All DNA samples were screened for c.35delG mutation by ARMS PCR. Samples from patients who were normal for c.35delG were analyzed for the other variations in GJB2 by direct sequencing. In the absence of mutation detection, GJB6 was screened for the del(GJB6-D13S1830) and del(GJB6-D13S1854). RESULT: Thirteen families demonstrated alteration in the Cx26 (26%). The 35delG mutation was the most common one, accounting for 69.2% (9 out of 13 families). All the detected families were homozygous for this mutation. Two families were homozygous for delE120 and 299-300delAT mutations. We also identified a novel mutation: c.463-464 delTA in 2 families resulting in a frame shift mutation. CONCLUSION: Our results suggest that c.35delG mutation in the GJB2 gene is the most important cause of GJB2 related deafness in Iranian Azeri population.