Identification of a Novel Variant in CC2D1A Gene Linked to Autosomal Recessive Intellectual Disability 3 in an Iranian Family and Investigating the Structure and Pleiotropic Effects of this Gene.

Objectives: Intellectual disability (ID) represents a significant health challenge due to its diverse and intricate nature. A multitude of genes play a role in brain development and function, with defects in these genes potentially leading to ID. Considering that many of these genes have yet to be identified, and those identified have only been found in a small number of patients, no complete description of the phenotype created by these genes is available. CC2D1A is one of the genes whose loss-of-function mutation leads to a rare form of non-syndromic ID-3(OMIM*610055), and four pathogenic variants have been reported in this gene so far. Materials & Methods: n the current study, two affected females were included with an initial diagnosis of ID who were from an Iranian family with consanguineous marriage. Whole-exome sequencing was used to identify the probable genetic defects. The Genotypic and phenotypic characteristics of the patients were compared with a mutation in the CC2D1A gene, and then the structure of the gene and its reported variants were investigated. Results: The patients carried a novel homozygous splicing variant (NM_017721, c.1641+1G>A) in intron 14, which is pathogenic according to the ACMG guideline. Loss-of-function mutations in CC2D1A have severe phenotypic consequences such as ID, autism spectrum disorder (ASD), and seizures. However, missense mutations lead to ASD with or without ID, and in some patients, they cause ciliopathy. Conclusion: This study reports the fifth novel, probably pathogenic variant in the CC2D1A gene. Comparing the clinical and molecular genetic features of the patients with loss-of-function mutation helped to describe the phenotype caused by this gene more precisely. Investigating the CC2D1A gene's mutations and structure revealed that it performs multiple functions. The DM14 domain appears more pivotal in triggering severe clinical symptoms, including ID, than the C2 domain.

The Spectrum of HBB Mutations among 2315 Beta Thalassemia Patients of a Reference Clinic in Tehran-Iran.

Beta Thalassemia is the most prevalent and well-studied single gene disorder in Iran. Here, we investigated the spectrum of HBB gene mutations, identified among 2315 patients, referred to a reference thalassemia clinic in Tehran, on the basis of suspicion to thalassemia major or intermedia. The patients were homozygous or compound heterozygous for HBB mutations, and were referred from various Iranian provinces, during 15 years (2001- 2016). The HBB mutations were classified based on their frequency, and the result was compared to a meta-analysis of 14,293 beta thalassemia cases in the Iranian population, within the same time period. The mutation spectrum in this study contained 43 HBB mutations, compared to the 90, presented by the meta-analysis. Similar to the meta-analysis, IVSII-1 (G > A) and IVSI-5 (G > C) were the most common mutations in this study. These two comprised 62.40% of the total HBB mutant alleles in the studied population, comparable to 51.92% of that in the meta-analysis. IVSII-1 (G > A) and IVSI-5 (G > C), followed by 17 other mutations that had frequencies ranging from 0.15% to 5.44%, were among the 20 common HBB mutations in Iran and neighboring countries, according to the meta-analysis. This study provided further evidence to support the spectrum of the most common HBB mutations in the Iranian population.

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.

When transcripts matter: delineating between non-syndromic hearing loss DFNB32 and hearing impairment infertile male syndrome (HIIMS).

Mutations in the CDC14A (Cell Division-Cycle 14A) gene, which encodes a conserved dual-specificity protein tyrosine phosphatase, have been identified as a cause of autosomal recessive non-syndromic hearing loss (DFNB32) and hearing impairment infertility male syndrome (HIIMS). We used next-generation sequencing to screen six deaf probands from six families segregating sensorineural moderate-to-profound hearing loss. Data analysis and variant prioritization were completed using a custom bioinformatics pipeline. We identified three homozygous loss of function variants (p.Arg345Ter, p.Arg376Ter, and p.Ala451Thrfs*43) in the CDC14A gene, segregating with deafness in each family. Of the six families, four segregated the p.Arg376Ter mutation, one family segregated the p.Arg345Ter mutation and one family segregated a novel frameshift (p.Ala451Thrfs*43) mutation. In-depth phenotyping of affected individuals ruled out secondary syndromic findings. This study implicates the p.Arg376Ter mutation might be as a founder mutation in the Iranian population. It also provides the first semen analysis for deaf males carrying mutations in exon 11 of CDC14A and reveals a genotype-phenotype correlation that delineates between DFNB32 and HIIMS. The clinical results from affected males suggest the NM_033313.2 transcript alone is sufficient for proper male fertility, but not for proper auditory function. We conclude that DFNB32 is a distinct phenotypic entity in males.

G130V de novo mutation in an Iranian pedigree with nonsyndromic hearing loss without palmoplantar keratoderma.

Mutations in the GJB2 gene encoding connexin 26 (Cx26) cause autosomal recessive and rarely dominant nonsyndromic sensorineural hearing loss as well as asyndromic hearing impairment with skin problems. A dominant GJB2 mutation, c.389G > T (p.G130V), has been reported previously in association with hearing impairment and palmoplantar keratoderm. Here we report the first de novo G130V mutation of GJB2 gene in a sporadic case of hearing loss in a consanguineous Iranian family which is not associated with skin disorder.

Novel Mutations in KCNQ4, LHFPL5 and COCH Genes in Iranian Families with Hearing Impairment.

BACKGROUND: Hearing loss (HL) is the most common sensory deficit in humans, and genetic factors contribute to about half of the cases. With 112 causative genes identified so far and a disproportionate share of the genes within different ethnic groups, HL has proven to be quite heterogeneous. METHODS: Twenty Iranian families having at least 2 children with hereditary HL were initially verified to be GJB2-negative and were then subjected to whole exome sequencing (WES). Sanger sequencing was used to confirm segregation of the variant identified in each family. RESULTS: In 3 families, WES revealed 3 novel variants in KCNQ4, LHFPL5 and COCH genes. The KCNQ4 gene (DFNA2A) encodes a potassium channel (KV7.4) and the heterozygous variant identified (c.1647C>G, p.F549L) resulted in the substitution of Phe549 residing in the KV7.4 cytoplasmic region. The homozygous variant (c.34A>T, p.K12X) was identified in the LHFPL5 gene (DFNB67) which encodes a transmembrane protein, and another variant in a homozygous state (c.116T>A, p.L39X) was identified in the COCH gene which encodes a secretory protein. Pathogenic variants in the COCH gene are associated with late onset autosomal dominant hearing loss (DFNA9) but the affected individuals displayed early onset HL with a recessive mode of inheritance. CONCLUSION: The 16% contribution of GJB2 to HL in the Iranian population necessitates the discovery of the remaining causal factors. This study is the first to report KCNQ4 and COCH related HL in the Iranian population and the second study, globally, to report HL due to biallelic inactivation of the COCH gene.

Novel mutations in MYTH4-FERM domains of myosin 15 are associated with autosomal recessive nonsyndromic hearing loss.

OBJECTIVE: Hereditary hearing loss is the most common neurosensory disorder in humans caused by myriad mutations in numerous genes. Autosomal recessive nonsyndromic hearing loss (ARNSHL) accounts for 80% of hearing impairments of genetic origin and is quite prevalent in societies with a high rate of consanguinity. In the current study, we investigated the causes of sensorineural hearing loss in 24 unrelated Iranian families who were mainly consanguineous and had at least two affected children. METHODS: All probands were initially screened for GJB2 mutations, as the most common causes of ARNSHL in Iran. Verified GJB2-negative samples were subsequently subjected to whole exome sequencing (WES) to identify the underlying causes of hearing impairment, and the variants identified in each family were further confirmed by Sanger sequencing. RESULTS: WES revealed three previously unreported mutations in MYO15A, the gene encoding the unconventional myosin 15 (Myo15). All variants identified, c.C6436T (p.R2146W), c.C9584G (p.P3195R) and c.G10266C (p.Q3422H), reside in the MYTH4 (myosin tail homology) and FERM (4.1 ezrin, radixin, moesin) domains of the protein. CONCLUSION: Globally, mutations in MYO15A are considered to be among the most prevalent genetic causes of ARNSHL, and they rank as the third leading cause of hearing loss in the Iranian population, below GJB2 and SLC26A4. Yet again, these results endorse the importance of MYO15 screening in hearing impaired populations, particularly in Iran.

SLC52A2 mutations cause SCABD2 phenotype: A second report.

INTRODUCTION: Autosomal recessive cerebellar ataxias (ARCAs) are a large group of neurodegenerative disorders that manifest mainly in children and young adults. Most ARCAs are heterogeneous with respect to age at onset, severity of disease progression, and frequency of extracerebellar and systemic signs. METHODS: The phenotype of a consanguineous Iranian family was characterized using clinical testing and pedigree analysis. Whole-exome sequencing was used to identify the disease-causing gene in this family. RESULTS AND CONCLUSION: Using whole exome sequencing (WES), a novel missense mutation in SLC52A2 gene is reported in a consanguineous Iranian family with progressive severe hearing loss, optic atrophy and ataxia. This is the second report of the genotype-phenotype correlation between this syndrome named spinocerebellar ataxia with blindness and deafness type 2 (SCABD2) and SLC52A2 gene.

Characterising the spectrum of autosomal recessive hereditary hearing loss in Iran.

BACKGROUND: Countries with culturally accepted consanguinity provide a unique resource for the study of rare recessively inherited genetic diseases. Although hereditary hearing loss (HHL) is not uncommon, it is genetically heterogeneous, with over 85 genes causally implicated in non-syndromic hearing loss (NSHL). This heterogeneity makes many gene-specific types of NSHL exceedingly rare. We sought to define the spectrum of autosomal recessive HHL in Iran by investigating both common and rarely diagnosed deafness-causing genes. DESIGN: Using a custom targeted genomic enrichment (TGE) panel, we simultaneously interrogated all known genetic causes of NSHL in a cohort of 302 GJB2-negative Iranian families. RESULTS: We established a genetic diagnosis for 67% of probands and their families, with over half of all diagnoses attributable to variants in five genes: SLC26A4, MYO15A, MYO7A, CDH23 and PCDH15. As a reflection of the power of consanguinity mapping, 26 genes were identified as causative for NSHL in the Iranian population for the first time. In total, 179 deafness-causing variants were identified in 40 genes in 201 probands, including 110 novel single nucleotide or small insertion-deletion variants and three novel CNV. Several variants represent founder mutations. CONCLUSION: This study attests to the power of TGE and massively parallel sequencing as a diagnostic tool for the evaluation of hearing loss in Iran, and expands on our understanding of the genetics of HHL in this country. Families negative for variants in the genes represented on this panel represent an excellent cohort for novel gene discovery.

Finding mutation within non-coding region of GJB2 reveals its importance in genetic testing of hearing loss in Iranian population.

OBJECTIVE: Hereditary hearing loss is the most common neurosensory disorder in humans. Half of the cases have genetic etiology with extraordinary genetic heterogeneity. Mutations in one gene, GJB2, are the most common cause for autosomal recessive non-syndromic hearing loss (ARNSHL) in many different populations. GJB2 encodes a gap junction channel protein (connexin 26), and is located on DFNB1 locus on chromosome 13q12.11 which also involve another connexin gene, GJB6. Mutation screening of GJB2 revealed that a high number of patients with deaf phenotype have heterozygous genotype and carry only one mutant allele. As the first comprehensive study in Iran, we have targeted GJB2-related Iranian heterozygotes, looking for second mutant allele which leads to hearing impairment. They bear first mutation in their coding exon of GJB2. METHOD: Using PCR-based direct sequencing, we assessed 103 patients with ARNSHL for variants in non-coding exon and promoter region of this gene, for the first time in Iran. RESULT: We have identified the second mutant allele in splice site of exon-1 of GJB2 which is known as IVS1+1G>A in 17 probands. We found no mutation in promoter region of GJB2. CONCLUSION: Our findings reveal that IVS1+1G>A mutation in noncoding exon of GJB2 is the most common mutation after 35delG within multi ethnical Iranian heterozygote samples. It emphasizes to approach exon1 of GJB2 in case of ARNSHL genetic diagnosis.

Association of polymorphisms at LDLR locus with coronary artery disease independently from lipid profile.

Coronary artery disease (CAD) is the leading cause of mortality in many parts of the world. Genome-wide association studies (GWAS) have identified several genetic variants associated with CAD in Low-density lipoprotein receptor (LDLR) locus. This study was evaluated the possible association of genetic markers at LDLR locus with CAD irrespective to lipid profile and as well as the association of these SNPs with severity of CAD in Iranian population. Sequencing of 2 exons in LDLR gene (Exon 2, 12) and part of intron 30 of SMARCA4 gene include rs1122608, was performed in 170 Iranian patients angiographically confirmed CAD and 104 healthy controls by direct sequencing. Sullivan's scoring system was used for determining the severity of CAD in cases. Our results showed that homozygote genotypes of rs1122608 (P<0.0001), rs4300767 (P<0.005) and rs10417578 (p<0.007) SNPs have strong protective effects on the CAD. In addition, we found that rs1122608 (GT or TT) was at higher risk of three vessel involvement compared to single vessels affecting (P=0.01).

A comprehensive study to determine heterogeneity of autosomal recessive nonsyndromic hearing loss in Iran.

Hearing loss is the most common sensory disorder worldwide and affects 1 of every 500 newborns. In developed countries, at least 50% of cases are genetic, most often resulting in nonsyndromic deafness (70%), which is usually autosomal recessive (∼80%). Although the cause of hearing loss is heterogeneous, mutations in GJB2 gene at DFNB1 locus are the major cause of autosomal recessive nonsyndromic hearing loss (ARNSHL) in many populations. Our previous study showed that mutations of GJB2 gene do not contribute to the major genetic load of deafness in the Iranian population (∼16%). Therefore, to define the importance of other genes in contributing to an ARNSHL phenotype in the Iranian population, we used homozygosity mapping to identify regions of autozygosity-by-descent in 144 families which two or more progeny had ARNSHL but were negative for GJB2 gene mutations. Using flanking or intragenic short-tandem repeat markers for 33 loci we identified 33 different homozygous variations in 10 genes, of which 9 are novel. In aggregate, these data explain ∼40% of genetic background of ARNHSL in the Iranian population.

Screening for MYO15A gene mutations in autosomal recessive nonsyndromic, GJB2 negative Iranian deaf population.

MYO15A is located at the DFNB3 locus on chromosome 17p11.2, and encodes myosin-XV, an unconventional myosin critical for the formation of stereocilia in hair cells of cochlea. Recessive mutations in this gene lead to profound autosomal recessive nonsyndromic hearing loss (ARNSHL) in humans and the shaker2 (sh2) phenotype in mice. Here, we performed a study on 140 Iranian families in order to determine mutations causing ARNSHL. The families, who were negative for mutations in GJB2, were subjected to linkage analysis. Eight of these families showed linkage to the DFNB3 locus, suggesting a MYO15A mutation frequency of 5.71% in our cohort of Iranian population. Subsequent sequencing of the MYO15A gene led to identification of 7 previously unreported mutations, including 4 missense mutations, 1 nonsense mutation, and 2 deletions in different regions of the myosin-XV protein.

The spectrum of GJB2 mutations in the Iranian population with non-syndromic hearing loss--a twelve year study.

OBJECTIVE: Mutations in GJB2, encoding connexin 26 (CX26), are causally related to autosomal recessive form of non-syndromic hearing loss (NSHL) at the DFNB1 locus and autosomal dominant NSHL at the DFNA3 locus. In this study, we investigated the prevalence of GJB2 mutations in the Iranian deaf population. METHODS: A total of 2322 deaf probands presenting the ethnically diverse Iranian population were screened for variants in GJB2. All persons were first screened for the c.35delG mutation, as this variant is the most prevalent GJB2-deafness causing mutation in the Iranian population. In all persons carrying zero or one c.35delG allele, exons 1 and 2 were then sequenced. RESULTS: In total, 374 (~16%) families segregated GJB2-related deafness caused by 45 different mutations and 5 novel variants. The c.35delG mutation was most commonly identified and accounts for ~65% of the GJB2 mutations found in population studied. CONCLUSION: Our data also show that there is a gradual decrease in the frequency of the c.35delG mutation and of GJB2-related deafness in general in a cline across Iran extending from the northwest to southeast.

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.

Did the GJB2 35delG mutation originate in Iran?

Mutations in GJB2 are a major cause of autosomal recessive non-syndromic hearing loss (ARNSHL) in many populations. A single mutation of this gene (35delG) accounts for approximately 70% of GJB2 mutations that are associated with ARNSHL in Caucasians in many European countries and also in Iranian. In this study, we used PCR and restriction digestion to genotype five single nucleotide polymorphisms (SNPs) that define the genetic background of the 35delG mutation over an interval of 98 Kbp that includes the coding and flanking regions of GJB2. Two microsatellite markers, D13S175 and D13S141, were also analyzed in patients and controls. These data suggest that the 35delG mutation originated in northern Iran.

Mutations in TMC1 are a common cause of DFNB7/11 hearing loss in the Iranian population.

OBJECTIVES: We investigated the cause of autosomal recessive nonsyndromic hearing loss (ARNSHL) that segregated in 2 consanguineous Iranian families. METHODS: Otologic and audiometric examinations were performed on affected members of each family. Genome-wide parametric multipoint linkage mapping using a recessive model was performed with Affymetrix 50K GeneChips or short tandem repeat polymorphisms. Direct sequencing was used to confirm the causative mutation in each family. RESULTS: In 2 Iranian families, L-1651 and L-8600606, with ARNSHL that mapped to the DFNB7/11 locus, homozygosity for a reported splice site mutation (c.776+1G>A), and a novel deletion (c.1589_1590delCT; p.S530*) were identified in the TMC1 gene, respectively. CONCLUSIONS: Consistent with the previously reported phenotype in DFNB7/11 families, the 2 Iranian families had segregated congenital, profound hearing impairment. However, in family L-1651, one affected family member (IV:3) has milder hearing impairment than expected, suggesting a potential genetic modifier effect. These results indicate that DFNB7/11 is a common form of genetic hearing loss in Iran, because this population is the source of 6 of the 29 TMC1 mutations reported worldwide.

Fragile X syndrome screening of families with consanguineous and non-consanguineous parents in the Iranian population.

Fragile X syndrome is the most common form of inherited mental retardation (MR). It is caused by the expansion of CGG triplet repeats in the fragile X mental retardation 1 (FMR1) gene. In mentally retarded males, the frequency of fragile X syndrome is approximately 2-3 percent, but little is known about its proportion in mentally retarded patients from countries where parental consanguinity is common. The objective of this study was to estimate the frequency of fragile X syndrome (FXS) in mentally retarded patients from Iran. We examined a total of 508 families with MR that had been referred to the Genetics Research Center (GRC) in Tehran of which 467 families had at least two mentally retarded children. In 384 families, the parents were related and in 124 they were not related of which most of them had putative or established X-linked inheritance pattern. Full FMR1 mutations were found in 32 of the 508 families studied (6.3%), in 19 out of 124 families with apparently unrelated parents (15.3%), and in 13 of the 384 consanguineous families (3.4%). Thus, in Iran, the relative frequency of FXS seems to be high, and in patients with unrelated parents is much higher. We also show that even in families with consanguineous parents, FXS has to be ruled out before assuming that familial MR is due to autosomal recessive gene defects. Molecular studies are in progress to explain the high proportion of FMR1 mutations in mentally retarded offspring of unrelated Iranian parents.

Molecular characterization of alpha-thalassemia in the Dohuk region of Iraq.

The molecular basis of alpha-thalassemia (alpha-thal) has been addressed by several studies from the eastern Mediterranean region, but not from Iraq. To address this issue, we studied 51 individuals with unexplained hypochromia and/or microcytosis, as well as nine patients with documented Hb H disease from the Dohuk region in northern Iraq. We used multiplex gap-polymerase chain reaction (gap-PCR), reverse hybridization, and sequencing for this purpose. It was found that the most common genotypes in those with unexplained hypochromia and/or microcytosis were -alpha(3.7)/alpha alpha, followed by - -(MED-I)/alpha alpha, then -alpha(3.7)/-alpha (3.7), respectively, detected in 84.3% of the above individuals. Other genotypes identified sporadically were -alpha(4.2)/alpha alpha, alpha(poly A1)alpha/alpha alpha (AATAAA>AATAAG), alpha(Adana)alpha/alpha alpha [Hb Adana, codon 59 (Gly-->Asp) or HBA1:c.179G>A], and alpha(Evanston)alpha/alpha alpha [Hb Evanston, codon 14 (Trp-->Arg) or HBA1:c.43 T>C]. Three cases (5.88%) remained uncharacterized even after sequencing. All nine Hb H cases carried the -alpha(3.7)/- -(MED-I) genotype. Such findings are rather different from those in other eastern Mediterranean populations, particularly with relevance to an Hb H molecular basis.