Clinical application of next generation sequencing for Mendelian disease diagnosis in the Iranian population.

Next-generation sequencing (NGS) has been proven to be one of the most powerful diagnostic tools for rare Mendelian disorders. Several studies on the clinical application of NGS in unselected cohorts of Middle Eastern patients have reported a high diagnostic yield of up to 48%, correlated with a high level of consanguinity in these populations. We evaluated the diagnostic utility of NGS-based testing across different clinical indications in 1436 patients from Iran, representing the first study of its kind in this highly consanguineous population. A total of 1075 exome sequencing and 361 targeted gene panel sequencing were performed over 8 years at a single clinical genetics laboratory, with the majority of cases tested as proband-only (91.6%). The overall diagnostic rate was 46.7%, ranging from 24% in patients with an abnormality of prenatal development to over 67% in patients with an abnormality of the skin. We identified 660 pathogenic or likely pathogenic variants, including 241 novel variants, associated with over 342 known genetic conditions. The highly consanguineous nature of this cohort led to the diagnosis of autosomal recessive disorders in the majority of patients (79.1%) and allowed us to determine the shared carrier status of couples for suspected recessive phenotypes in their deceased child(ren) when direct testing was not possible. We also highlight the observations of recessive inheritance of genes previously associated only with dominant disorders and provide an expanded genotype-phenotype spectrum for multiple less-characterized genes. We present the largest mutational spectrum of known Mendelian disease, including possible founder variants, throughout the Iranian population, which can serve as a unique resource for clinical genomic studies locally and beyond.

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.

Brief Report of Variants Detected in Hereditary Hearing Loss Cases in Iran over a 3-Year Period.

BACKGROUND: Diagnosis of hereditary hearing loss (HHL) as a heterogeneous disorder is very important especially in countries with high rates of consanguinity where the autosomal recessive pattern of inheritance is prevalent. Techniques such as next-generation sequencing, a comprehensive genetic test using targeted genomic enrichment and massively parallel sequencing (TGE + MPS), have made the diagnosis more cost-effective. The aim of this study was to determine HHL variants with comprehensive genetic testing in our country. METHODS: Fifty GJB2 negative individuals with HHL were referred to the Kariminejad-Najmabadi Pathology and Genetics Center, Tehran, one of the reference diagnostic genetic laboratories in Iran, during a 3-year period between 2014 and 2017. They were screened with the OtoSCOPE test, the targeted genomic enrichment and massively parallel sequencing (TGE + MPS) platform after a detailed history had been taken along with clinical evaluation. RESULTS: Among 32 out of 50 GJB2 negative patients (64%), 34 known pathogenic and novel variants were detected of which 16 (47%) were novel, identified in 10 genes of which the most prevalent were CDH23, MYO7A and MYO15A. CONCLUSION: These results provide a foundation from which to make appropriate recommendations for the use of comprehensive genetic testing in the evaluation of Iranian patients with hereditary hearing loss.

Exome-wide copy number variation analysis identifies a COL9A1 in frame deletion that is associated with hearing loss.

Pathogenic variants in COL9A1 are primarily associated with autosomal recessive Stickler syndrome. Patients with COL9A1-associated Stickler syndrome (STL) present hearing loss (HL), ophthalmic manifestations and skeletal abnormalities. However, the clinical spectrum of patients with COL9A1 variants can also include multiple epiphyseal dysplasia, as well as non-syndromic HL that was observed in one previously reported proband. Exome sequencing was performed on the genomic DNA of an Iranian patient and his affected brother who both report non-syndromic HL. A 44.6 kb homozygous in-frame deletion spanning exons 6 to 33 of COL9A1 was detected via exome-based copy number variation analysis. The deleted exons were confirmed by PCR in the patient and his affected brother, who both have non-syndromic HL. Segregation analysis via qPCR confirmed the parents as heterozygous deletion carriers. Breakpoint analysis mapped the homozygous deletion spanning introns 5 to 33 (g.70,948,188_70,997,277del, NM_001851.4(COL9A1):c.697-3754_2112+769del, p.(Phe233_Ser704del), with an additional 67 bp of inserted intronic sequence that may have originated due to a fork stalling and template switching/microhomology-mediated break-induced replication (FoSTeS/MMBIR) mechanism. This mechanism has not been previously implicated in HL or STL. This is also the first reported copy number variation in COL9A1 that was identified through an exome data set in an Iranian family with apparent non-syndromic HL. The present study emphasizes the importance of exome-wide copy number variation analysis in molecular diagnosis and provides supporting evidence to associate COL9A1 with autosomal recessive non-syndromic HL.

Effect of inbreeding on intellectual disability revisited by trio sequencing.

In outbred Western populations, most individuals with intellectual disability (ID) are sporadic cases, dominant de novo mutations (DNM) are frequent, and autosomal recessive ID (ARID) is very rare. Because of the high rate of parental consanguinity, which raises the risk for ARID and other recessive disorders, the prevalence of ID is significantly higher in near- and middle-east countries. Indeed, homozygosity mapping and sequencing in consanguineous families have already identified a plethora of ARID genes, but because of the design of these studies, DNMs could not be systematically assessed, and the proportion of cases that are potentially preventable by avoiding consanguineous marriages or through carrier testing is hitherto unknown. This prompted us to perform whole-exome sequencing in 100 sporadic ID patients from Iran and their healthy consanguineous parents. In 61 patients, we identified apparently causative changes in known ID genes. Of these, 44 were homozygous recessive and 17 dominant DNMs. Assuming that the DNM rate is stable, these results suggest that parental consanguinity raises the ID risk about 3.6-fold, and about 4.1 to 4.25-fold for children of first-cousin unions. These results do not rhyme with recent opinions that consanguinity-related health risks are generally small and have been "overstated" in the past.

Parental Whole-Exome Sequencing Enables Sialidosis Type II Diagnosis due to an NEU1 Missense Mutation as an Underlying Cause of Nephrotic Syndrome in the Child.

INTRODUCTION: Monogenetic renal diseases, including recessively inherited nephrotic syndromes, represent a significant health burden despite being rare conditions. Precise diagnosis, including identification of the underlying molecular cause, is especially difficult in low-income countries and/or if affected individuals are unavailable for biochemical testing. Whole-exome sequencing (WES) has opened up novel diagnostic perspectives for these settings. However, sometimes the DNA of affected individuals is not suitable for WES due to low amounts or degradation. METHODS: We report on the use of parental WES with implementation of specific stepwise variant filtering to identify the underlying molecular cause of the childhood-onset nephrotic syndrome as nephrosialidosis resulting from a mutation in NEU1. RESULTS: Sequencing both parents enabled a nephrosialidosis diagnosis in the deceased child. To date, only 16 other cases of nephrosialidosis have been reported in the literature, with only 1 genetically confirmed case. After we reviewed the clinical information of all reported cases, we found that most patients presented with proteinuria, which started at between 2 and 3 years of age. Renal pathology showed mainly focal segmental glomerulosclerosis (FSGS)with vacuolated cells, and steroid treatment was always unsuccessful. Hepatomegaly was present in nearly all cases, whereas corneal clouding and a cherry red spot on the macula was observed in only approximately 50% of cases. Fourteen of 16 previously reported cases were no longer alive at the time of reporting. CONCLUSIONS: Our findings demonstrate the power of parental WES to diagnose rare genetic diseases, such as childhood-onset nephrotic syndrome. We further provide a comprehensive overview of the clinical course of nephrosialidosis and raise awareness of this ultra-rare condition as an underlying cause of FSGS.

Molecular genetic study of Calpainopathy in Iran.

INTRODUCTION: Calpainopathy is an autosomal recessive form of limb girdle muscular dystrophies (LGMDs) caused by mutations in the CAPN3 gene. CAPN3 is a Ca2+-dependent cystein protease consisting of 821 amino acids. LGMD is a highly heterogeneous disorder and mutation identification of this disease by Sanger sequencing of all genes is expensive and time consuming. Using autozygosity mapping is an effective approach to address this issue. METHODS: We used two sets of multiplex STR (Short tandem repeat) markers linked to CAPN3, DYSF, SGCA, SGCB, SGCG, SGCD genes following sequencing of the CAPN3 gene. In silico analysis and mutation detection in one hundred ethnically matched healthy individuals were carried out to determine the pathogenicity of novel mutations. Sequence variant interpretation was performed using the American College of Medical Genetics and Genomics (ACMG) guideline. RESULTS: Sixteen out of 50 families linked to the CAPN3 gene. In this study, mutations were found in 14 out of 16 families including 4 novel (c.1894A > T, c.567delG, c.2254-2256delAAC, and c.2373C > T) and 9 previously reported mutations consisting of 5 missense (c.2105C > T, c.2243G > A, c.1714C > T, c.291C > A, c.956C > T), 3 splice site (c.2380 + 2 T > G, c.946-2A > G, c.380G > A), and one indel (c.2257delinsAA) mutations. DISCUSSION: The c.2105C > T was found to be the most frequent mutation in this study. The results of this study revealed that most cases with splicing, frame shift and nonsense mutations experienced more severe clinical manifestations. Nonetheless, this should be confirmed by further studies on larger sample size.

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.

The assessment of affected factors on cytomegalovirus and rubella virus prevalence in females in Hamadan, Iran.

Cytomegalovirus (CMV) and rubella are considered as dangerous viral infections to the fetus. The findings of this research can clear the possible progress made thus far toward prevention in this part of the country. The data of all referees to genetic center of Shahid Beheshti Hospital in Hamadan, including the rubella and CMV tests were recorded in questionnaires and analyzed by logistic regression models. Univariate and multivariate logistic regression were utilized to assess the affected factors on CMV and Rubella separately. STATA and SPSS16 statistical software were used with setting P-value as 0.05. Logistic regression analysis indicates a statistically significant relationship between CMV IgM and on occupation (P=0.045), pregnancy (P=0.03) and years of referring the patients (P<0.001). The results of multivariate logistic regression analysis showed that job was significantly affected on the CMV infection [OR (95% C.I) = 1.71(1.1-2.83)]. Univariate logistic regression showed that age (P=0.001), the residential area (P=0.03), pregnancy (P=0.03), the marital status (P=0.022) and years of referring the patients (P<0.0001) has a significant effect on rubella IgG. However, multivariate logistic regression analysis also showed that residential status (OR=1.77) and age (OR=0.63) were significantly affected on the Rubella infection. The high level of IgG positivity against rubella in females may highlight the considerable impact of increasing public vaccination in this part of Iran. Also, the current data demonstrating frequency of primary infections with CMV in females which support the conclusion that regular prenatal screening tests is justified.

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.

A defect in the TUSC3 gene is associated with autosomal recessive mental retardation.

Recent studies have shown that autosomal recessive mental retardation (ARMR) is extremely heterogeneous, and there is reason to believe that the number of underlying gene defects goes into the thousands. To date, however, only four genes have been implicated in nonsyndromic ARMR (NS-ARMR): PRSS12 (neurotrypsin), CRBN (cereblon), CC2D1A, and GRIK2. As part of an ongoing systematic study aiming to identify ARMR genes, we investigated a large consanguineous family comprising seven patients with nonsyndromic ARMR in four sibships. Genome-wide SNP typing enabled us to map the relevant genetic defect to a 4.6 Mbp interval on chromosome 8. Haplotype analyses and copy-number studies led to the identification of a homozygous deletion partly removing TUSC3 (N33) in all patients. All obligate carriers of this family were heterozygous, but none of 192 unrelated healthy individuals from the same population carried this deletion. We excluded other disease-causing mutations in the coding regions of all genes within the linkage interval by sequencing; moreover, we verified the complete absence of a functional TUSC3 transcript in all patients through RT-PCR. TUSC3 is thought to encode a subunit of the endoplasmic reticulum-bound oligosaccharyltransferase complex that catalyzes a pivotal step in the protein N-glycosylation process. Our data suggest that in contrast to other genetic defects of glycosylation, inactivation of TUSC3 causes nonsyndromic MR, a conclusion that is supported by a separate report in this issue of AJHG. TUSC3 is only the fifth gene implicated in NS-ARMR and the first for which mutations have been reported in more than one family.