Bi-allelic variants in OGDHL cause a neurodevelopmental spectrum disease featuring epilepsy, hearing loss, visual impairment, and ataxia.

The 2-oxoglutarate dehydrogenase-like (OGDHL) protein is a rate-limiting enzyme in the Krebs cycle that plays a pivotal role in mitochondrial metabolism. OGDHL expression is restricted mainly to the brain in humans. Here, we report nine individuals from eight unrelated families carrying bi-allelic variants in OGDHL with a range of neurological and neurodevelopmental phenotypes including epilepsy, hearing loss, visual impairment, gait ataxia, microcephaly, and hypoplastic corpus callosum. The variants include three homozygous missense variants (p.Pro852Ala, p.Arg244Trp, and p.Arg299Gly), three compound heterozygous single-nucleotide variants (p.Arg673Gln/p.Val488Val, p.Phe734Ser/p.Ala327Val, and p.Trp220Cys/p.Asp491Val), one homozygous frameshift variant (p.Cys553Leufs∗16), and one homozygous stop-gain variant (p.Arg440Ter). To support the pathogenicity of the variants, we developed a novel CRISPR-Cas9-mediated tissue-specific knockout with cDNA rescue system for dOgdh, the Drosophila ortholog of human OGDHL. Pan-neuronal knockout of dOgdh led to developmental lethality as well as defects in Krebs cycle metabolism, which was fully rescued by expression of wild-type dOgdh. Studies using the Drosophila system indicate that p.Arg673Gln, p.Phe734Ser, and p.Arg299Gly are severe loss-of-function alleles, leading to developmental lethality, whereas p.Pro852Ala, p.Ala327Val, p.Trp220Cys, p.Asp491Val, and p.Arg244Trp are hypomorphic alleles, causing behavioral defects. Transcript analysis from fibroblasts obtained from the individual carrying the synonymous variant (c.1464T>C [p.Val488Val]) in family 2 showed that the synonymous variant affects splicing of exon 11 in OGDHL. Human neuronal cells with OGDHL knockout exhibited defects in mitochondrial respiration, indicating the essential role of OGDHL in mitochondrial metabolism in humans. Together, our data establish that the bi-allelic variants in OGDHL are pathogenic, leading to a Mendelian neurodevelopmental disease in humans.

Identification and characterization of the largest deletion in the PCCA gene causing severe acute early-onset form of propionic acidemia.

Whole-exome sequencing (WES) is an excellent method for the diagnosis of diseases of uncertain or heterogeneous genetic origin. However, it has limitations for detecting structural variations such as InDels, which the bioinformatics analyzers must be aware of. This study aimed at using WES to evaluate the genetic cause of the metabolic crisis in a 3-day-old neonate admitted to the neonatal intensive care unit (NICU) and deceased after a few days. Tandem mass spectrometry (MS/MS) showed a significant increase in propionyl carnitine (C3), proposing methylmalonic acidemia (MMA) or propionic acidemia (PA). WES demonstrated a homozygous missense variant in exon 4 of the BTD gene (NM_000060.4(BTD):c.1330G > C), responsible for partial biotinidase deficiency. Segregation analysis of the BTD variant revealed the homozygous status of the asymptomatic mother. Furthermore, observation of the bam file, around genes responsible for PA or MMA, by Integrative Genomics Viewer (IGV) software displayed a homozygous large deletion in the PCCA gene. Comprehensive confirmatory studies identified and segregated a novel outframe deletion of 217,877 bp length, "NG_008768.1:g.185211_403087delinsTA", extended from intron 11 to 21 of the PCCA, inducing a premature termination codon and activation of nonsense-mediated mRNA decay (NMD). Homology modeling of the mutant PCCA demonstrated eliminating the protein's active site and critical functional domains. Thereupon, this novel variant is suggested as the largest deletion in the PCCA gene, causing an acute early-onset PA. These results could expand the PCCA variants spectrum, and improve the existing knowledge on the molecular basis of PA, as well as provide new evidence of pathogenicity of the variant (NM_000060.4(BTD):c.1330G > C.

Identifying and predicting the pathogenic effects of a novel variant inducing severe early onset MMA: a bioinformatics approach.

BACKGROUND: Methylmalonic acidemia (MMA) is a rare metabolic disorder resulting from functional defects in methylmalonyl-CoA mutase. Mutations in the MMAB gene are responsible for the cblB type of vitamin B12-responsive MMA. RESULTS: This study used Whole-exome sequencing (WES), Sanger sequencing, linkage analysis, and in-silico evaluation of the variants' effect on protein structure and function to confirm their pathogenicity in a 2-day-old neonate presenting an early-onset metabolic crisis and death. WES revealed a homozygous missense variant on chromosome 12, the NM_052845.4 (MMAB):c.557G > A, p.Arg186Gln, in exon 7, a highly conserved and hot spot region for pathogenic variants. After being confirmed by Sanger sequencing, the wild-type and mutant proteins' structure and function were modeled and examined using in-silico bioinformatics tools and compared to the variant NM_052845.4 (MMAB):c.556C > T, p.Arg186Trp, a known pathogenic variant at the same position. Comprehensive bioinformatics analysis showed a significant reduction in the stability of variants and changes in protein-protein and ligand-protein interactions. Interestingly, the variant c.557G > A, p.Arg186Gln depicted more variations in the secondary structure and less binding to the ATP and B12 ligands compared to the c.556C > T, p.Arg186Trp, the known pathogenic variant. CONCLUSION: This study succeeded in expanding the variant spectra of the MMAB, forasmuch as the variant c.557G > A, p.Arg186Gln is suggested as a pathogenic variant and the cause of severe MMA and neonatal death. These results benefit the prenatal diagnosis of MMA in the subsequent pregnancies and carrier screening of the family members. Furthermore, as an auxiliary technique, homology modeling and protein structure and function evaluations could provide geneticists with a more accurate interpretation of variants' pathogenicity.

Development of a Quantitative Multiplex PCR to Detect Three Common Alpha Thalassemia Deletions.

Alpha thalassemia is an autosomal recessive genetic disorder with a high prevalence in the Middle East. The severe form of alpha-thalassemia is incompatible with life and can cause significant obstetric complications in the mother. Therefore, it is important to determine the genotype in parents who have a chance of having a fetus with one of the severe forms of this disease. A total of 112 samples that were previously analyzed for common alpha thalassemia mutations in Iran were used in this study. A new multiplex PCR including quantitative polymerase chain reaction to amplify the homologous regions of the alpha-globin gene cluster and fluorescent gap PCR was designed to identify -α3.7, -α4.2, --MED deletions. The ROC curve was used to determine the optimum cutoff points. Statistical analysis showed that there is a significant difference between the peak height ratios for different genotypes. The peak corresponding to the 297 bp fragment resulting from the amplification of the allele with MED-I deletion was detected in all the samples with this deletion. Different cutoffs for a range of sensitivities and specificities were determined by the ROC curve. The suggested method can identify three common large deletions in the alpha-globin gene cluster. A study with a larger sample size can provide more accurate information about the sensitivity and specificity of this test.

Regulatory Mutation Study in Cases with Unsolved Hypochromic Microcytic Anemia and α-Major Regulatory Element Haplotype Analysis in Iran.

α-Thalassemia (α-thal) is an inherited blood disorder with different clinical manifestations. Although genetic causes of anemia are identified routinely in the majority of α-thal cases, a pathogenic variant in a few cases remains undiagnosed. In this study, some reported regulatory mutations have been investigated in five unsolved α-thal carriers. α-Major regulatory element (α-MRE) haplotype analysis has also been performed in Iran for the first time. Four regions, including the HBA2 core promoter, the highly conserved sequence of hypersensitive-40 (HS-40), a region containing regulatory single nucleotide polymorphism (SNP) CR062116, and a region containing rs7203560, were screened for changes by Sanger sequencing in a total of five unsolved suspected α-thal carriers. The frequencies of α-MRE haplotypes B and C were also determined in control samples with normal hematological indices. No pathogenic variant was found in the investigated regions. Haplotype frequencies observed for B and C haplotypes fell into the range of frequencies observed in previous studies. The investigated genotypes in the control group were in the Hardy-Weinberg equilibrium. This study can provide evidence that there is no association between the B haplotype and microcytic hypochromic anemia. The cause of anemia remains a mystery in our unsolved cases, which demonstrates the need for further studies on the causes of hypochromic microcytic anemia in individuals with intact α- and ß-globin genes without iron deficiency.

Identification of a novel mutation in the ST14 gene in an Iranian family with ichthyosis and hypotrichosis.

Inherited ichthyosis is a heterogeneous group of rare cutaneous disorders characterized by hyperkeratosis and scaly skin. So far, only a few genetic studies on ichthyosis have been performed in Iran. Herein, we reported a family with two cases of ichthyosis and hypotrichosis that were investigated by whole exome sequencing. Targeted data analysis identified a novel nonsense variant c.1243C>T (p.Gln415Ter) located at exon 11 of the ST14 gene in the proband. Sanger sequencing showed co-segregation of this mutation with the disease in this family. Further studies are needed to develop knowledge about the spectrum of changes in this gene and their effects on protein function and disease phenotype.

Characterization of Niemann-Pick diseases genes mutation spectrum in Iran and identification of a novel mutation in SMPD1 gene.

Background: Niemann-Pick diseases are rare inherited lipid storage disorders caused by mutations in the SMPD1, NPC1, and NPC2 genes. The aim of this study was to assess the mutation spectrum of a cohort of Iranian Niemann-Pick patients. Methods: A consanguineous couple with a child suspected of having Niemann-Pick disease type A (died at age 2) was screened for gene mutations in the SMPD1 gene. Sanger sequencing was performed for all exons and exon-intron boundary regions. A literature review on SMPD1, NPC1, and NPC2 genes mutations in Iran was conducted using published original papers on this subject. Results: A novel frameshift c.762delG (p.Leu256fs*) at a heterozygous state was identified in the parents. According to the review study, identified mutations in 39 Iranian patients were concentrated in exon 2 of the SMPD1 gene and exons 8 and 9 of the NPC1 gene. Conclusion: Niemann-Pick diseases genes mutation analysis (SMPD1, NPC1, and NPC2) in Iran shows the genetic heterogeneity of these diseases in this country. More studies with larger sample sizes should be conducted to further examine genetic changes associated with Niemann-Pick diseases in Iran.

The first successful application of preimplantation genetic diagnosis for hearing loss in Iran.

Hearing impairment (HI) caused by mutations in the connexin-26 gene (GJB2) accounts for the majority of cases with inherited, nonsyndromic sensorineural hearing loss. Due to the illegality of the abortion of deaf fetuses in Islamic countries, preimplantation genetic diagnosis (PGD) is a possible solution for afflicted families to have a healthy offspring. This study describes the first use of PGD for GJB2 associated non-syndromic deafness in Iran. GJB2 donor splicing site IVS1+1G>A mutation analysis was performed using Sanger sequencing for a total of 71 Iranian families with at least 1 deaf child diagnosed with non-syndromic deafness. In Vitro Fertilization (IVF) was performed, followed by PGD for a cousin couple with a 50% chance of having an affected child. Bi-allelic pathogenic mutations were found in a total of 12 families (~17 %); of which a couple was a PGD volunteer. The deaf woman in this family was homozygous and her husband was a carrier of the IVS1+1G>A gene mutation. Among 8 biopsied embryos, two healthy embryos were implanted which resulted in a single pregnancy and subsequent birth of a healthy baby boy. This is the first report of a successful application of PGD for hearing loss in Iran. Having a baby with a severe hearing impairment often imposes families with long-term disease burden and heavy therapy costs. Today PGD has provided an opportunity for high-risk individuals to avoid the birth of a deaf child.

FLT3 Gene Mutation Profile and Prognosis in Adult Acute Myeloid Leukemia.

BACKGROUND: Internal tandem duplication (ITD) of FMS-related tyrosine kinase 3 (FLT3) gene, which occurs in exons 14 and 15, is one of the most prevalent somatic mutations in adult acute myeloid leukemia (AML) and has biological, prognostic, and therapeutic implications. The prognostic importance of codon 835 tyrosine kinase domain (TKD) mutation (exon 20), which occurs relatively frequently in adult AML, is often debated. We aimed to study the FLT3 gene mutation profile and prognosis in 139 adult Iranian patients with newly diagnosed AML. METHODS: We determined the status of ITD and TKD mutations using fragment analysis and the polymerase chain reaction-restriction fragment polymorphism method, respectively. In addition, we used direct sequencing to confirm the results of TKD mutation analysis. RESULTS: Twenty five percent of the patients had an ITD mutation. The mean size of the inserted fragment was 63.5 base pairs. Twenty percent of the ITD-positive patients showed more than one mutated population upon polymerase chain reaction. Statistical analyses showed that the ITD mutation was associated with a decreased overall survival among patients in the intermediate cytogenetic risk group (p-value = 0.013). The size of the ITD was not correlated with overall survival. Eight out of 139 patients (5.7%) had the codon 835 mutation. One new mutation of the insertion/deletion type was discovered. Analyses did not show any relationship between the TKD mutation and overall survival. Two patients (1.4%) showed concurrent TKD and ITD mutations. The TKD and ITD mutation rates of the FLT3 gene were consistent with previous studies on AML patients. CONCLUSIONS: This study supports the results of previous studies regarding the association of the FLT3-ITD mutation and poor prognosis.

GJB2 c.-23+1G>A mutation is second most common mutation among Iranian individuals with autosomal recessive hearing loss.

GJB2 mutation analysis is used routinely as a first step in genetic testing for autosomal recessive non-syndromic sensorineural hearing loss. Although most GJB2 mutations can be detected by sequencing of the exon 2 of this gene, a prevalent splice mutation, c.-23+1G>A (IVS1+1G>A), is not usually included in the analyzed region. In this study, we have developed an ARMS-PCR strategy for detection of this mutation among Iranian deaf individuals. A total of 418 Iranian individuals with hearing loss consistent with autosomal recessive non-syndromic sensorineural hearing loss based on audiological test result, medical history, physical examination and pedigree of the family, were included in this study. c.35delG and c.-23+1G>A mutations were detected by using ARMS-PCR. Direct sequencing of the exon 2 of the GJB2 gene was performed for mutation analysis of the coding region of this gene. Among 418 investigated cases, a total of 81 patients (~19.4 %) with biallelic pathogenic mutations in the GJB2 gene and 13 cases with only one pathogenic mutant allele were identified. The total allele frequencies of the two most frequent mutations, c.35delG and c.-23+1G>A, among mutated alleles were found to be around 59 and 15.7 %, respectively. High frequency of the c.35delG and c.-23+1G>A mutations among Iranian deaf individuals shows the importance of developing rapid and cost-effective methods for primary mutation screening methods before performing direct sequencing.

Reporting the presence of three different diseases causing GJB2 mutations in a consanguineous deaf family.

OBJECTIVE: This paper reports a consanguineous deaf family with three different mutations in the GJB2 gene. DESIGN: Four members of an Iranian deaf family were recruited in this study. The GJB2 coding region and exon-intron boundaries were investigated using direct sequencing. STUDY SAMPLE: The proposita was a 12-year-old girl with congenital non-syndromic hearing loss. She was born to consanguineous parents. The proposita, her parents and deaf maternal uncle were screened for GJB2 mutations. RESULTS: Sequencing demonstrated the presence of the c.176_191del and c.327_328delGGinsA mutations in the proposita, the c.176_191del mutation in her father, and the c.35delG and c.327_328delGGinsA mutations in trans in her apparently unaffected mother as well as in her congenitally deaf uncle. Follow-up pure-tone audiometry revealed moderate to severe mid- and high-frequency hearing loss in the mother. CONCLUSIONS: This study shows the complexity of genetic testing and counseling for hearing loss.

In silico Analysis of Two Novel Variants in the Pyruvate Carboxylase (PC) Gene Associated with the Severe Form of PC Deficiency.

Background: Inborne errors of metabolism are a common cause of neonatal death. This study evaluated the acute early-onset metabolic derangement and death in two unrelated neonates. Methods: Whole-exome sequencing (WES), Sanger sequencing, homology modeling, and in silico bioinformatics analysis were employed to assess the effects of variants on protein structure and function. Results: WES revealed a novel homozygous variant, p.G303Afs*40 and p.R156P, in the pyruvate carboxylase (PC) gene of each neonate, which both were confirmed by Sanger sequencing. Based on the American College of Medical Genetics and Genomics guidelines, the p.G303Afs*40 was likely pathogenic, and the p.R156P was a variant of uncertain significance (VUS). Nevertheless, a known variant at position 156, the p.R156Q, was also a VUS. Protein secondary structure prediction showed changes in p.R156P and p.R156Q variants compared to the wild-type protein. However, p.G303Afs*40 depicted significant changes at C-terminal. Furthermore, comparing the interaction of wild-type and variant proteins with the ATP ligand during simulations, revealed a decreased affinity to the ATP in all the variants. Moreover, analysis of Single nucleotide polymorphism impacts on PC protein using Polyphen-2, SNAP2, FATHMM, and SNPs&GO servers predicted both R156P and R156Q as damaging variants. Likewise, free energy calculations demonstrated the destabilizing effect of both variants on PC. Conclusion: This study confirmed the pathogenicity of both variants and suggested them as a cause of type B Pyruvate carboxylase deficiency. The results of this study would provide the family with prenatal diagnosis and expand the variant spectrum in the PC gene,which is beneficial for geneticists and endocrinologists.

The Spectrum of Pathogenic Variants in Iranian Families with Hemophilia A.

BACKGROUND: Hemophilia A (HA) is an X-linked recessive bleeding disorder with a high rate of genetic heterogeneity. The present study was conducted on a large cohort of Iranian HA patients and data obtained from databases. METHODS: A total of 622 Iranian HA patients from 329 unrelated families who had been referred to a medical genetics laboratory in Tehran from 2005 to 2019, were enrolled in this retrospective, observational study. Genetic screening of pathogenic variants of the F8 gene was performed using inverse shifting PCR, direct sequencing, and multiplex ligation-dependent amplification (MLPA). Point mutation frequencies in different exons were analyzed for our samples as well as 6031 HA patients whose data were recorded in a database. RESULTS: A total of 144 different pathogenic or likely pathogenic variants including 29 novel variants were identified. A strategy to decrease costs of genetic testing of HA was suggested based on this finding. CONCLUSION: This study provides comprehensive information on F8 pathogenic/likely pathogenic variants in Iranian HA patients which improves the spectrum of causative mutations and can be helpful to clinicians and medical geneticists in counseling and molecular diagnosis of HA.

Once in a Blue Moon, a Very Rare Coexistence of Glutaric Acidemia Type I and Mucopolysaccharidosis Type IIIB in a Patient

Background: Glutaric acidemia (GAI) and mucopolysaccharidosis type IIIB (MPSIIIB) are two rare genetic disorders caused by pathogenic variants in two different genes. Here, we report a coexistence of these two different rare disorders in an individual. Methods: A four-year-old Iranian boy born to first-cousin parents suspected to have MPSIIIB and/or GAI was investigated in this study. Targeted genomic enrichment and next-generation sequencing were used to examine genes related to MPS and GA. Sanger sequencing was performed to confirm the results. Results: Two homozygous likely pathogenic variants in α-N-acetylglucosaminidase (NAGLU) and GCDH genes were found and confirmed in the proband. Conclusion: A combination of specific features of two different diseases in a patient has been reported here. More studies on this case and similar cases can provide more information about the effect of simultaneous pathogenic variants in different genes.

Allelic heterogeneity among Iranian DFNB7/11 families: report of a new Iranian deaf family with TMC1 mutation identified by next-generation sequencing.

CONCLUSION: Co-segregation of c.2030T>C mutation with hearing loss in an Iranian family and absence of this mutation in 100 Iranian controls confirms the pathogenicity of this mutation. Allelic heterogeneity among Iranian DFNB7/11 families has been shown by the identification of six different mutations in eight families. OBJECTIVES: Transmembrane channel-like 1 (TMC1) gene encodes an integral membrane protein such that its mutations can cause DFNB7/11 hearing loss. To date, several TMC1 mutations have been reported from Iran. Here we report a new DFNB7/11 Iranian family with an unreported TMC1 mutation in Iran. METHODS: A total of 66 genes related to hearing loss were analyzed using the OtoSCOPE platform in an affected member of an Iranian deaf family (Irn-Deaf-6866). Sanger sequencing was performed to confirm next-generation sequencing findings. RESULTS: A mutation, c.2030T>C, was identified in exon 21 of the TMC1 gene in the investigated member of the family. Sequencing results in all members of the family confirmed association of this mutation with hearing loss. None of 100 ethnically matched healthy controls had this mutation.

A transversion mutation in non-coding exon 3 of the TMC1 gene in two ethnically related Iranian deaf families from different geographical regions; evidence for founder effect.

OBJECTIVES: Transmembrane channel-like 1 (TMC1) gene is a member of the transmembrane channel-like (TMC) gene family that encodes an integral membrane protein of the inner ear. It is suggested that mutation in this gene is one of the main causes of autosomal recessive non-syndromic hearing loss (ARNSHL) in different populations. The aim of this study was to determine the contribution of the TMC1 gene mutations in causing hearing loss in Iran. METHODS: In total 54 unrelated Iranian families containing 159 affected individuals with ARNSHL detected by audiometric and otologic examinations were analyzed. Haplotype analysis of all members of 45 GJB2- & GJB6-negative families, using four microsatellite markers linked to DFNB7/11 was performed. RESULTS: Co-segregation of hearing loss with all investigated markers for the DFNB7/11 locus was found in one family. DNA sequencing of all coding and non-coding exons and intron boundaries of the TMC1 gene identified c.-258A>C mutation in non-coding exon 3 only in individuals with hearing loss. This mutation has been previously reported in another Iranian family (G9) that share similar ethnicity. This variant was not detected in 300 ethnically matched healthy controls. CONCLUSIONS: These results increase the probability that this nucleotide variation may be a pathogenic mutation. This study showed that the ethnicity may be more useful than geographical location to design research strategy for determining which genes should be considered when a heterogeneous disorder is under investigation.

Sex chromosome aneuploidy in cytogenetic findings of referral patients from south of Iran.

BACKGROUND: Chromosome abnormality (CA) including Sex chromosomes abnormality (SCAs) is one of the most important causes of disordered sexual development and infertility. SCAs formed by numerical or structural alteration in X and Y chromosomes, are the most frequently CA encountered at both prenatal diagnosis and at birth. OBJECTIVE: This study describes cytogenetic findings of cases suspected with CA referred for cytogenetic study. MATERIALS AND METHODS: Blood samples of 4151 patients referred for cytogenetic analysis were cultured for chromosome preparation. Karyotypes were prepared for all samples and G-Banded chromosomes were analyzed using x100 objective lens. Sex chromosome aneuploidy cases were analyzed and categorized in two groups of Turners and Klinefelter's syndrome (KFS). RESULTS: Out of 230 (5.54%) cases with chromosomally abnormal karyotype, 122 (30%) cases suspected of sexual disorder showed SCA including 46% Turner's syndrome, 46% KFS and the remaining other sex chromosome abnormalities. The frequency of classic and mosaic form of Turner's syndrome was 33% and 67%, this was 55% and 45% for KFS, respectively. CONCLUSION: This study shows a relatively high sex chromosome abnormality in this region and provides cytogenetic data to assist clinicians and genetic counselors to determine the priority of requesting cytogenetic study. Differences between results from various reports can be due to different genetic background or ethnicity.