First report of a patient with homozygous hemoglobin Ernz: Evidence to support a non-pathogenic variant.

Hemoglobin Ernz (Hb Ernz) is a missense variant in ß-globin caused by a Threonine to Asparagine substitution at the 123rd amino acid position and HBB c.371C > A in gene level. Hb Ernz has been classified as Uncertain Significance (VUS) by ACMG due to limited reports and the absence of any homozygote genotypes. In our study, we found eight cases of Hb Ernz by DNA sequencing of the ß-globin gene during >20 years of Thalassemia Screening in individuals with borderline hematological parameters who were possible carriers of thalassemia or their spouses. We also report the first homozygote variant of Hb Ernz. Our findings suggest that the changes in hematological parameters observed in individuals with Hb Ernz are likely due to α-globin gene mutations rather than Hb Ernz itself. These findings support the reclassification of Hb Ernz as a benign variant in variant classification.

A decade of molecular preimplantation genetic diagnosis of 350 blastomeres for beta-thalassemia combined with HLA typing, aneuploidy screening and sex selection in Iran.

BACKGROUND: Preimplantation genetic diagnosis (PGD) has been developed to detect genetic disorders before pregnancy which is usually done on blastomeres biopsied from 8-cell stage embryos obtained from in vitro fertilization method (IVF). Here we report molecular PGD results for diagnosing of beta thalassemia (beta-thal) which are usually accompanied with evaluating chromosomal aneuploidies, HLA typing and sex selection. METHODS: In this study, haplotype analysis was performed using short tandem repeats (STRs) in a multiplex nested PCR and the causative mutation was detected by Sanger sequencing. RESULTS: We have performed PGDs on 350 blastomeres from 55 carrier couples; 142 blastomeres for beta-thal only, 75 for beta-thal and HLA typing, 76 for beta-thal in combination with sex selection, and 57 for beta-thal and aneuploidy screening. 150 blastomeres were transferable, 15 pregnancies were happened, and 11 babies born. We used 6 markers for beta-thal, 36 for aneuploidy screening, 32 for sex selection, and 35 for HLA typing. To our knowledge combining all these markers together and the number of STR markers are much more than any other studies which have ever done. CONCLUSIONS: PGD is a powerful diagnostic tool for carrier couples who desire to have a healthy child and wish to avoid medical abortion.

Identification of a novel mutation in congenital afibrinogenemia in Iranian patients.

Congenital afibrinogenemia is a rare autosomal recessive disorder that is caused by defects in the fibrinogen. Fibrinogen is a hexameric glycoprotein made of two pairs of three homologous polypeptide chains including Aα, Bß, and γ that are encoded by three genes named FGA, FGB, and FGG. We aim to study four Iranian families who were referred to our lab for molecular diagnosis of afibrinogenemia. Genomic DNA was extracted from whole blood and Sanger sequencing was performed using primers for all exons and exon-intron junctions of FGA, FGB, and FGG genes. Pathogenicity of the variants was predicted using different in-silico tools and was interpreted according to the American College of Medical Genetics and Genomics guideline. We found three types of mutations in the studied families; two were in the FGA gene and one was in the FGB gene including a nonsense, a novel splicing mutation, and two deletion ones. The nonsense and the deletion mutations may cause a truncated protein and are likely pathogenic and pathogenic, respectively. The novel mutation of splicing found in the FGB gene is a pathogenic one and can break the wild-type acceptor site. Studying mutations in afibrinogenemia patients can expand our knowledge about this disease in Iran.

Comprehensive Mutation Analysis and Report of 12 Novel Mutations in a Cohort of Patients with Spinal Muscular Atrophy in Iran.

Spinal muscular atrophies (SMAs) are a heterogeneous group of neuromuscular diseases characterized by loss of motor neurons, muscle weakness, hypotonia and muscle atrophy, with different modes of inheritance; however, the survival motor neuron 1 (SMN1) gene is predominantly involved. The aims of the current study were to clarify the genetic basis of SMA and determine the mutation spectrum of SMN1 and other associated genes, in order to provide molecular information for more accurate diagnosis and future prospects for treatment. We performed a comprehensive analysis of 5q SMA in 1765 individuals including 528 patients from 432 unrelated families with at least one child with suspected clinical presentation of SMA. Copy number variations of the SMN1 and SMN2 genes and linkage analysis were performed using multiplex ligation-dependent probe amplification (MLPA) and short tandem repeat (STR) markers linked to the SMN1 gene. Cases without mutation in the SMA locus on 5q were analyzed for the DNAJB2, IGHMBP2, SIGMAR1 and PLEKHG5 genes using linked STR markers. Sanger sequencing of whole genes was performed for cases with homozygous haplotypes. Whole-genome sequencing (WGS) and whole-exome analysis was conducted for some of the remaining cases. Mutations in the SMN1 gene were identified in 287 (66.43%) families including 269 patients (62.26%) with homozygous deletion of the entire SMN1 gene. Only one of the patients had a homozygous point mutation in the SMN1 gene. Among the remaining families, three families showed mutations in either the DNAJB2, SIGMAR1 or PLEKHG5 genes, which were linked using STR analysis and Sanger sequencing. From 10 families who underwent WGS, we found six homozygous point mutations in six families for either the TNNT1, TPM3, TTN, SACS or COL6A2 genes. Two mutations in the PLA2G6 gene were also found in another patient as compound heterozygous. This rather large cohort allowed us to identify genotype patterns in Iranian 5q SMA patients. The process of identifying 11 mutations (9 novel) in 9 different genes among non-5q SMA patients shows the diversity of genes involved in non-5q SMA in Iranians. Genotyping of patients with SMA is essential for prenatal and preimplantation genetic diagnosis (PGD), and may be very helpful for guiding treatment, with the advent of new, more effective, albeit very expensive, therapies. Also, combining linkage analysis was shown to be beneficial in many ways, including sample authenticity and segregation analysis, and for ruling out maternal cell contamination during prenatal diagnosis (PND).