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.

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.

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.

Molecular Characterization of δ-Thalassemia in Iran.

δ-Thalassemia (δ-thal) (OMIM #142000) resulting from mutations on the HBD gene usually has no clinical consequences. However, it may cause the misdiagnosis of ß-thalassemia (ß-thal) carriers by lowering the Hb A2 level to the normal range. Therefore, a study for δ-thal should be considered as a step in the detection of at-risk couple in our region. The aim of the present study was to characterize the mutations of the HBD gene in ß-thal carriers with normal Hb A2 levels, and also in normal individuals with Hb A2 of less than 2.0%. Four ß-thal carriers with normal Hb A2 and 39 individuals with Hb A2 of less than 2.0% were enrolled. Genomic DNA was extracted by the salting out method and the HBD gene was investigated by polymerase chain reaction (PCR) and direct DNA sequencing. Hb A2-Yialousa (HBD: c.82 G > T) was the most common variant found in the HBD gene, but the following mutations were also found: Hb A2-NYU (HBD: c.39 T > A), Hb A2-Coburg (HBD: c.350 G > A), Hb A2-Etolia (HBD: c.257 T > C), Hb A2-Fitzroy (HBD: c.428 C > A) and the δ-IVS-I-5 (G > T) (HBD: c.92 + 5 G > T). One case was a compound heterozygote for δ-IVS-I-5/Hb A2-Fitzroy. The results of this single center study suggest that the mutations in the HBD gene in the Iranian population are heterogeneous and should be considered in genetic counseling of families.

A Large Cohort Study of Genotype and Phenotype Correlations of Beta- Thalassemia in Iranian Population.

BACKGROUND: Thalassemia syndromes are the most prevalent single gene disorders in Iran. This study aimed to evaluate the effect of different types of beta-globin gene mutations, co-inheritance of alpha-globin gene mutations and/or Xmn1 SNP on disease phenotype in a large cohort of Iranian patients. SUBJECTS AND METHODS: In total, 433 patients were clinically classified into ß-thalassemia major (TM) or intermedia (TI). Multiplex PCR, ARMS-PCR, RFLP-PCR and DNA sequencing were performed to identify both α- and ß-globin gene mutations and Xmn1 polymorphism as well. All data were compared and analyzed by SPSS software in TM and TI groups, consequently. RESULTS: A total of 39 different ß-globin mutations were identified. Among them, the most common were IVS IInt1 (40.33%) followed by IVS Int5 (9.56%), C30 (7.22%) and Fr8-9(7%). All patients were subjected to evaluate common α-globin gene deletions. The patients inherited concomitant mutations of α- and ß-globin, showed no clinical modifications compared with those who had only ß-globin mutation. The TI patients showed a significant increase in frequency of both heterozygous and homozygous form of the Xmn1 polymorphism. It was also found that ß(0)/ß(0) genotype patients, inherited the Xmn1 polymorphism required lesser blood transfusion. CONCLUSION: No significant differences were observed, on the severity of disease, between patient's inherited defective α- and ß-globin genes and ones with just ß-globin gene mutation. Taking the results of this research into account, Xmn1 polymorphism can be considered as an important genetic factor modulating the severity of disease.

Comparisons between RT-PCR, real-time PCR, and in vitro globin chain synthesis by α/ß ratio calculation for diagnosis of α- from ß-thalassemia carriers.

BACKGROUND: Thalassemia, which may be due to point mutations, translocations, and deletions involving the α or ßglobin gene, is the most prevalent single gene disorder in Iran.This study aims to calculate the α/ß ratio in normal cases, α- and ß-thalassemia carriers by RT-PCR, real-time PCR, and in vitro globin chain synthesis (GCS) in order to establish the most accurate technique to distinguish between α- and ß-thalassemia carriers in suspicious cases. METHODS: The α/ß ratios were calculated in all samples by RT-PCR, real-time RT-PCR, and in vitro GCS. RESULTS: Using RT-PCR, the ratios were 1.09 ± 0.07 in normal samples, 1.2 ± 0.17 in ß-thalassemia, 1.08 ± 0.19 in mild α-thalassemia, and 0.96 ± 0.19 in severe α-thalassemia carriers. In real-time RT-PCR, the ratios were 2.21 ± 1.36 in normal samples, 5.12 ± 1.83 in ß-thalassemia, 2.88 ± 0.81 in mild α-thalassemia, and 1.18 ± 0.52 in severe α-thalassemia carriers. With GCS, the ratios were 1.03 ± 0.1 in normal samples, 1.9 ± 0.37 in ß-thalassemia, 0.8 ± 0.13 in mild α-thalassemia, and 0.59 ± 0.12 in severe α-thalassemia carriers. CONCLUSION: To determine the most accurate technique, we statistically analyzed the α/ß ratios obtained from the three standard methods. The ratio obtained by GCS and real-time PCR were helpful in distinguishing between α and ß carriers in suspicious patients in whom the mutation detection was limited and the risk for offspring was not clear. The use of this technique is more obvious when time is restricted (i.e. during the pregnancy period).

Validation and comparison of two quantitative real-time PCR assays for direct detection of DMD/BMD carriers.

OBJECTIVES: To develop a robust and reliable assay for direct identification of female carriers of deletions in the dystrophin gene. DESIGN AND METHODS: We compared two quantitative real-time PCR approaches for the detection of the deletions of exons 4, 17, 47, and 50 in DMD/BMD carriers. One hundred and ten individuals from 26 unrelated families, including 8 large pedigrees characterized by having at least two DMD affected males, were studied. Carrier status of the subjects was also evaluated by MLPA. RESULTS: The results showed the gene dosage ratio of 0.99+/-0.14 and 1.09+/-0.19 for normal individuals and 0.48+/-0.06 and 0.50+/-0.10 for carriers in SYBR green and TaqMan probe assays, respectively. Carrier status was accurately attributed in 100% of cases and confirmed by MLPA. CONCLUSION: Quantitative real-time PCR can be used as a direct method for carrier detection in female relatives of DMD patients with known deletions. The results are comparable to the MLPA data.

Detection of unknown deletions in beta-globin gene cluster using relative quantitative PCR methods.

beta-Thalassemia is mainly caused by mutations involving single base substitution and small deletions. However, a considerable number of carriers are suspected to have large deletions in beta-globin gene cluster. Common strategy for identifying deletions with definite breakpoints is based on Gap PCR. There are, however, some cases with indefinite breakpoints which usually cannot be detected by this method. We developed and optimized a quantitative real-time PCR assay for copy number analysis of beta-globin gene cluster. The copy number of target fragments (i.e. beta, delta or (G)gamma-globin genes) was determined using comparative threshold cycle method. In addition, gene dosage was analyzed using multiplex ligation-dependent probe amplification (MLPA) method in all suspected carriers. Using these relative quantitative assays, normal or carrier statuses of all 26 unknown samples were successfully determined according to the ranges obtained from the ratios of normal and definite carrier samples. Interestingly, large deletions involving the entire beta-globin gene cluster were observed in six carrier individuals. This study showed that the MLPA as a preliminary screening test can be followed by SYBR Green real-time PCR for analysis of copy number variations in beta-globin gene cluster. Combination of these relative quantitative PCR methods could be an appropriate approach for accurate diagnosis of unknown beta-thalassemia deletions in routine diagnosis of beta-thalassemia mutations.