Identifying genetic variants and pathways associated with extreme levels of fetal hemoglobin in Sickle Cell Disease in Tanzania.
Siana NkyaLiberata MwitaJosephine MgayaHappiness KumburuMarco van ZwetselaarStephan MenzelGaston K. MazanduRaphael Zozimus SangedaEmile R. ChimusaJulie Makani
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Abstract Background: Sickle cell disease (SCD) is a blood disorder caused by a point mutation on the beta globin gene resulting in the synthesis of abnormal hemoglobin. Fetal hemoglobin (HbF) reduces disease severity, but the levels vary from one individual to another. Most research has focused on common variants which differ across populations and hence do not fully account for HbF variation. Methods: We investigated rare and common genetic variants that influence HbF levels in 14 SCD patients to elucidate variants and pathways in SCD patients with extreme HbF levels (≥7.7% for high HbF) and (≤2.5% for low HbF) in Tanzania. We performed targeted next generation sequencing (Illumina_Miseq) covering exonic and other significant fetal hemoglobin-associated loci, including BCL11A , MYB , HOXA9 , HBB , HBG1 , HBG2 , CHD4 , KLF1 , MBD3 , ZBTB7A and PGLYRP1 . Results: Results revealed a range of genetic variants, including bi-allelic and multi-allelic SNPs, frameshift insertions and deletions, some of which have functional importance. Notably, there were significantly more deletions in individuals with high HbF levels (11% vs 0.9%). We identified deletions with high HbF levels and frameshift insertions in individuals with low HbF. CHD4 and MBD3 genes, interacting in the same sub-network, were identified to have a significant number of pathogenic or non-synonymous mutations in individuals with low HbF levels, suggesting an important role of epigenetic pathways in the regulation of HbF synthesis. Conclusions: This study provides new insights in selecting essential variants and identifying potential biological pathways associated with extreme HbF levels in SCD using multiple genomic variants associated with HbF in SCD.Mendelian inheritance
Phosphoglucomutase
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Despite the large number of genes that are expected to be involved in non-syndromal, recessive deafness, only a few have been cloned. One of these genes is GJB2, which encodes connexin 26. A frameshift mutation in this gene has been reported to be common in several populations and a carrier frequency of about 1 in 30 people has been detected in Italy and Spain. Mutation 35delG is difficult to detect because it lies within a stretch of six guanines flanked by thymines, so the deletion of one G does not create or destroy a restriction site and mutagenesis primers are not useful for this mutation. We have generated an allele specific oligonucleotide method that uses 12-mer oligonucleotides and easily discriminates between the normal and 35delG alleles. The method should permit a rapid analysis of this mutation in congenital cases (recessive or sporadic), including diagnosis and carrier detection of 35delG in the population.
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Lac repressor
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Abstract We have examined the effects of mutations in the six allele-specific modifier genes su(Hw), e(we), su(f), su(s), su(wa), and su(pr) on the expression of 18 modifiable alleles, situated at 11 loci. Ten of the modifiable alleles are associated with insertions of the gypsy retrotransposon and the others include alleles associated with insertions of copia and 412. We tested or retested 90 of the 108 possible combinations and examined the expression of modifiable alleles in flies mutant for pairs of modifier genes in various heterozygous and homozygous configurations. Our principal findings are: (1) a screen of 40,000 mutagenized X chromosomes yielded three new mutations in known modifier genes, but revealed no new modifier genes; (2) the modification effects of different mutations in a given modifier gene were qualitatively similar; (3) each of the six modifiers suppressed some modifiable alleles, enhanced others, and had no noticeable effect on still others; (4) the modifier genes could be placed in four classes, according to their effects on the gypsy-insertion alleles; and (5) the effects of mutations in different modifier genes combined additively. Implications of these results for models of modifier gene action are discussed.
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To investigate further the genetic basis of hereditary multiple exostoses (EXT) and provide useful information for gene diagnosis of the disease.Polymerase chain reaction-single strand conformation polymorphism was used to examine the entire coding regions of EXT(1) gene on chromosome 8 and EXT(2) gene on chromosome 11 for mutation in thirty EXT families. Mutations were further identified by sequencing.Two frameshift mutations were identified in two unrelated EXT families. One was the deletion of one base(T) in exon 6 of the EXT(1) gene, and the other was the deletion of four bases (tgtt) in exon 2 of the EXT(2) gene. Both of the mutations resulted in a frameshift and premature termination of translation.EXT is a genetically heterogeneous bone disorder caused by the mutation of EXT tumor suppressor gene. These results could be directly applied in the genetic counseling and prenatal genetic diagnosis of EXT.
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A form of autosomal recessive spastic ataxia (ARSACS) has been described in the Charlevoix and Saguenay regions of Quebec. So far a frameshift and a nonsense mutation have been identified in the SACS gene. The authors report a new mutation (1859insC), leading to a frameshift with a premature termination of the gene product sacsin, in two sisters from consanguineous parents. The phenotype is similar to previously described patients with ARSACS.
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Nonsense
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Waardenburg syndrome type 1 (WS1) is an autosomal dominant disorder characterized by deafness, dystopia canthorum, heterochromia iridis, white forelock, and premature greying. A similar phenotype is caused in the mouse by mutations in the Pax-3 gene. This observation, together with comparisons of conserved syntenies in the murine and human genetic maps, suggested that at least some WS1 mutations should occur in HuP2, the probable human homolog of Pax-3. Two mutations in the HuP2 sequence of individuals with WS1 have been reported recently. Both of them occur in the highly conserved paired box region of the gene, which encodes a DNA binding domain. The functional consequences of these mutations are at present speculative. We report here a 14 bp deletion in the paired domain encoded by exon 2 of HuP2 in an Indonesian family segregating for WS1. This frameshift mutation results in a premature termination codon in exon 3. The HuP2 product is a truncated protein lacking most of the paired domain and all of the predicted homeo domain. We propose that the WS1 phenotype in this family is due to loss of function of HuP2 and discuss two mechanisms for the dominant effect of this mutation.
Waardenburg syndrome
Stop codon
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A novel heterozygous mutation (c.325dup) was identified in EXT1 gene from the proband and the affected family members; this mutation was absent in all the unaffected family members. The identification of the novel frameshift insertion mutation (c.325dup) expands the mutation spectrum of HME, which provides new evidence for HME diagnosis.
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Hereditary multiple exostoses
Insertion
Chinese family
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Mutation Testing
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ABSTRACT Programmed translational frameshifts have been identified in genes from a broad range of organisms, but typically only a very few genes in a given organism require a frameshift for expression. In contrast, a recent analysis of gene sequences available in GenBank from ciliates in the genus Euplotes indicated that >5% required one or more +1 translational frameshifts to produce their predicted protein products. However, this sample of genes was nonrandom, biased, and derived from multiple Euplotes species. To test whether there truly is an abundance of frameshift genes in Euplotes , and to more accurately assess their frequency, we sequenced a random sample of 25 cloned genes/macronuclear DNA molecules from Euplotes crassus . Three new candidate +1 frameshift genes were identified in the sample that encode a membrane occupation and recognition nexus (MORN) repeat protein, a C 2 H 2 -type zinc finger protein, and a Ser/Thr protein kinase. Reverse transcription-PCR analyses indicate that all three genes are expressed in vegetatively proliferating cells and that the mRNAs retain the requirement of a frameshift. Although the sample of sequenced genes is relatively small, the results indicate that the frequency of genes requiring frameshifts in E. crassus is between 3.7% and 31.7% (at a 95% confidence interval). The current and past data also indicate that frameshift sites are found predominantly in genes that likely encode nonabundant proteins in the cell.
Translational frameshift
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Objective To investigate EXT gene sequence in a pedigree with hereditary multiple exostoses (HME) and identify the causative mutation in the pedigree. Methods All the exons and their flanking sequences of EXT1 and EXT2 genes were amplified by PCR from proband′ s genomic DNA, PCR products were purified and sent for direct sequencing. Results A novel insertion-deletion (651 - 664 delins TTT) mutation in exon 1 of EXT1 gene was found, the mutation resulted in a frameshift at amino acid position 218 and a premature stop codon at position 220 (K218 fs X220) in the EXT1 protein. The mutation also lead to a truncated protein. Family study results showed that this mutation came from the proband′s mother. No variant was found in EXT2 gene of this proband. Conclusions 651 - 664 delins TTT heterozygous mutation in EXT1 gene was the molecular mechanism of HME for this pedigree.
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Hereditary multiple exostoses
genomic DNA
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