Codon Research Articles

Creation of a functional unc-11/PICALM GFP knock-in by CRISPR.

unc-11 is the only C. elegans ortholog of mammalian PICALM/AP180, paralogs that play an important role in Clathrin-Mediated Endocytosis (CME) and the recycling of a subset of SNAREs, the vesicle-associated membrane proteins (VAMPs). In this publication we report the creation of a new unc-11 allele that is endogenously-tagged with GFP just upstream of the stop codon. Moreover, we demonstrate that the UNC-11::GFP fusion protein functions like wild type with an expression pattern similar to UNC-11 antibody staining described previously.

Erratum: CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy.

An erratum was issued for: Studying TGF-β Signaling and TGF-β-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells. The phrases"surveyor assay"and"SurveyorNuclease" havebeen updated to "T7E1 assay" to " T7endonuclease I" respectively. Step 1.2 inthe Protocol has been updated from: Surveyor nucleaseassay of sgRNA NOTE: The targeting efficiency of the sgRNA used for the knock-in experiment is evaluated by surveyor nuclease assay (also known as T7 endonuclease I (T7EI) assay)17. Select the sgRNA with high DNA cleavage efficiency and a low distance between the sgRNA cutting site and the stop codon. to: T7 endonuclease assay of sgRNA NOTE: The targeting efficiency of the sgRNA used for the knock-in experiment is evaluated by T7 endonuclease (T7EI)assay17. Select the sgRNA with high DNA cleavage efficiency and a low distance between the sgRNA cutting site and the stop codon. Figure 1 in the Representative Results has been updatedfrom: Figure 1: HMEJ-mediated targeted integration in vitro. (A) Experimental scheme for selection of sgRNAs: Six different sgRNAs (Cdx2-sgRNA1~Cdx2-sgRNA6) around the stop codon of the Cdx2 locus with a higher rank and off-target potential were chosen based on online CRISPR design tool. The protospacer adjacent motif (PAM) sequence is in red. (B) Experimental design: The Cas9-CMV-GFP expression plasmids expressing sgRNA, Cas9, and GFP were introduced into N2acells. GFP+ cells were sorted at day 3 for surveyor assay. (C) Surveyor assay for Cdx2 targeting: 6 different sgRNAs were designed for surveyor assay. Normal N2a cell genomic DNA serves as control. *, the sgRNA used for Cdx2-2A-mCherry knock-in experiment. (D) Schematic overview of construction of HMEJ donors using Gibson assembly. (E) Schematic overview of HMEJ-mediated gene targeting strategy at Cdx2 locus. HAL/HAR, left/right homology arm; triangles, sgRNA target sites; OF/OR, outer forward/reverse primer; IF/IR, inner forward/reverse primer. Figure modified from previous report10.Please click here to view a larger version of this figure. to: Figure 1: HMEJ-mediated targeted integration in vitro. (A) Experimental scheme for selection of sgRNAs: Six different sgRNAs (Cdx2-sgRNA1~Cdx2-sgRNA6) around the stop codon of the Cdx2 locus with a higher rank and off-target potential were chosen based on online CRISPR design tool. The protospacer adjacent motif (PAM) sequence is in red. (B) Experimental design: The Cas9-CMV-GFP expression plasmids expressing sgRNA, Cas9, and GFP were introduced into N2acells. GFP+ cells were sorted at day 3 for T7EIassay. (C) T7EI assay for Cdx2 targeting: 6 different sgRNAs were designed for T7EI assay. Normal N2a cell genomic DNA serves as control. *, the sgRNA used for Cdx2-2A-mCherry knock-in experiment. (D) Schematic overview of construction of HMEJ donors using Gibson assembly. (E) Schematic overview of HMEJ-mediated gene targeting strategy at Cdx2 locus. HAL/HAR, left/right homology arm; triangles, sgRNA target sites; OF/OR, outer forward/reverse primer; IF/IR, inner forward/reverse primer. Figure modified from previous report10.Please click here to view a larger version of this figure.

BNT162b2 Vaccine: Possible Codons Misreading, Errors in Protein Synthesis and Alternative Splicing's Anomalies

BNT162b2 vaccine against Covid-19 is composed of an RNA having 4284 nucleotides, divided into 6 sections, which bring the information to create a factory of S Spike proteins, the ones used by Sars-CoV-2 (Covid-19) to infect the host. After that, these proteins are directed outside the cell, triggering the immune reaction and antibody production. The problem is the heavy alteration of the mRNA: Uracil is replaced to fool the immune system with Ψ (Pseudouridine); the letters of all codon triplets are replaced by a C or a G, to extremely increase the speed of protein synthesis; replacement of some amino acids with Proline; addition of a sequence (3'-UTR) with unknown alteration. These impairments could cause strong doubts about the presence of codon usage errors. An eventual mistranslation has consequences on the pathophysiology of a variety of diseases. In addition, mRNA injected is a pre-mRNA, which can lead to the multiple mature mRNAs; these are alternative splicing anomalies, direct source of serious long-term harm on the human health. In essence, what will be created may not be identical with protein S Spike: just an error in translational decoding, codons misreading, production of different amino acids, then proteins, to cause serious long-term damage to human health, despite the DNA is not modified, being instead in the cell nucleus and not in the cytoplasm, where the modified mRNA arrives. However, in this case, the correlation between speed of synthesis and protein expression with synthesis errors, as well as the mechanism that could affect the translation of the sequence remain obscure, many trials have not yet been performed.

A novel homozygous TPM1 mutation in familial pediatric hypertrophic cardiomyopathy and in silico screening of potential targeting drugs.

Familial hypertrophic cardiomyopathy (HCM) is the most common genetic cardiac disease. While sarcomeric gene mutations explain many HCM cases, the genetic basis of about half of HCM cases remains elusive. Here we aimed to identify the gene causing HCM in a non-consanguineous Saudi Arabian family with affected family members and a history of sudden death. The impact of the identified mutation on protein structure and potential drug targets were evaluated in silico. Triplets (two HCM subjects and one patent ductus arteriosus (PDA) case) and unaffected parents were screened by targeted next-generation sequencing (NGS) for 181 candidate cardiomyopathy genes. In silico structural and functional analyses, including protein modeling, structure prediction, drug screening, drug binding, and dynamic simulations were performed to explore the potential pathogenicity of the variant and to identify candidate drugs. A homozygous missense mutation in exon 1 of TMP1 (assembly GRCh37-chr15: 63340781; G>A) was identified in the triplets [two HCM and one patent ductus arteriosus (PDA)] that substituted glycine for arginine at codon 3 (p.Gly3Arg). The parents were heterozygous for the variant. The mutation was predicted to cause a significant and deleterious change in the TPM1 protein structure that slightly affected drug binding, stability, and conformation. In addition, we identified several putative TPM1-targeting drugs through structure-based in silico screening. TPM1 mutations are a common cause of HCM and other congenital heart defects. To date, TPM1 has not been associated with isolated PDA; to our knowledge, this is the first report of the homozygous missense variation p.Gly3Arg in TPM1 associated with familial autosomal recessive pediatric HCM and PDA. The identified candidate TPM1 inhibitors warrant further prospective investigation.

Shine & Lal index as a predictor for early detection of \u03b2-thalassemia carriers in a limited resource area in Bandung, Indonesia

sBackgroundThalassemia is the most common inherited disease in the world, involving α- or β-globin in red blood cells. Thalassemia cases rank fifth in the list of national catastrophic diseases in Indonesia; however, nationwide screening for thalassemia carriers is not yet mandatory. This study aimed to assess whether blood count metrics, such as the Shine & Lal index (SLI; MCV*MCV*MCH/100), might serve as a predictor to screen thalassemia carriers in a limited resource area where molecular methods are not readily available.MethodsDuring a family gathering of thalassemia patients, family members (n196) underwent a complete blood count test. Those with MCV < 80 fL and/or MCH < 27 pg and/or SLI < 1530 were further examined for Hb analysis. Only samples with HbA2 fraction > 4% or with a peak in the HbE fraction were sequenced to confirm β-globin gene mutations.ResultsOf 196 family members, 117 (59.6%) had low MCV and/or low MCH and/or low SLI. The HbE fraction (mean 24.06% ± 0.95, range 22.4–26.5) was found in 27 (13.7%) cases, and all had a mutation at codon (CD)26 (c.79G > A). The mean HbA2 fraction in these samples was 3.18% ± 0.62 (range 2.6–3.8). For samples with HbA2 > 4% (n30; 15.3%), all had mutations at IVS1nt5 (c.92 + 5 G > C; n28), CD8/9 (c.27_28insG; n1) and CD19 (c.59A > G; n1). The mean HbA2 fraction with a mutation at IVS1nt5 (c.92 + 5 G > C) was 4.65% ± 0.77 (range 4.0–5.6). Interestingly, anaemia was only present in 25 and 57% of β-thalassemia carriers with mutations at CD26 (c.79G > A) and at IVS1nt5 (c.92 + 5 G > C), respectively.ConclusionsThe Shine & Lal index is helpful in the early screening of β-thalassemia carriers, since this index confirms mutations at CD-26 (c.79G > A) and at IVS1nt5 (c.92 + 5 G > C), which are both common mutations in Bandung, Indonesia. Further DNA analysis is a topic of interest to map variants in globin genes and their distribution across populations.

Survival outcomes in the PRIME study for patients (pts) with RAS/BRAF wild-type (WT) metastatic colorectal cancer (mCRC), by baseline Eastern Cooperative Oncology Group (ECOG) performance status (PS).

3557^ Background: In PRIME, panitumumab (pmab) + FOLFOX4 improved overall survival (OS) for RAS WT mCRC pts vs FOLFOX4 alone (Douillard et al 2013). Prespecified subgroup analysis identified only E...

Characterisation of a new HLA-DRB1 allele:DRB1*03:85

HLA-DRB1*03:85 differs from HLA-DRB1*03:06 by two nucleotides, position 257 A>T and position 258T>C, resulting in Valine at codon 57.

Use of denaturing gradient gel electrophoresis in screening unknown β-thalassemia mutations in Egyptian patients

The molecular defects resulting in a β-thalassemia phenotype, in the Egyptian population show a clear heterogenic pattern. Many studies have embarked on the molecular detection and characterization of these mutations, using a wide array of the available techniques with successful detection of both known and unknown mutations. PCR based techniques, as well as direct DNA sequencing are effective with some limitations as regards the time, effort and high cost to reach a final diagnosis. Intermediary screening techniques have proved to be effective tools to overcome these drawbacks. This study aims to assess the use of the denaturing gradient gel electrophoresis (DGGE)1DGGE1: denaturing gradient gel electrophoresis.1 to detect β-thalassemia mutations prior to the performance of direct sequencing to minimize the cost and workload involved in the process. In this study, forty-two previously genotyped patients in a study by El-Gawhary et al. in 2007, have been analyzed by DGGE for fragment 2 then 1. These are the β-globin gene fragments showing the majority of the β-thalassemia mutations. Sixty-eight alleles out of 79 mutant alleles in total were detected within these two fragments. The 11 undetected alleles comprise 9 alleles that require further examination using other DGGE fragments (0, 4 and 5) and correspond to -87(C>G), intervening sequence (IVS)II-1(G>A), IVS II-745, and IVS II-848(C>A). The remaining two that failed detection correspond to codon (CD) 37(G>A). Although, IVS-II 745(C>G) is undetectable within these two fragments, its 100% linkage polymorphism (+20 C>T) was detected in fragment 1 gel. DGGE is a sensitive technique to screen for β-thalassemia mutations. For simultaneous analysis of multiple samples with unknown mutations, it is recommended that direct DNA sequencing be coupled with DGGE whenever available to reduce time, effort and cost.

Characterization of a novel allele, HLA‐A*03:132

A*03:132 differs from A*03:01:01:01 at nucleotide 853 (codon 261) in exon 4.

Association of the p53 codon 72 polymorphism with colorectal cancer in South West of Iran

The p53 tumor suppressor gene plays two important roles in genomic stability: blocking cell proliferation after DNA damage until it has been repaired, and starting apoptosis if the damage is too critical. Codon 72 exon4 polymorphism (Arg72Pro) of the P53 gene has been implicated in cancer risk. Various studies have been done to investigate the status of p53 at codon 72 for arginine (Arg) and proline (Pro) alleles in different populations and also the association of this codon 72 polymorphism with various tumors. Our objective was to investigate the possible association between P53 Arg72Pro polymorphism and susceptibility to colorectal cancer among Isfahan and Chaharmahal Va Bakhtiari (a part of south west of Iran) population. We investigated the status of p53 at codon 72 for Arg/Arg, Arg/Pro and Pro/Pro allele polymorphisms in blood samples from 145 colorectal cancer patients and 140 controls by Nested-PCR of p53 exon 4 and digestion with BstUI restriction enzyme and the DNA fragments were then resolved by electrophoresis in 2% agarose gel. The Pro allele was 279 bp, while the Arg allele was restricted into two fragments of 160 and 119 bp. Among the 145 colorectal cancer cases 49 cases (33.79%) were homozygous for the Arg72 allele (Arg/Arg), 18 cases (12.41%) were homozygous for the Pro72 allele (Pro/Pro) and 78 cases (53.8%) found in heterozygous (Arg/Pro). In conclusion, it can be said that p53Arg/Arggenotype may be correlated with possible increased risk of this kind of cancers in south west of Iran. Key words: Colorectal cancer, nested-PCR, p53 codon 72, polymorphism, Iran.

SEQUENCIAMENTO E ANÁLISE DOS GENES DAS SUBUNIDADES ALFA E BETA DO HORMÔNIO FOLÍCULO ESTIMULANTE DE BOVINO (Bos taurus indicus)

The aim this work was to promove the cloning and analysis sequencing of the subunits alpha and beta of the Bos taurus indicus follicle stimulated hormone (FSH). It to compare the results of sequencing these subunits between subunits aplha and beta from swine and Bos taurus taurus previouly published in GenBank. There was a high similarity between nucleotides and predicted amino acids in the ?FSH chain of Bos taurus indicus and those of swine and buffalo. In the compare the sequence of the subunit of ?FSH of Bos taurus indicus with swine showed differences in three aminoacid residues with sFSH there was a modification in the first base of the codon, which had to an alteration in the 83 amino acid residue which in Bos taurus indicus and a glycin, this was serine in Bos taurus taurus. This modification, as well as those indentyfied in cDNA of the ?FSH and sFSH chains were confirmed by cloning. The modification of serine for glycine in position 83 was the only substitute that altered the residue in the comparson between sFSH subunit of Bos taurus indicus and Bos taurus taurus . Nevertheles this modification showld not significantly alter the physiological properties of FSH as the glycine residue was also found in the swine sFSH, it is therefore a specific modification which distinguishes between the sFSH of Bos taurus taurus and Bos taurus indicus. KEY WORDS: Bovine, cloning, FSH, hormone.

Current status of thalassemia in minority populations in Guangxi, China

Thalassemia is one of the most common monogenic disorders in the world. In order to develop a community-based prevention program, we screened 12,900 individuals for alpha- and beta-thalassemia in Baise City, Guangxi, China, with hematological methods and molecular assays. We found that the frequency of carriers in this area for alpha-thalassemia is 15%. Beta-thalassemia carriers comprise 4.8% of the populations. Five mutations account for 98% of alpha-thalassemia [--SEA 46.7%; -alpha/4.2, 23.9%; -alpha/3.7, 21.7%; hemoglobin (Hb) Constant Spring, 6.5%; Hb Quong Sze, 1.1%]. Seven mutations in the beta-globin gene account for 99% of the mutations [codon (CD) 41/42 (-TCTT) (39.4%), CD 17(A-->T) (32%), CD 71/72 (+A) (7.4%), -28 (A-->G) (5.8%), IVS-2-654 (C-->T) (5.8%), CD26 (Hb E) (4%), IVS-1 (G-->A) (3.7%), and CD 43(G-->T) (1.9%)]. Most individuals with alpha-thalassemia major die in the uterus or shortly after birth. Among 106 patients with beta-thalassemia major followed by our clinic, the majority died before 5 years of age. Knowledge surveys about thalassemia were conducted. Our results show a severe lack of knowledge about thalassemia in both medical professionals and in the general populations. This study shows that thalassemia is a very severe public health issue in minority populations in Baise City, China. Identification of the common mutations will allow us to design cost-effective molecular tests. There is an urgent need to educate the general population and the medical community for a successful community-based prevention program.

Song: The codon song (to the tune of “when I'm sixty‐four”)*

Song: The codon song (to the tune of “when I'm sixty‐four”)*

Rapid, simultaneous genotyping of five common Southeast Asian beta-thalassemia mutations by multiplex minisequencing and denaturing HPLC.

β-Thalassemias are inherited hemoglobin disorders characterized by reduced production of β-globin chain. The severe forms of β-thalassemia produce marked anemia starting a few months after birth, and survival relies on regular blood transfusion and the lifelong use of drugs to prevent iron accumulation (1). β-Thalassemias are among the commonest genetic disorders in the world, and more than 200 mutations have been described to date. However, each at-risk population has a few common mutations (usually four or five) and a variable number of rare alleles (1). Common Southeast Asian β-thalassemia mutations include −28 A>G, codon (CD) 26 G>A, CD 41/42 −CTTT, CD 71/72 +A, IVS2 + 654 C>T, and others. The mutation CD 26 G>A produces hemoglobin E, presents like thalassemia, and is particularly common in Thailand, Cambodia, Vietnam, and Malaysia (1). There are many methods for genotyping known β-thalassemia mutations (2). Here we describe a rapid method for simultaneous genotyping of these five common mutations. The study was approved by the Human Subject Ethics Subcommittee of the Hong Kong Polytechnic University. The method is based on multiplex minisequencing followed by analysis of the single-base-extended products by denaturing HPLC (DHPLC). In minisequencing, a primer anneals to a DNA template immediately upstream of a mutation site to be analyzed and is extended by a single base as a result of incorporating a dideoxyribonucleotide triphosphate (ddNTP) (3). The single-stranded extension products are then separated by ion-pair reversed-phase liquid chromatography under completely denaturing condition, and the retention time is a function of both size and base composition (4). The DNA template for minisequencing was first generated by amplifying the whole human β-globin gene using primers HBBpF1 and HBBpR1 (Table 1⇓ ). The 25-μL reaction mixture contained 0.1 μM each of the primers, 50 ng of genomic DNA, 2.0 mM MgCl2 …

The molecular basis for the thalassaemias in Sri Lanka.

The beta-globin gene mutations and the alpha-globin genes of 620 patients with the phenotype of severe to moderate thalassaemia from seven centres in Sri Lanka were analysed. Twenty-four beta-globin gene mutations were identified, three accounting for 84.5% of the 1240 alleles studied: IVSI-5 (G-->C) 56.2%; IVSI-1 (G-->A) 15.2%; and haemoglobin E (codon (CD)26 GAG-->GAA) 13.1%. Three new mutations were found; a 13-bp deletion removing the last nucleotide in CD6 to CD10 inclusively, IVSI-129 (A-->C) in the consensus splice site, and a frame shift, CD55 (-A). The allele frequency of alpha+ thalassaemia was 6.5% and 1.1% for -alpha3.7 and -alpha4.2 deletions respectively. Non-deletion alpha-thalassaemia was not observed. Triplicate or quadruplicate alpha-globin genes were unusually common. In 1.5% of cases it was impossible to identify beta-thalassaemia alleles, but in Kurunegala detailed family studies led to an explanation for the severe thalassaemia phenotype in every case, including a previously unreported instance of homozygosity for a quadruplicated alpha-globin gene together with beta-thalassaemia trait. These findings have implications for the control of thalassaemia in high-frequency populations with complex ethnic histories.

Co‐existence of the Codon 16 (–C) (β0) and Codon 10 (C→A) (β+) Mutations on the Same β‐Globin Gene

Co‐existence of the Codon 16 (–C) (β0) and Codon 10 (C→A) (β+) Mutations on the Same β‐Globin Gene

Beta-thalassemia in the Korean population.

Beta-thalassemia is uncommon in the Korean population, however it must be considered in the differential diagnosis of hypochromic anemia. The molecular characterization of beta-thalassemia is absolutely necessary for molecular diagnosis as well as any genetic epidemiological study in this region. We analyzed the molecular basis of beta-thalassemia in 47 Korean families. Using direct sequencing of genomic DNA amplified through PCR and haplotype analysis, 44 beta-thalassemia genes were characterized, all of which were heterozygous. Fourteen different mutations were identified. The common mutations noted included the initiation codon (CD) ATG-->AGG (23.4%), CD 17 A-->T (21.2%), and IVS-II-1 G-->A (12.7%). Interestingly, mutations causing dominantly inherited beta-thalassemia were common (17.0%). All cases of IVS-II-1 G-->A mutations were linked to the silent mutation of CD 91 C-->T of the -globin gene. The initiation CD ATG-->AGG and IVS-II-1 G-->A with CD 91 C-->T were found in the Far East only, and may be inherited from a common origin for each mutation, at least in Koreans. CD17 A-->T and CDs 41/42-TTCT were suggested to be introduced by gene-flow from southern China. Otherwise, Hb Korea, CDs 89/90 -GT and a novel beta-thalassemia mutation, CD 131 CAG-->TAG, were only identified in Koreans. This mutation spectrum is characteristic of the low prevalent area of beta-thalassemia, however it is quite different even from the adjacent countries, Japan or China.

Identification of novel Asian Indian and Japanese mutations causing beta-thalassaemia in the Egyptian population.

beta-thalassaemia is a major health problem in Egypt. It has been estimated that of the 1.5 million live births. 1000 children with beta-thalassaemia major are born annually. Although the available treatment has increased the life expectancy of patients, it is still unsatisfactory and represents a significant drain on the country's resources. National screening and prenatal diagnosis programmes can be provided in Egypt once the spectrum of beta-thalassaemia mutations has been identified within the Egyptian population. We have examined 16 DNA samples with 21 beta-thalassaemia mutations that remained unidentified in a study of 54 patients reported by Rady and colleagues in 1996. Using the polymerase chain reaction and single strand conformation analysis we identified the following changes: frameshift (FS) codon (CD) 8/9 (+G), 4 FS CD 29 (-G) and 2 novel mutations in exon I (15 CD 22 A-C and 1 FS CD 28 -C). In addition, a silent, probably polymorphic mutation, CD 17 G-A was present in all chromosomes.

HB Valletta [β87(F3)THR →PRO] DUE to an A→C Substitution at Codon 87 in a Calabrian Family with α-Thalassemia

HB Valletta [β87(F3)THR →PRO] DUE to an A→C Substitution at Codon 87 in a Calabrian Family with α-Thalassemia

Globin mRNA in beta-thalassemia heterozygotes with different beta-thalassemia alleles and in heterozygotes for hereditary persistence of fetal hemoglobin.

Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to determine the alpha 2/alpha 1-, alpha/beta-, and gamma/beta-mRNA ratios in subjects with beta-thalassemia (beta-thal), hereditary persistence of fetal hemoglobin (HPFH), and normal adults. The alpha- and beta-globin gene mutations were characterized with gene mapping, PCR, and DNA sequencing. The average alpha 2/alpha 1-mRNA ratio was the same in normal adults and beta-thal heterozygotes with four alpha-globin genes (2.61-2.63) or with an alpha-thal-2 trait (1.48-1.55). The average alpha/beta-mRNA ratios were 4.47 and 3.84 in normal adults with four alpha-globin genes and with alpha-thal-2 trait (-alpha/alpha alpha), respectively. There was an increase of approximately 50% in beta-thal heterozygotes with transcriptional mutants [-88 (C-->T) and -29 (A-->G)] with lower values (approximately 25%) in those with alpha-thal-2 trait (-alpha/alpha alpha). High alpha/beta ratios were also observed for heterozygotes for nonsense or frameshift mutants located in exon 1 or exon 2. Increases of approximately 150-165% were seen in subjects with RNA processing defects; an exception was the IVS-1-110 (G-->A) mutation with a normal value in the heterozygote. The increases were also less pronounced in heterozygotes for the codon (CD) 121 (G-->T) mutation and the CDs 134-137 insertion/deletion. Normal alpha/(gamma + beta) values were seen in 3 heterozygotes each with a different deletion involving part of the beta-globin gene. The presence of the silent beta-thal allele, -101 (C-->T), in trans to a CD 8 (-AA) allele has a minor effect on the alpha/beta-mRNA ratio. The alpha/beta-mRNA ratio in HPFH heterozygotes was approximately 145% of normal, but with a gamma-mRNA level of 35.4-44.7% the calculated alpha/(gamma + beta) ratio became as in normal adults. The RT-PCR methodology appears useful in expression studies in beta-thal (and HPFH) and values of mRNA appear to correspond to the type of prevailing mutation(s) and concomitant alpha-thal.