The OsMYC2-JA feedback loop regulates diurnal flower-opening time via cell wall loosening in rice.

Diurnal flower-opening time (DFOT), the time of spikelet opening during the day, is an important trait for hybrid rice (Oryza sativa L.) seed production. Hybrids between indica and japonica rice varieties have strong heterosis, but the parental lines usually have different, nonoverlapping DFOTs. This reduces the success of hybrid seed production in crosses between indica and japonica subspecies, thus hindering the utilization of indica and japonica inter-subspecies heterosis. However, little is known about the molecular mechanisms regulating DFOT in rice. Here, we obtained japonica rice lines with a DFOT 1.5 h earlier than the wild type by overexpressing OsMYC2, a gene encoding a key transcription factor in the jasmonate (JA) signaling pathway. OsMYC2 is activated by JA signaling and directly regulates the transcription of genes related to JA biosynthesis and cell wall metabolism. Overexpressing OsMYC2 led to significantly increased JA contents and decreased cellulose and hemicellulose contents in lodicule cells, as well as the softening of lodicule cell walls. This may facilitate the swelling of lodicules, resulting in early diurnal flower-opening. These results suggest that the OsMYC2-JA feedback loop regulates DFOT in rice via cell wall remodeling. These findings shed light on the understanding of regulatory mechanism of the DFOT of plants, which should promote the development of indica and japonica varieties suitable for hybrid rice breeding.

Controlling diurnal flower-opening time by manipulating the jasmonate pathway accelerates development of indica-japonica hybrid rice breeding.

Inter-subspecific indica-japonica hybrid rice (Oryza sativa) has the potential for increased yields over traditional indica intra-subspecies hybrid rice, but limited yield of F1 hybrid seed production (FHSP) hinders the development of indica-japonica hybrid rice breeding. Diurnal flower-opening time (DFOT) divergence between indica and japonica rice has been a major contributing factor to this issue, but few DFOT genes have been cloned. Here, we found that manipulating the expression of jasmonate (JA) pathway genes can effectively modulate DFOT to improve the yield of FHSP in rice. Treating japonica cultivar Zhonghua 11 (ZH11) with methyl jasmonate (MeJA) substantially advanced DFOT. Furthermore, overexpressing the JA biosynthesis gene OPDA REDUCTASE 7 (OsOPR7) and knocking out the JA inactivation gene CHILLING TOLERANCE 1 (OsHAN1) in ZH11 advanced DFOT by 1- and 2-h respectively; and knockout of the JA signal suppressor genes JASMONATE ZIM-DOMAIN PROTEIN 7 (OsJAZ7) and OsJAZ9 resulted in 50-min and 1.5-h earlier DFOT respectively. The yields of FHSP using japonica male-sterile lines GAZS with manipulated JA pathway genes were significantly higher than that of GAZS wildtype. Transcriptome analysis, cytological observations, measurements of elastic modulus and determination of cell wall components indicated that the JA pathway could affect the loosening of the lodicule cell walls by regulating their composition through controlling sugar metabolism, which in turn influences DFOT. This research has vital implications for breeding japonica rice cultivars with early DFOT to facilitate indica-japonica hybrid rice breeding.

Integrated full-length transcriptome and metabolome analysis reveals the defence response of melon to gummy stem blight.

Gummy stem blight (GSB), a widespread disease causing great loss to cucurbit production, has become a major threat to melon cultivation. However, the melon-GSB interaction remains largely unknown. Here, full-length transcriptome and widely targeted metabolome were used to investigate the defence responses of resistant (PI511089) and susceptible (Payzawat) melon accessions to GSB pathogen infection at 24 h. The biosynthesis of secondary metabolites and MAPK signalling pathway were specifically enriched for differentially expressed genes in PI511890, while carbohydrate metabolism and amino acid metabolism were specifically enriched in Payzawat. More than 1000 novel genes were identified and MAPK signalling pathway was specifically enriched for them in PI511890. There were 11 793 alternative splicing events involving in the defence response to GSB. Totally, 910 metabolites were identified in Payzawat and PI511890, and flavonoids were the dominant metabolites. Integrated full-length transcriptome and metabolome analysis showed eriodictyol and oxalic acid were the potential marker metabolites for GSB resistance in melon. Moreover, posttranscription regulation was widely involved in the defence response of melon to GSB pathogen infection. These results not only improve our understanding on the interaction between melon and GSB, but also facilitate the genetic improvement of melon with GSB resistance.

Human TDP1, APE1 and TREX1 repair 3'-DNA-peptide/protein cross-links arising from abasic sites in vitro.

Histones and many other proteins react with abundant endogenous DNA lesions, apurinic/apyrimidinic (abasic, AP) sites and/or 3'-phospho-α,ß-unsaturated aldehyde (3'-PUA), to form unstable but long-lived Schiff base DNA-protein cross-links at 3'-DNA termini (3'-PUA-protein DPCs). Poly (ADP-ribose) polymerase 1 (PARP1) cross-links to the AP site in a similar manner but the Schiff base is reduced by PARP1's intrinsic redox capacity, yielding a stable 3'-PUA-PARP1 DPC. Eradicating these DPCs is critical for maintaining the genome integrity because 3'-hydroxyl is required for DNA synthesis and ligation. But how they are repaired is not well understood. Herein, we chemically synthesized 3'-PUA-aminooxylysine-peptide adducts that closely resemble the proteolytic 3'-PUA-protein DPCs, and found that they can be repaired by human tyrosyl-DNA phosphodiesterase 1 (TDP1), AP endonuclease 1 (APE1) and three-prime repair exonuclease 1 (TREX1). We characterized these novel repair pathways by measuring the kinetic constants and determining the effect of cross-linked peptide length, flanking DNA structure, and the opposite nucleobase. We further found that these nucleases can directly repair 3'-PUA-histone DPCs, but not 3'-PUA-PARP1 DPCs unless proteolysis occurs initially. Collectively, we demonstrated that in vitro 3'-PUA-protein DPCs can be repaired by TDP1, APE1, and TREX1 following proteolysis, but the proteolysis is not absolutely required for smaller DPCs.

PARP1 Incises Abasic Sites and Covalently Cross-links to 3'-DNA Termini via Cysteine Addition Not Reductive Amination.

Poly [ADP-ribose] polymerase 1 (PARP1) is a ubiquitous nuclear enzyme that plays multifaceted roles in the cellular response to DNA damage. Previous studies demonstrated that PARP1 incises the most frequently formed DNA lesion, the apurinic/apyrimidinic (AP) site, and in the process is trapped as a DNA-PARP1 cross-link at the 3'-terminus. The covalent linkage was proposed to be composed of a secondary amine resulting from formal reductive amination of an initially formed incision product. PARP1 cysteine residues were proposed to reduce the initially formed Schiff base. Here, we report evidence to support a different mechanism in which DNA-PARP1 cross-links result from cysteine addition to incised AP sites.

In vitro eradication of abasic site-mediated DNA-peptide/protein cross-links by Escherichia coli long-patch base excision repair.

Apurinic/apyrimidinic (AP or abasic) sites are among the most abundant DNA lesions. Numerous proteins within different organisms ranging from bacteria to human have been demonstrated to react with AP sites to form covalent Schiff base DNA-protein cross-links (DPCs). These DPCs are unstable due to their spontaneous hydrolysis, but the half-lives of these cross-links can be as long as several hours. Such long-lived DPCs are extremely toxic due to their large sizes, which physically block DNA replication. Therefore, these adducts must be promptly eradicated to maintain genome integrity. Herein, we used in vitro reconstitution experiments with chemically synthesized, stable, and site-specific Schiff base AP-peptide/protein cross-link analogs to demonstrate for the first time that this type of DPC can be repaired by Escherichia coli (E. coli) long-patch base excision repair. We demonstrated that the repair process requires a minimum of three enzymes and five consecutive steps, including: (1) 5'-DNA strand incision of the DPC by endonuclease IV; (2 to 4) strand-displacement DNA synthesis, removal of the 5'-deoxyribose phosphate-peptide/protein adduct-containing flap, and gap-filling DNA synthesis by DNA polymerase I; and (5) strand ligation by a ligase. We further demonstrated that endonuclease IV plays a major role in incising an AP-peptide cross-link within E. coli cell extracts. We also report that eradicating model AP-protein (11.2-36.1 kDa) DPCs is less efficient than that of an AP-peptide10mer cross-link, supporting the emerging model that proteolysis is likely required for efficient DPC repair.

Synthesis and Excision Repair of Site-Specific 3'-End DNA-Histone Cross-Links Derived from Abasic Sites.

Histones catalyze the DNA strand incision at apurinic/apyrimidinic (AP) sites accompanied by formation of reversible but long-lived DNA-protein cross-links (DPCs) at 3'-DNA termini within single-strand breaks. These DPCs need to be removed because 3'-hydroxyl is required for gap-filling DNA repair synthesis but are challenging to study because of their reversible nature. Here we report a chemical approach to synthesize stable and site-specific 3'-histone-DPCs and their repair by three nucleases, human AP endonuclease 1, tyrosyl-DNA phosphodiesterase 1, and three-prime repair exonuclease 1. Our method employs oxime ligation to install an alkyne to 3'-DNA terminus, genetic incorporation of an azidohomoalanine to histone H4 at a defined position, and click reaction to conjugate DNA to H4 site-specifically. Using these model DPC substrates, we demonstrated that the DPC repair efficiency is highly affected by the local protein environment, and prior DPC proteolysis facilitates the repair.

Predictive and prognostic markers from endoscopic ultrasound with biopsies during definitive chemoradiation therapy in esophageal squamous cell carcinoma.

INTRODUCTION: Endoscopic ultrasound (EUS) may play a role in evaluating treatment response after definitive chemoradiation therapy (dCRT) for esophageal squamous cell carcinoma (ESCC). This study explored the prognostic markers of EUS with biopsies and developed two nomograms for survival prediction. METHODS: A total of 821 patients newly diagnosed with ESCC between January 2015 and December 2019 were reviewed. We investigated the prognostic value of the changes in tumor imaging characteristics and histopathological markers by an interim response evaluation, including presence of stenosis, ulceration, tumor length, tumor thickness, lumen involvement, and tumor remission. Independent prognostic factors of progression-free survival (PFS) and overall survival (OS) were determined using Cox regression analysis and further selected to build two nomogram models for survival prediction. The receiver operating characteristic (ROC) curve, calibration curve, and decision curve analysis (DCA) were used to respectively assess its discriminatory capacity, predictive accuracy, and clinical usefulness. RESULTS: A total of 155 patients were enrolled in this study and divided into the training (109 cases) and testing (46 cases) cohorts. Tumor length, residual tumor thickness, reduction in tumor thickness, lumen involvement, and excellent remission (ER) of spatial luminal involvement in ESCC (ER/SLI) differed significantly between responders and non-responders. For patients undergoing dCRT, tumor stage (P = 0.001, 0.002), tumor length (P = 0.013, 0.008), > 0.36 reduction in tumor thickness (P = 0.004, 0.004) and ER/SLI (P = 0.041, 0.031) were independent prognostic markers for both PFS and OS. Time-dependent ROC curves, calibration curves, and DCA indicated that the predicted survival rates of our two established nomogram models were highly accurate. CONCLUSION: Our nomogram showed high accuracy in predicting PFS and OS for ESCC after dCRT. External validation and complementation of other biomarkers are needed in further studies.

Predation stress experienced as immature mites extends their lifespan.

The early-life experience is important in modulating the late-life performance of individuals. It has been predicted that there were trade-offs between early-life fitness and late-life success. Most of the studies on senescence have focused on the trade-offs between the reproduction and lifespan, and the influences of diet, mating, and other factors. Because the negative, non-consumptive effects of predators could also modulate the behaviour and underlying mechanisms of the prey, this study aimed to examine the different effects of predator-induced stress experienced in the early life compared with later life of the prey. The prey (Tyrophagus putrescentiae) was exposed to predation stress from the predator (Neoseiulus cucumeris) during different periods of its life (immature, oviposition period, and post-oviposition period). The results showed that the predation stress experienced during immature stages delayed development by 7.3% and prolonged lifespan by 9.7%, while predation stress experienced in the adult stage (both oviposition and post-oviposition periods) decreased lifespans of T. putrescentiae (by 24.8% and 28.7%, respectively). Predation stress experienced during immature stages also reduced female fecundity by 7.3%, whereas that experienced during the oviposition period reduced fecundity of the prey by 50.7%. This study demonstrated for the first time lifespan extension by exposure to predation stress when young and highlighted the importance of early-life experience to aging and lifespan.

Long noncoding RNA PVT1 accelerates the growth of placental trophoblasts in preeclampsia through the microRNA-24-3p/HSD11B2 axis.

Long noncoding RNA plasmacytoma variant translocation 1 (PVT1) is essential for the maintenance of normal functions of trophoblasts in preeclampsia (PE). This study aims to decipher the concrete mechanism of PVT1 with the microRNA-24-3p/Type-2 11ß-hydroxysteroid dehydrogenase (miR-24-3p/HSD11B2) axis in PE. PVT1, miR-24-3p, and HSD11B2 expression levels in normal placental tissues and PE placental tissues were defined. HTR-8/SVneo cells were transfected to determine the effects of PVT1, miR-24-3p, and HSD11B2 on the growth of HTR-8/SVneo cells. The relationships among PVT1/miR-24-3p/HSD11B2 in HTR-8/SVneo cells were identified. PVT1 and HSD11B2 were downregulated, while miR-24-3p was upregulated in the placenta of PE. Upregulated/downregulated PVT1 promoted/impeded the growth of human placental trophoblast (HTR-8/SVneo) cells in PE. Restored/knocked down miR-24-3p impeded/enhanced the growth of HTR-8/SVneo cells in PE. PVT1 inhibited miR-24-3p to mediate HSD11B2. PVT1 sponges miR-24-3p to regulate HSD11B2; thereby, the growth of placental trophoblasts is promoted in PE.

Human TREX1 Repairs 3'-End DNA Lesions in Vitro.

Human three-prime repair exonuclease 1 (TREX1) is the major 3' to 5' exonuclease that functions to deplete the cytosolic DNA to prevent the autoimmune response. TREX1 is upregulated and translocates from cytoplasm to the nucleus in response to genotoxic stress, but the function of nuclear TREX1 is not well understood. Herein, we wish to report our in vitro finding that TREX1 efficiently excises 3'-phospho-α,ß-unsaturated aldehyde and 3'-deoxyribose phosphate that are commonly produced as base excision repair intermediates and also from the nonenzymatic strand incision at abasic sites.

Level-dependent effects of predation stress on prey development, lifespan and reproduction in mites.

In predator-prey interactions, non-consumptive effects of predators have been less studied than consumptive effects. However, non-consumptive effects may have significant influences on prey and can change different aspects of their life history such as development, reproduction and lifespan. The odour and other cues associated with a predator, without direct contact, could induce stress in prey, leading to phenotypic changes in life history traits. In this study, we investigate how mild and strong predator-induced stress could affect prey life history. The prey (Tyrophagus putrescentiae) was exposed, from hatching to death, to three different levels of predation stress from its predator (Neoseiulus cucumeris) (1, 3 or 5 predator adults in an adjacent cage separated by a mesh screen). Compared with the control, both males and females under predator-induced stress had longer developmental time and shorter lifespan when the level of predation stress increased, showing significant level-dependence. In addition, females had reduced fecundity under predation stress. Sex-specific response to predation stress was observed under a low level of predation stress: females had greater reduction in lifespan than males. Furthermore, the reduction in female lifespan was due more from the decrease in the post-oviposition period than the decrease in the oviposition period. Future studies applying even milder levels of predation press, such as exposure of prey to predator cues only during part of the prey lifespan, may provide additional insights.

The SlTCP26 promoting lateral branches development in tomato.

KEY MESSAGE: The SlTCP26 negatively regulated auxin signal to relieve the apical dominance and suppressed abscisic acid signal to remove the lateral bud dormancy, promoting lateral branches development. Lateral branches formation from lateral buds is a complex regulatory process in higher plants, and the interaction between transcription factors and hormones is indispensable during this process. TCP transcription factors have been reported to regulate lateral branches development, while the detailed function, especially interacting with auxin and ABA during this process, was still ambiguous in tomato. In this study, a branch regulatory gene, SlTCP26, was identified in tomato, and its role along with its interaction to hormones during branch development, as investigated. The results indicated that overexpression of SlTCP26 would promote lateral branches development, and could suppress the expressing of the genes associated with IAA signaling, presenting similar effects in decapitated plants. Conversely, the exogenous IAA application could inhibit the expression of SlTCP26. Furthermore, the expressing of the ABA signaling-related genes was inhibited in SlTCP26 overexpressed tomato, similar to that in decapitated tomato. Our findings suggested that SlTCP26 may be a crucial adjuster for synergistic action between ABA and IAA signals during the development of lateral branches, and it could promote the lateral buds grow into lateral shoots, via inhibiting IAA signal to relieve the apical dominance and suppressing ABA signal to remove the lateral bud dormancy. Our study provided some insights for the development of tomato lateral branches to understand the apical dominance regulatory network.

Detection and characterization of anterior loop, accessory mental foramen, and lateral lingual foramen by using cone beam computed tomography.

STATEMENT OF PROBLEM: The presence of an anterior loop (AL), accessory mental foramen (AMF), or lateral lingual foramen (LLF) adds complexity to the mental foraminal region, and consequently, implant placement in this region can damage the nerves and blood vessels if the structures are not carefully identified. PURPOSE: The purpose of this retrospective study was to evaluate the characteristics of the AL, AMF, and LLF to provide recommendations for surgeons operating in the mandibular mental foraminal region. MATERIAL AND METHODS: Reconstructed cone beam computed tomography (CBCT) images in 306 Southern Chinese patients were retrospectively analyzed. The prevalence of the anterior loop was assessed, and the anterior loop length (ALL) was measured from the most anterior point of the mental foramen to the most mesial part of the anterior loop. The prevalence, location of the AMF as related to the mental foramen, and adjacent teeth were analyzed. Additionally, the distance from the mental foramen to AMF centers were measured. The prevalence, diameter, and location of the LLF were evaluated. The chi-square test was used for prevalence analysis (α=.05). RESULTS: Anterior loops were identified in 67.8% of the 612 hemimandibles, with a mean length of 3.3 ±1.2 mm, ranging from 1.2 to 7.3 mm. A total of 5.56% of the hemimandibles had an ALL ≥5 mm, and male patients had a statistically longer loop length (P=.006). The ALL on the right side was found to be statistically longer than that on the left side (P=.002). No statistically significant differences in the ALL were found among different age groups (P=.124). AMFs were observed in 10.5% of the patients, with most located in the posterosuperior position of the mental foramen. The mean distance between the center of the AMF and the mental foramen was 5.1 ±1.4 mm. LLFs presented in 69.9% of patients, with 74.0% located in the premolar region. The mean vertical diameter of the LLF was 0.9 ±0.3 mm, and the mean distance from LLF to the mandibular inferior border was 7.1 ±1.9 mm. CONCLUSIONS: This study revealed that ALs, AMFs, and LLFs occurred often in the Southern Chinese population. Identifying the presence of the AL, AMF, and LLF is important before surgery in the mental foraminal region.

Influence of pathogenic fungi on the life history and predation rate of mites attacking a psyllid pest.

The two biological control agents, predatory mite Amblydromalus limonicus Garman & McGregor (Acari: Phytoseiidae) and entomopathogenic fungi (EPF) Beauveria bassiana (Balsamo) Vuillemin (Hypocreales: Cordycipitaceae), have the potential to independently suppress the invasive tomato potato psyllid (TPP), Bactericera cockerelli (Sulc), in New Zealand. The integrated strategy of combining the release of predatory mites and EPF may further promote biocontrol of pests. To examine the compatibility of A. limonicus and B. bassiana, we tested the lethal effects of B. bassiana on A. limonicus females on B. cockerelli and calculated the sublethal concentrations of B. bassiana. The effects of sublethal concentrations (LC10 and LC30) of EPF on predatory mite females were assessed on the reproduction parameters of the parental generation (F0). We also evaluated the transgenerational effects of EPF on life table parameters and predation rates of the offspring generation (F1) that was fed on the psyllids. Our results showed that A. limonicus females were susceptible to B. bassiana and the LC50 was 2.2 × 105 conidia mL-1. Sublethal concentrations of EPF (LC30) significantly reduced the fecundity, longevity, oviposition period and predation rates of F0 predatory mite females. However, life table and predation rates of F1 predatory mites were not influenced by sublethal concentrations (LC30), except for the reduction of the preadult and total pre-oviposition period, and the predation rate of F1 males. Also, F1 population parameters including the intrinsic rate of increase (rm), finite rate of increase (λ), gross reproduction rate (GRR), and net reproduction rate (R0) were not significantly influenced by sublethal concentrations (LC10 and LC30). This result suggests that the transgenerational effects of entomopathogenic fungi cannot be carried over to F1. In conclusion, the simultaneous application of both biological control agents does have the potential to control TPP at appropriate intervals during the crop season. Further evaluation in the field will be needed to confirm the viability of this approach to control TPP.

Regulatory T-cell deficiency leads to pathogenic bullous pemphigoid antigen 230 autoantibody and autoimmune bullous disease.

BACKGROUND: Autoimmune bullous diseases/dermatoses (AIBDs) are severe autoantibody-mediated skin diseases. The pathogenic relevance of autoreactive CD4+ T cells for the induction of autoantibody production remains to be fully evaluated. Scurfy mice lack functional regulatory T (Treg) cells, experience spontaneous activation of autoreactive CD4+ T cells, and display severe erosive skin lesions suggestive of AIBDs. OBJECTIVE: We sought to determine whether AIBDs develop in Treg cell-deficient scurfy mice. METHODS: Histology, indirect immunofluorescence (IF) microscopy, direct IF, and ELISA were used to prove the presence of AIBDs in scurfy mice. Monoclonal autoantibodies from sera of scurfy mice were screened by using indirect IF on murine skin, and immunoprecipitation and mass spectrometry were used for target antigen identification, followed by confirmation in modified human embryonic kidney cells and murine keratinocytes. Pathogenicity was determined by injecting the autoantibody into neonatal mice and transferring scurfy CD4+ T cells into nu/nu mice. RESULTS: Autoantibodies against different known autoantigens of AIBDs spontaneously develop in scurfy mice. Histology reveals subepidermal blisters, and direct IF of skin of scurfy mice shows a predominant linear staining pattern. The mAb 20B12 shows a linear staining pattern in indirect IF, recognizes the murine hemidesmosomal protein bullous pemphigoid antigen 230 (BP230) as the target antigen, and cross-reacts with human BP230. Purified mAb 20B12 induces subepidermal blisters in neonatal mice. Transfer of scurfy CD4+ T cells is sufficient to induce antibodies with reactivity to AIBD autoantigens and subepidermal blisters in the skin of recipient T cell-deficient nu/nu mice. CONCLUSION: We show that the absence of Treg cells leads to AIBDs by pathogenic autoantibodies targeting BP230.

Identification of the -α(2.4) Deletion in One Family and in One Hb H Disease Patient in Guangxi, People's Republic of China.

The 2.4 kb (or -α(2.4)) deletion in the α-globin gene cluster (NG_000006.1) is an α(+)-thalassemia (α(+)-thal) allele. The molecular basis of -α(2.4) is a deletion from 36860 to 39251 of the α-globin gene cluster. It was reported by three research groups in 2005, 2012 and 2014, respectively. In routine thalassemia screening studies by this research group, we found an individual with the -α(2.4)/αα genotype and an Hb H (ß4) disease patient whose genotype was - -(SEA)/-α(2.4). Samples from the parents of the carrier of the -α(2.4)/αα genotype were collected to perform pedigree analysis, and the proband's mother's genotype was diagnosed to be - -(SEA)/-α(2.4). The research revealed that the -α(2.4) allele exists in the population of southern Guangxi, People's Republic of China.

Diagnosis of a Family with the Novel -α(21.9) Thalassemia Deletion.

The Qinzhou α-thalassemia (α-thal) or -α(21.9) deletion was first described at the Qinzhou Maternal and Child Health Care Hospital, Qinzhou, Guangxi, People's Republic of China (PRC) in 2013. The molecular biological mechanism by which this allele leads to α-thal involves the deletion of a 21.9 kb DNA fragment of the α-globin gene cluster (NG_000006.1), designated as -α(21.9). During routine screening, a new family with -α(21.9) was found by the research group. This is the first time that an adult patient with the -α(21.9)/αα genotype and a 6-month-old baby with the -α(21.9)/- -(SEA) (Southeast Asian) genotype were detected in one family. The discovery of this family demonstrates that there is a certain risk for the Qinzhou α-thal deletion in the southern regions of Guangxi Province, PRC. The detection of the adult patient with the -α(21.9)/αα genotype and the analysis of hematological data are important supplements for -α(21.9) research. Additionally, Hb Bart's (γ4) and Hb H (ß4) were detected in the 6-month-old, confirming that the baby with the -α(21.9)/- -(SEA) genotype also carries Hb H disease. The analysis of this family verifies that the -α(21.9) deletion is an α(+)-thal allele.