We have isolated and characterised the nuclear gene that codes for the 30.4-kDa subunit of the peripheral arm of complex I from Neurospora crassa. The single-copy gene was localised on chromosome VI of the fungal genome by restriction fragment length polymorphism mapping. An extra copy of the gene was introduced into a strain of N. crassa by transformation. This strain was crossed with another strain in order to inactivate, by repeat-induced point mutations, both copies of the duplication carried by the parental transformant. Ascospore progeny from the cross were analysed and a mutant strain lacking the 30.4-kDa protein, nuo30.4, was isolated and further characterised. The mutant appears to assemble the membrane arm of complex I, while formation of the peripheral arm is prevented. Nevertheless, the mutant grows reasonably well--indicating that this well conserved protein is not essential for vegetative growth--and is able to mate with other strains both as male or female. Strains with multiple mutations are readily obtained from heterozygous crosses between different complex I mutants of N. crassa. On the other hand, homozygous crosses between several mutants, including nuo30.4, fail to produce ascospores. These results suggest that complex I plays an essential role during the sexual phase of the life cycle of the fungus.

The nuclear gene coding for the 20.8-kDa subunit of the membrane arm of respiratory chain NADH: ubiquinone reductase (Complex I) from Neurospora crassa, nuo-20.8, was localized on linkage group I of the fungal genome. A genomic DNA fragment containing this gene was cloned and a duplication was created in a strain of N. crassa by transformation. To generate RIP (repeat-induced point) mutations in the duplicated sequence, the transformant was crossed with another strain carrying an auxotrophic marker on chromosome I. To increase the chance of finding an isolate with a non-functional nuo-20.8 gene, random progeny from the cross were selected against this auxotrophy since RIP of the target gene will only occur in the nucleus carrying the duplication. Among these, we isolated and characterised a mutant strain that lacks the 20.8 kDa mitochondrial protein, indicating that this cysteine-rich polypeptide is not essential. Nevertheless, the absence of the 20.8-kDa subunit prevents the full assembly of complex I. It appears that the peripheral arm and two intermediates of the membrane arm of the enzyme are still formed in the mutant mitochondria. The NADH: ubiquinone reductase activity of sonicated mitochondria from the mutant is rotenone insensitive. Electron microscopy of mutant mitochondria does not reveal any alteration in the structure or numbers of the organelles.

Alpha-thalassemias are among the most common genetic diseases in the world. They are characterized by hypochromic and microcytic anemia and great clinical variability, ranging from a practically asymptomatic phenotype to severe anemia, which can lead to intrauterine or early neonatal death. Deletions affecting the α-globin genes, located on chromosome 16p13.3, are the main causes of α-thalassemia. Multiplex ligation-dependent probe amplification (MLPA) can be used to detect rearrangements that cause α-thalassemia, particularly large deletions involving the whole α cluster and/or deletions in the HS-40 region. Here, MLPA was used to investigate the molecular basis of α-thalassemia in five unrelated patients, three of whom had Hb H disease. In addition to the -α3.7 deletion identified in the patients with Hb H disease, four different α0 deletions removing 15 to 225 kb DNA segments were found: two of them remove both the α genes, one affects only the regulatory element (HS-40) region, and another one extends over the entire α cluster and the HS-40 region. These results illustrate the diversity of α-thalassemia deletions in the Brazilian population and highlight the importance of molecular investigation in cases that present with microcytosis and hypochromia without iron deficiency and normal or reduced Hb A2 levels..

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Alpha thalassemias are the commonest group of hereditary hemoglobin disorders in the world, the most common cause being deletions involving the α globin genes on 16p13.3. We found a new α0 deletionassociated with the -α3.7 deletion in a 3-month-old brown-skinned female Brazilian patient with Hb H disease. The deletion was identified by Multiplex Ligation-dependent Probe Amplification (MLPA) using the SALSA MLPA P140 C1 HBA kit (MRC Holland, Amsterdam, Holland). An approximately 360 kb-region of DNA extending from the telomeric region of the short arm of chromosome 16 to the DECR2 gene was analyzed, and the fragments generated were compared with Coffalyser.Net. In addition to the α3.7 deletion, an extensive trans deletion encompassing 70 to 105 kb of DNA and involving the α2and α1 genes and the contiguous LUC7L and ITFG3 genes was detected (Figure 1). The 5' breakpoint of the deletion is located between the ψα1and the α2 gene, while the 3' breakpoint region is between the ITFG3 and RGS11 genes. Familial analysis revealed that while the patient's father was heterozygous for the -α3.7 deletion, her mother and oldest sister were heterozygous for the α0 deletion and her middle sister also had Hb H disease. A polymorphism (C > T) in the 220 probe binding region in the hybrid gene (α2α1) (-α3.7 deletion) was also detected in the patient and her father, causing this probe to have a different pattern from that expected in MLPA. To our knowledge, this deletion does not have any similarities in terms of its breakpoint regions with other deletions previously described in the literature. It was named --ATB because the family came from the town of Atibaia in the state of São Paulo in southeast Brazil. Molecular characterization of α-thalassemia deletions is essential both for correct diagnosis of carriers of these deletions and for genetic counselling so that new cases of Hb H disease or Hb Bart's Hydrops Fetalis can be prevented. The hematologic and molecular data for the family studied here are shown in Table 1. Financial Support: FAPESP - 2014/00984-3, CNPq and CAPES/Brazil. Table 1. Hematologic and molecular data for the family members. Family Proband (3 months) Mother Father Sister (2 years) Sister (3 years) RBC RV: M:4,5-6,1/F: 4,2-5,4 4.55 5.81 5.39 5.50 5.07 Hb RV: M: 14-18/F: 12-16 7.75 12.1 14.8 7.6 10.4 MCH RV: 27-32 17 20.8 27.4 13.8 20.6 MCV RV: 80-99 54.1 64.2 79.6 41.5 61.4 A2 (%) VR: 1,6-4 0.78 2.13 2.80 2.40 2.80 F (%) RV: < 2 23.70 0.70 0.90 4.70 1 Hb profile A2, F, A, Bart's + H (10%) A2, A A2, A A2, A, H A2, A Genotype -α3.7/--ATB αα/--ATB αα/-α3.7 -α3.7/--ATB αα/--ATB RV: Reference values RBC: red blood cells (106/mm3); Hb: hemoglobin (g/dL); MCH: mean corpuscular hemoglobin (pg); MCV: mean corpuscular volume (fl). Figure 1. Figure 1. (A) Graph generated by the Coffalyser.Net software with the result for the proband. The x-axis represents the probes, and the y-axis the ratio of the signal intensity of the probes for the proband to the mean signal intensity for the normal controls. A ratio of 1 indicates the presence of both alleles, a ratio of 0.5 a loss of 1 allele and 0 the loss of that region in both alleles. (B) Schematic representation of 16p13.3. The oval represents the telomeric region, the arrows the locations of the probes and the boxes the genes. The blue line corresponds to the deleted fragment, the dotted lines denote the first and last deleted probes and the blank regions show where the breakpoints may be (adapted from MRC-Holland, 2014). Disclosures No relevant conflicts of interest to declare.