Mutations of the Mitochondrial Holocytochrome c–Type Synthase in X-Linked Dominant Microphthalmia with Linear Skin Defects Syndrome
2006; Elsevier BV; Volume: 79; Issue: 5 Linguagem: Inglês
10.1086/508474
ISSN1537-6605
AutoresIsabella Wimplinger, Manuela Morleo, Georg Rosenberger, Daniela Iaconis, Ulrike Orth, Peter Meinecke, Israela Lerer, Andrea Ballabio, Andreas Gal, Brunella Franco, Kerstin Kutsche,
Tópico(s)RNA regulation and disease
ResumoThe microphthalmia with linear skin defects syndrome (MLS, or MIDAS) is an X-linked dominant male-lethal disorder almost invariably associated with segmental monosomy of the Xp22 region. In two female patients, from two families, with MLS and a normal karyotype, we identified heterozygous de novo point mutations—a missense mutation (p.R217C) and a nonsense mutation (p.R197X)—in the HCCS gene. HCCS encodes the mitochondrial holocytochrome c–type synthase that functions as heme lyase by covalently adding the prosthetic heme group to both apocytochrome c and c1. We investigated a third family, displaying phenotypic variability, in which the mother and two of her daughters carry an 8.6-kb submicroscopic deletion encompassing part of the HCCS gene. Functional analysis demonstrates that both mutant proteins (R217C and Δ197–268) were unable to complement a Saccharomyces cerevisiae mutant deficient for the HCCS orthologue Cyc3p, in contrast to wild-type HCCS. Moreover, ectopically expressed HCCS wild-type and the R217C mutant protein are targeted to mitochondria in CHO-K1 cells, whereas the C-terminal–truncated Δ197–268 mutant failed to be sorted to mitochondria. Cytochrome c, the final product of holocytochrome c–type synthase activity, is implicated in both oxidative phosphorylation (OXPHOS) and apoptosis. We hypothesize that the inability of HCCS-deficient cells to undergo cytochrome c–mediated apoptosis may push cell death toward necrosis that gives rise to severe deterioration of the affected tissues. In summary, we suggest that disturbance of both OXPHOS and the balance between apoptosis and necrosis, as well as the X-inactivation pattern, may contribute to the variable phenotype observed in patients with MLS. The microphthalmia with linear skin defects syndrome (MLS, or MIDAS) is an X-linked dominant male-lethal disorder almost invariably associated with segmental monosomy of the Xp22 region. In two female patients, from two families, with MLS and a normal karyotype, we identified heterozygous de novo point mutations—a missense mutation (p.R217C) and a nonsense mutation (p.R197X)—in the HCCS gene. HCCS encodes the mitochondrial holocytochrome c–type synthase that functions as heme lyase by covalently adding the prosthetic heme group to both apocytochrome c and c1. We investigated a third family, displaying phenotypic variability, in which the mother and two of her daughters carry an 8.6-kb submicroscopic deletion encompassing part of the HCCS gene. Functional analysis demonstrates that both mutant proteins (R217C and Δ197–268) were unable to complement a Saccharomyces cerevisiae mutant deficient for the HCCS orthologue Cyc3p, in contrast to wild-type HCCS. Moreover, ectopically expressed HCCS wild-type and the R217C mutant protein are targeted to mitochondria in CHO-K1 cells, whereas the C-terminal–truncated Δ197–268 mutant failed to be sorted to mitochondria. Cytochrome c, the final product of holocytochrome c–type synthase activity, is implicated in both oxidative phosphorylation (OXPHOS) and apoptosis. We hypothesize that the inability of HCCS-deficient cells to undergo cytochrome c–mediated apoptosis may push cell death toward necrosis that gives rise to severe deterioration of the affected tissues. In summary, we suggest that disturbance of both OXPHOS and the balance between apoptosis and necrosis, as well as the X-inactivation pattern, may contribute to the variable phenotype observed in patients with MLS. Microphthalmia with linear skin defects syndrome (MLS [MIM #309801]), also known as "MIDAS" (microphthalmia, dermal aplasia, and sclerocornea), is a rare X-linked dominant condition characterized by unilateral or bilateral microphthalmia and linear skin defects—which are limited to the face and neck, consisting of areas of aplastic skin that heal with age to form hyperpigmented areas—in affected females and in utero lethality for males. Additional features in female patients include agenesis of the corpus callosum, sclerocornea, chorioretinal abnormalities, infantile seizures, congenital heart defect, mental retardation, and diaphragmatic hernia.1Van den Veyver IB Microphthalmia with linear skin defects (MLS), Aicardi, and Goltz syndromes: are they related X-linked dominant male-lethal disorders?.Cytogenet Genome Res. 2002; 99: 289-296Crossref PubMed Scopus (36) Google Scholar In the majority of cases, patients carry a chromosomal aberration that results in segmental monosomy of the Xp22 chromosomal region (>11 Mb). To date, eight patients with an apparently normal female karyotype have been described.2Bird LM Krous HF Eichenfield LF Swalwell CI Jones MC Female infant with oncocytic cardiomyopathy and microphthalmia with linear skin defects (MLS): a clue to the pathogenesis of oncocytic cardiomyopathy?.Am J Med Genet. 1994; 53: 141-148Crossref PubMed Scopus (56) Google Scholar, 3Happle R Daniels O Koopman RJ MIDAS syndrome (microphthalmia, dermal aplasia, and sclerocornea): an X-linked phenotype distinct from Goltz syndrome.Am J Med Genet. 1993; 47: 710-713Crossref PubMed Scopus (84) Google Scholar, 4Kherbaoui-Redouani L Eschard C Bednarek N Morville P [Cutaneous aplasia, non compaction of the left ventricle and severe cardiac arrhythmia: a new case of MLS syndrome (microphthalmia with linear skin defects)].Arch Pediatr. 2003; 10: 224-226Crossref PubMed Scopus (17) Google Scholar, 5Morleo M Pramparo T Perone L Gregato G Le Caignec C Mueller RF Ogata T Raas-Rothschild A de Blois MC Wilson LC Zaidman G Zuffardi O Ballabio A Franco B Microphthalmia with linear skin defects (MLS) syndrome: clinical, cytogenetic, and molecular characterization of 11 cases.Am J Med Genet A. 2005; 137: 190-198Crossref PubMed Scopus (29) Google Scholar, 6Zvulunov A Kachko L Manor E Shinwell E Carmi R Reticulolinear aplasia cutis congenita of the face and neck: a distinctive cutaneous manifestation in several syndromes linked to Xp22.Br J Dermatol. 1998; 138: 1046-1052Crossref PubMed Scopus (22) Google Scholar The MLS minimal critical region has been delineated to 610 kb in Xp22.2, through breakpoint mapping of two X;Y translocations of patients not affected with MLS and of the smallest deletion characterized in a female with the typical MLS phenotype.7Wapenaar MC Schiaffino MV Bassi MT Schaefer L Chinault AC Zoghbi HY Ballabio A A YAC-based binning strategy facilitating the rapid assembly of cosmid contigs: 1.6 Mb of overlapping cosmids in Xp22.Hum Mol Genet. 1994; 3: 1155-1161Crossref PubMed Scopus (38) Google Scholar Three genes are located in the critical interval, including MID1, HCCS, and ARHGAP67Wapenaar MC Schiaffino MV Bassi MT Schaefer L Chinault AC Zoghbi HY Ballabio A A YAC-based binning strategy facilitating the rapid assembly of cosmid contigs: 1.6 Mb of overlapping cosmids in Xp22.Hum Mol Genet. 1994; 3: 1155-1161Crossref PubMed Scopus (38) Google Scholar (fig. 1A). MID1 is mutated in Opitz G/BBB syndrome,8Quaderi NA Schweiger S Gaudenz K Franco B Rugarli EI Berger W Feldman GJ Volta M Andolfi G Gilgenkrantz S Marion RW Hennekam RC Opitz JM Muenke M Ropers HH Ballabio A Opitz G/BBB syndrome, a defect of midline development, is due to mutations in a new RING finger gene on Xp22.Nat Genet. 1997; 17: 285-291Crossref PubMed Scopus (296) Google Scholar whereas no disease-associated mutations have yet been described for HCCS and ARHGAP6. The ARHGAP6 gene codes for a Rho GTPase–activating protein (Rho GAP) that functions as a GAP for the small GTPase RhoA, as well as a protein implicated in reorganization of the actin cytoskeleton.9Schaefer L Prakash S Zoghbi HY Cloning and characterization of a novel rho-type GTPase-activating protein gene (ARHGAP6) from the critical region for microphthalmia with linear skin defects.Genomics. 1997; 46: 268-277Crossref PubMed Scopus (38) Google Scholar, 10Prakash SK Paylor R Jenna S Lamarche-Vane N Armstrong DL Xu B Mancini MA Zoghbi HY Functional analysis of ARHGAP6, a novel GTPase-activating protein for RhoA.Hum Mol Genet. 2000; 9: 477-488Crossref PubMed Scopus (57) Google ScholarHCCS encodes a mitochondrial holocytochrome c–type synthase, also known as "heme lyase," composed of 268 aa.11Schaefer L Ballabio A Zoghbi HY Cloning and characterization of a putative human holocytochrome c-type synthetase gene (HCCS) isolated from the critical region for microphthalmia with linear skin defects (MLS).Genomics. 1996; 34: 166-172Crossref PubMed Scopus (49) Google Scholar, 12Schwarz QP Cox TC Complementation of a yeast CYC3 deficiency identifies an X-linked mammalian activator of apocytochrome c.Genomics. 2002; 79: 51-57Crossref PubMed Scopus (23) Google Scholar It catalyzes the covalent attachment of heme to both apocytochrome c and c1, the precursor forms, thereby leading to the mature forms, holocytochrome c and c1, which are necessary for proper functioning of the mitochondrial respiratory chain.13Bernard DG Gabilly ST Dujardin G Merchant S Hamel PP Overlapping specificities of the mitochondrial cytochrome c and c1 heme lyases.J Biol Chem. 2003; 278: 49732-49742Crossref PubMed Scopus (61) Google Scholar, 14Moraes CT Diaz F Barrientos A Defects in the biosynthesis of mitochondrial heme c and heme a in yeast and mammals.Biochim Biophys Acta. 2004; 1659: 153-159Crossref PubMed Scopus (43) Google Scholar In addition to the well-known role of cytochrome c in oxidative phosphorylation (OXPHOS), cytochrome c is released from mitochondria in response to a variety of intrinsic death-promoting stimuli that, in turn, result in caspase-dependent cell death, namely "apoptosis."15Jiang X Wang X Cytochrome c-mediated apoptosis.Annu Rev Biochem. 2004; 73: 87-106Crossref PubMed Scopus (1035) Google Scholar The majority of patients display the classic phenotypic features of MLS; however, a high intra- as well as interfamilial clinical variability that is not correlated with the extent of the chromosomal deletion has been reported. For example, a few patients show the typical skin defects but no ocular manifestation,2Bird LM Krous HF Eichenfield LF Swalwell CI Jones MC Female infant with oncocytic cardiomyopathy and microphthalmia with linear skin defects (MLS): a clue to the pathogenesis of oncocytic cardiomyopathy?.Am J Med Genet. 1994; 53: 141-148Crossref PubMed Scopus (56) Google Scholar, 16Allanson J Richter S Linear skin defects and congenital microphthalmia: a new syndrome at Xp22.2.J Med Genet. 1991; 28: 143-144Crossref PubMed Scopus (35) Google Scholar, 17Kutsche K Werner W Bartsch O von der Wense A Meinecke P Gal A Microphthalmia with linear skin defects syndrome (MLS): a male with a mosaic paracentric inversion of Xp.Cytogenet Genome Res. 2002; 99: 297-302Crossref PubMed Scopus (17) Google Scholar, 18Lindsay EA Grillo A Ferrero GB Roth EJ Magenis E Grompe M Hulten M Gould C Baldini A Zoghbi HY Ballabio A Microphthalmia with linear skin defects (MLS) syndrome: clinical, cytogenetic, and molecular characterization.Am J Med Genet. 1994; 49: 229-234Crossref PubMed Scopus (84) Google Scholar whereas others present with only eye abnormalities, and dermal lesions are absent.19Cape CJ Zaidman GW Beck AD Kaufman AH Phenotypic variation in ophthalmic manifestations of MIDAS syndrome (microphthalmia, dermal aplasia, and sclerocornea).Arch Ophthalmol. 2004; 122: 1070-1074Crossref PubMed Scopus (24) Google Scholar, 20Kobayashi M Kiyosawa M Toyoura T Tokoro T An XX male with microphthalmos and sclerocornea.J Pediatr Ophthalmol Strabismus. 1998; 35: 122-124PubMed Google Scholar, 21Kono T Migita T Koyama S Seki I Another observation of microphthalmia in an XX male: microphthalmia with linear skin defects syndrome without linear skin lesions.J Hum Genet. 1999; 44: 63-68Crossref PubMed Scopus (24) Google Scholar It has been suggested that the pattern of X inactivation may play a role in the development of the various symptoms seen in patients with MLS.22Franco B Ballabio A X-inactivation and human disease: X-linked dominant male-lethal disorders.Curr Opin Genet Dev. 2006; 16: 254-259Crossref PubMed Scopus (59) Google Scholar, 23Van den Veyver IB Skewed X inactivation in X-linked disorders.Semin Reprod Med. 2001; 19: 183-191Crossref PubMed Scopus (86) Google Scholar The identification of the genetic defect for MLS has been hampered by the absence of patients with a full-blown MLS phenotype and a normal karyotype. In 2005, Morleo and colleagues5Morleo M Pramparo T Perone L Gregato G Le Caignec C Mueller RF Ogata T Raas-Rothschild A de Blois MC Wilson LC Zaidman G Zuffardi O Ballabio A Franco B Microphthalmia with linear skin defects (MLS) syndrome: clinical, cytogenetic, and molecular characterization of 11 cases.Am J Med Genet A. 2005; 137: 190-198Crossref PubMed Scopus (29) Google Scholar undertook the first attempt at a detailed characterization of four patients with MLS and no obvious chromosomal rearrangements. With use of FISH with genomic clones spanning the MLS critical region and a genomewide analysis with BAC microarrays, no microdeletion or duplication could be detected in these patients. Similarly, direct sequencing of coding regions and exon-intron boundaries of MID1, HCCS, and ARHGAP6 revealed no pathogenic sequence alteration or small rearrangement that would suggest that these patients carry cryptic rearrangements that had been missed by the techniques applied.5Morleo M Pramparo T Perone L Gregato G Le Caignec C Mueller RF Ogata T Raas-Rothschild A de Blois MC Wilson LC Zaidman G Zuffardi O Ballabio A Franco B Microphthalmia with linear skin defects (MLS) syndrome: clinical, cytogenetic, and molecular characterization of 11 cases.Am J Med Genet A. 2005; 137: 190-198Crossref PubMed Scopus (29) Google Scholar We examined the family of proband II.7. She is the youngest daughter of healthy and unrelated parents. There is no maternal history of skin defect or any other pathology, nor is there any family history of genetic disease or malformations. The couple has three healthy sons and one healthy daughter. Three pregnancies ended with spontaneous abortions early in the first trimester (fig. 1B). The proband was born with a left anophthalmia, sclerocornea, and lateral skin defect on her cheek. At age 6 mo, she presented with junctional ectopic tachycardia, which was successfully treated by catheter ablation. At age 3 years, she had normal psychomotor development. Her eldest sister (II.1) was born with the following manifestations: a left opaque cornea, congenital glaucoma with total anterior synechia, and a white anterior cataract. On the right side, she presented with corneal leukoma, which has resolved with time. She showed no other malformations, and her psychomotor development is normal. The couple had another baby girl, with bilateral anophthalmia, who died at age 6 h from complications of a left diaphragmatic hernia. High-resolution karyotype of both affected sisters was normal. Patient MS1, a 5-year-old girl, is the first child of healthy parents. Pregnancy was uneventful, and delivery was without any complication; birth measurements were in the upper-normal range. The newborn showed bilateral microphthalmia (more severe on the right), with bilateral cloudy and vascular cornea. In addition, linear and patchy erythrodermia of the patient's cheeks and right lateral neck was noticed immediately after birth. At age 3 mo, complete sclerocornea was noticed; however, erythrodermia had faded gradually. Reexamination at age 6 mo revealed poor vision but only very mild facial erythrodermia and only mild developmental delay. Cranial magnetic-resonance-imaging studies at age 1 year demonstrated hypoplasia of the corpus callosum, lack of the septum pellucidum, and slightly dilated third ventricle. The patient's further development was characterized by normal growth, mild-to-moderate developmental delay, and very severe visual impairment. The happy-natured girl is now able to speak short sentences. Patient MS2, a 9-year-old girl, was born to healthy unrelated parents after an uneventful pregnancy. Birth measurements were within the normal range. After birth, she presented with bilateral microphthalmia and sclerocornea. A small mandible, a single palmar crease, and a sandal gap but no erythematous skin lesions were noticed. At the end of her 1st year of life, she developed idiopathic ventricular tachycardia, which was converted to normal sinus rhythm by amiodaron and ajmalin. After extubation and end of sedation, she suffered an occlusion of her right arteria cerebri media and subsequently developed hemiparesis on the left side. Neurologic symptoms declined after physiotherapy. At age 8 years, she suffered a sole tonic-clonic seizure. Our ethics committees approved this study, and written informed consent was obtained from all participants or their legal guardians. Genomic DNA was isolated by standard procedures. We amplified the coding region (exons 2–7) of HCCS (GenBank accession number NM_005333), including the flanking intronic sequences, from genomic DNA. Primer sequences and PCR conditions are available on request. PCR products were directly sequenced with the Big Dye Terminator ready reaction kit (PE Applied Biosystems) on an ABI Prism 377 (PE Applied Biosystems). Examination of the methylation pattern at the androgen receptor (AR) locus was performed according to the procedures of Allen et al.,24Allen RC Zoghbi HY Moseley AB Rosenblatt HM Belmont JW Methylation of HpaII and HhaI sites near the polymorphic CAG repeat in the human androgen-receptor gene correlates with X chromosome inactivation.Am J Hum Genet. 1992; 51: 1229-1239PubMed Google Scholar with minor modifications. For each DNA sample, two reactions were prepared: in the first, 400 ng of DNA was digested with 8 U HpaII in a total volume of 10 μl, for 30 min at 37°C; in the second, 400 ng DNA was incubated with the enzyme reaction buffer, but without enzyme. To confirm complete digestion, DNA samples from a male and from a female with unilateral X inactivation were included as controls. Subsequent PCR amplification of the AR locus with primers AR-A_FAM (5′-CTTTCCAGAATCTGTTCCAG-3′; labeled with 5′ FAM) and AR-B (5′-AAGGTTGCTGTTCCTCATC-3′) was performed in a 25−μl reaction volume for 40 ng of the undigested DNA and in a total of 50 μl volume for 250 ng of HpaII-cleaved DNA. The PCR contained both oligonucleotide primers at a concentration of 0.4 μM each, 0.2 mM dinucleotide triphosphates (dNTPs), and 0.5 U Taq polymerase (QIAGEN). Samples were denatured, with use of a PTC thermocycler (MJ Research), at 95°C for 3 min, followed by 35 cycles at 95°C for 1 min, at 55°C for 1 min, and at 72°C for 1 min, followed by a final incubation at 72°C for 10 min. A quantity of 0.4–0.8 μl of the amplicons was mixed with 20 μl deionized formamide and 0.4 μl TAMRA (red fluorescent dye) Size Standard (PE Applied Biosystems) and was denatured at 95°C for 5 min. PCR products were analyzed on an ABI Prism 310 Genetic Analyzer (PE Applied Biosystems). Data were taken as a ratio of peak areas of the shorter-to-longer alleles. Real-time quantitative PCR on genomic DNA was performed using an ABI Prism 7000 (PE Applied Biosystems) in a 96-well optical plate, with a final reaction of 20 μl. All reactions were prepared with 10 μl of 2× SYBR green I fluorescent dye PCR Master Mix and 400 nM forward and reverse primers. Primers for real-time experiments were designed using the Primer Express software, in accordance with the PE Applied Biosystems guidelines; primers sequences are available on request. The RPP30 gene (GenBank accession number NM_006413) coding for a ribonuclease P was used as an internal reference. A total of 100 ng of DNA was used as a template for each sample; each was analyzed in quadruplicate. Thermal cycling conditions included a prerun of 2 min at 50°C and of 10 min at 95°C. Cycle conditions were 40 cycles at 95°C for 15 s and at 60°C for 1 min, in accordance with the PCR protocol (PE Applied Biosystems). Relative quantification of exon-copy numbers on genomic DNA was performed using the comparative threshold cycle (ddCt) method: the starting copy number of exons in patients was determined in comparison with the known copy number of the calibrator sample (healthy male control), with use of the formula ddCt=dCt HCCS (patient with MLS)−dCt RPP30 (patient with MLS)−dCt HCCS (healthy male)−dCt RPP30 (healthy male) .The relative exon-copy number was calculated by the expression 2∧-(ddCt±SE) that is of ∼2 for a diploid sample and ∼1 for a haploid sample. Haplotype analysis of family members of patient II.7 was performed using five polymorphic microsatellite markers (tel-DXS7108-CxM06-CxM09-DXS8019-DXS999-cen) in the Xp22.2-p22.13 region, which contains the HCCS gene. PCR products were amplified using FAM (blue fluorescent)–labeled primers, following standard methods, and were electrophoresed on an ABI Prism 3100 Genetic Analyzer. Results were processed by GENESCAN software (PE Applied Biosystems). For analysis of polymorphism rs5901444, exon 1 containing the trinucleotide CGG repeat was amplified using forward (5′-CCTGCCACCGCCACATTTTG-3′) and reverse (5′-ATGAATAGGAATTCGAAGAAACGAAGG-3′) primers. The PCR contained both primers, each at a concentration of 0.2 μM, 0.2 mM dNTPs, and 1 U AmpliTaq Gold (PE Applied Biosystems), in a total volume of 30 μl. Samples were amplified using a GeneAmp PCR System 9700 thermocycler (PE Applied Biosystems), with an initial denaturation at 95°C for 7 min and 35 cycles at 98°C for 1 min, at 58°C for 1 min, and at 72°C for 1 min. Total RNA was extracted from Epstein Barr virus–transformed lymphoblastoid cells of patient MS1 with the RNeasy kit (QIAGEN), according to the manufacturer's instructions. For first-strand cDNA synthesis, 1 μg of total RNA was reverse transcribed by using the Omniscript RT Kit (QIAGEN) and random hexamers, according to the protocol provided. Of each first-strand reaction, 1 μl cDNA was taken as template in PCRs, with use of Advantage cDNA-Polymerase Mix (BD Biosciences Clontech) and primers HCCS_RT_4F (5′-ATATCATTAGAATTCACAATCAG-3′) and HCCS_RT_3R (5′-AAACTGCAAGGTACAACACAAGTC-3′). The PCR condition was as follows: 35 cycles, each comprising 15 s at 95°C, 10 s at 58°C, and 40 s at 72°C, with an initial denaturation at 95°C for 3 min. For Southern-blot analysis, 10 μg of genomic DNA was digested with the appropriate restriction enzymes: SpeI and EcoRV (double digestion), NdeI and SacI (double digestion), and TaqI. DNA was separated by electrophoresis in 0.8% agarose gel, was transferred to Hybond-N (Amersham), and was hybridized to a probe covering the coding and the 5′ UTR regions of the HCCS transcript. This probe was generated by RT-PCR on cDNA obtained from lymphoblastoid cell lines, with use of primers RTF1 (5′-CGTGAAGTCACTGCTGCTCTG-3′) and RTR1 (5′-TCTGAAACAGTGCTTTACGAGGTC-3′). HCCS cDNA clone DKFZp779I1858 (GenBank accession number CR749578) was provided by the RZPD German Resource Center for Genome Research (Berlin). The DNA insert of this cDNA clone was sequenced by SP6 and T7 primers and primer walking (primer sequences are available on request). Wild-type HCCS and HCCS (Δ197–268) cDNA inserts were generated using specific PCR primers and the DKFZp779I1858 clone as template. The HCCS (R217C) insert was established by PCR-mediated mutagenesis.25Ito W Ishiguro H Kurosawa Y A general method for introducing a series of mutations into cloned DNA using the polymerase chain reaction.Gene. 1991; 102: 67-70Crossref PubMed Scopus (260) Google Scholar Purified PCR products were cloned into pENTR/d-TOPO (Invitrogen) according to the protocol provided. Constructs were sequenced for integrity and then were used for cloning the HCCS-coding region into plasmids pcDNA-DEST53 (N-terminal EGFP epitope [Invitrogen]), pcDNA-DEST47 (C-terminal EGFP epitope [Invitrogen]), pMT2SM-HA-DEST (N-terminal HA epitope), and pcDNA3.2/V5-DEST (C-terminal V5 epitope [Invitrogen]) via left-right reaction, following the manufacturer's instructions. Plasmid pMT2SM-HA-DEST was generated by blunt-end ligation of the GATEWAY Cloning Reading Frame Cassette C (Invitrogen) into the multiple cloning site of vector pMT2SM-HA. All yeast-expression constructs were generated in pYEX4Tps, which drives expression of the glutathione-S-transferase (GST)–fusion proteins from the Saccharomyces cerevisiae CUP1 promoter and carries the selectable markers leu2-d, URA3, and Ampr. To generate pYEX4Tps-HCCS wild type and pYEX4Tps-HCCS Δ197–268, PCR products were amplified by using specific PCR primers and the DKFZp779I1858 clone as template. pYEX4Tps-HCCS-R217C was established by PCR-mediated mutagenesis.25Ito W Ishiguro H Kurosawa Y A general method for introducing a series of mutations into cloned DNA using the polymerase chain reaction.Gene. 1991; 102: 67-70Crossref PubMed Scopus (260) Google Scholar After purification of PCR products, each amplicon was restricted with BamHI and NotI and was cloned unidirectionally into pYEX4Tps. To establish the control construct pYEX4Tps-CYC3, a PCR product was amplified by using specific primers and pAB256 carrying yeast CYC3 as template. Subsequently, the amplicon was cloned as BamHI-NotI fragment into pYEX4Tps. Primer sequences and PCR conditions are available on request. All constructs were sequenced for integrity, and large and pure amounts of plasmid DNA were prepared by using a plasmid maxikit (QIAGEN). CHO-K1 cells were cultured on fibronectin-coated (10 μg/ml) coverslips in F12-Ham's Nutrient Mixture supplemented with 10% fetal calf serum, 1% l-glutamine, and penicillin-streptomycin, at 37°C in 5% CO2. Cells were transfected with pcDNA-DEST53-HCCS wild type, pcDNA-DEST53-HCCS R217C, pcDNA-DEST53-HCCS Δ197–268, pcDNA-DEST47-HCCS wild type, pcDNA-DEST47-HCCS R217C, pcDNA-DEST47-HCCS Δ197–268, pMT2SM-HA-DEST-HCCS wild type, pMT2SM-HA-DEST-HCCS R217C, pMT2SM-HA-DEST-HCCS Δ197–268, pcDNA3.2/V5-DEST-HCCS wild type, pcDNA3.2/V5-DEST-HCCS R217C, and pcDNA3.2/V5-DEST-HCCS Δ197–268 (each with 1 μg of DNA), with use of Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's protocol, and were incubated overnight. The next day, cells were incubated in normal growth medium supplemented with 50 nM MitoTracker Red CMXRos (Invitrogen) for 20 min at 37°C, were rinsed in PBS, and were fixed with 4% paraformaldehyde in PBS. After washing three times with PBS, EGFP-transfected cells on coverslips were directly mounted in glycerol gelatin (Sigma). Cells transfected with HA- or V5-tagged HCCS constructs were incubated in permeabilization/blocking solution (2% BSA, 3% goat serum, and 0.5% Nonidet P-40 in PBS) for 60 min. Subsequently, HA- and V5-tagged HCCS proteins were detected with rat monoclonal anti-HA-Flourescein antibody (1.25 μg/ml) (Roche) and mouse monoclonal anti-V5-FITC antibody (2.0 μg/ml) (Invitrogen), respectively, both diluted in antibody solution (3% goat serum and 0.1% Nonidet P-40 in 1× PBS). After extensive washing with PBS, cells were mounted in glycerol gelatin on microscopic slides. Images were acquired with use of a Zeiss Axiovert 200 M confocal microscope equipped with a 63× Planapochromat/1.4 DIC lens. For complementation studies, we used S. cerevisiae strain B-8025 (MATαcan1-100 CYC1 cyc3-Δcyc7-Δ::CYH2 cyh2 his3-Δ1 leu2-3,112 trp1-289), which carries a deletion of CYC3,26Tong J Margoliash E Cytochrome c heme lyase activity of yeast mitochondria.J Biol Chem. 1998; 273: 25695-25702Crossref PubMed Scopus (18) Google Scholar as well as B-7553 (MATαcan1-100 CYC1 cyc7::CYH2 cyh2 his3-Δ1 leu2-3,112 trp1-289) as a growth control.27Dumont ME Schlichter JB Cardillo TS Hayes MK Bethlendy G Sherman F CYC2 encodes a factor involved in mitochondrial import of yeast cytochrome c.Mol Cell Biol. 1993; 13: 6442-6451Crossref PubMed Scopus (37) Google Scholar Both B-8025 and B-7553 carry a deletion of CYC7, which encodes cytochrome c1, one of the two yeast cytochrome c proteins. Strains lacking CYC3 but still harboring CYC7 may still show residual respiratory growth due to conversion of apocytochrome c1 to its holoform by the second yeast heme lyase Cyt2p. Thus, the CYC3− mutant strain lacking CYC7 is not able to grow at all on nonfermentable carbon sources and needs complementation to show respiratory growth. Yeast cells were maintained in complete medium (YPD). Transformation of cells was done by the standard lithium-acetate method. Positive transformants were selected by growth in minimal medium, supplemented with 2% glucose but lacking leucine. Colonies that showed growth under this condition were grown in liquid minimal medium until saturation and subsequently were tested for growth on nonfermentable carbon sources (YPG medium containing 3% glycerol, 2% peptone, 1% yeast extract, 2% agar, and 0.2 mM CuSO4). Therefore, we generated two dilutions (0.6-fold and 0.2-fold) from a saturated culture of each strain and spotted 5 μl of these cultures on YPG plates and in parallel, as growth control, on plates containing minimal medium with glucose. Plates were incubated for 5 d at 30°C. Expression of various GST-HCCS and GST-CYC3–fusion proteins was confirmed by immunoblotting of lysates from saturated yeast cultures (grown in minimal medium or YPG medium with copper) with use of horseradish peroxidase–conjugated goat polyclonal anti-GST antibody (0.3 μg/ml) (Amersham Pharmacia Biotech). We examined a family in which the youngest daughter (II.7) shows the classic MLS phenotype and a normal karyotype. However, a pathogenic mutation in MID1, ARHGAP6, and HCCS in patient II.7 has not yet been identified.5Morleo M Pramparo T Perone L Gregato G Le Caignec C Mueller RF Ogata T Raas-Rothschild A de Blois MC Wilson LC Zaidman G Zuffardi O Ballabio A Franco B Microphthalmia with linear skin defects (MLS) syndrome: clinical, cytogenetic, and molecular characterization of 11 cases.Am J Med Genet A. 2005; 137: 190-198Crossref PubMed Scopus (29) Google Scholar The older sister (II.1) presents a milder phenotype, a third a
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