Mutation C11994T in the mitochondrial ND4 gene is not a cause of low sperm motility in Portugal
2007; Elsevier BV; Volume: 89; Issue: 3 Linguagem: Inglês
10.1016/j.fertnstert.2007.03.048
ISSN1556-5653
AutoresLuı́sa Pereira, João Gonçalves, Hans‐Jürgen Bandelt,
Tópico(s)Sperm and Testicular Function
ResumoIt has recently been suggested that a hitherto unobserved mutation, C11994T, causes oligoasthenozoospermia in men from India but at the same time does not affect systems other than the motility of the sperm. There are good reasons to question this proposition, in view of the worldwide mtDNA database and the Indian record in particular. We have further analyzed the oligoasthenozoospermic samples from a previous systematic study of infertile Portuguese men and found no instance of C11994T. It has recently been suggested that a hitherto unobserved mutation, C11994T, causes oligoasthenozoospermia in men from India but at the same time does not affect systems other than the motility of the sperm. There are good reasons to question this proposition, in view of the worldwide mtDNA database and the Indian record in particular. We have further analyzed the oligoasthenozoospermic samples from a previous systematic study of infertile Portuguese men and found no instance of C11994T. "Defects of the mitochondrial genome are recognized as common causes of genetic disease. … However, establishing whether a detected sequence change is polymorphic or pathogenic is still a major difficulty owing to its highly polymorphic nature" (1Mitchell A.L. Elson J.L. Howell N. Taylor R.W. Turnbull D.M. Genes: mutation or polymorphism? Sequence variation in mitochondrial complex I.J Med Genet. 2006; 43: 175-179Crossref PubMed Scopus (100) Google Scholar). Homoplasmic substitutions in mitochondrial DNA may, in some cases, be responsible for a mitochondrial disorder. In other instances, a whole suite of mutations may be associated with a disorder or influence its phenotype. Many diseases involving impaired energy production of the cell have come into suspicion, at least temporarily, for having a mitochondrial DNA component. It therefore should not come as a surprise that subfertility of men has also become a target of mtDNA studies seeking pathogenic mutations.Ruiz-Pesini et al. (2Ruiz-Pesini E. Lapeña A.C. Díez-Sánchez C. Pérez-Martos A. Montoya J. Alvarez E. et al.Human mtDNA haplogroups associated with high or reduced spermatozoa motility.Am J Hum Genet. 2000; 67: 682-696Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar) from Spain have claimed that mtDNA "haplogroups H and T are significantly more abundant in nonasthenozoospermic and asthenozoospermic populations, respectively." It has, however, been demonstrated that such a seeming association can be spurious, even in a small country such as Portugal (3Pereira L. Gonçalves J. Goios A. Rocha T. Amorim A. Human mtDNA haplogroups and reduced male fertility: real association or hidden population substructuring.Int J Androl. 2005; 28: 241-247Crossref PubMed Scopus (30) Google Scholar). Indeed, as has been emphasized many times on similar occasions, "association studies can be confounded if patients and control subjects are not well matched" (4Carelli V. Achilli A. Valentino M.L. Rengo C. Semino O. Pala M. et al.Haplogroup effects and recombination of mitochondrial DNA: novel clues from the analysis of Leber hereditary optic neuropathy pedigrees.Am J Hum Genet. 2006; 78: 564-574Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Further, the claim by Holyoake et al. (5Holyoake J.A. McHugh P. Wu M. O'Carroll S. Benny P. Sin I.L. Sin F.Y.T. High incidence of single nucleotide substitutions in the mitochondrial genome is associated with poor semen parameters in men.Int J Androl. 2001; 24: 175-182Crossref PubMed Scopus (98) Google Scholar) that the mtDNA mutations G9055A (characteristic of haplogroup K) and G11719A (signifying nonR0 haplogroup status) would compromise the semen quality of those men who possess any of these mutations in their mtDNAs was evidently based on some single-stranded conformation polymorphism (SSCP) misanalysis (6Bandelt H.-J. Misanalysis gave false association of mtDNA mutations with infertility.Int J Androl. 2007; (in press)Google Scholar). Therefore one cannot yet posit that haplogroups play any role in low sperm motility. This may parallel the situation with Alzheimer's disease and Parkinson's disease, where claims of mtDNA association seem to have been premature (7Elson J.L. Herrnstadt C. Preston G. Thal L. Morris C.M. Edwardson J.A. et al.Does the mitochondrial genome play a role in the etiology of Alzheimer's disease?.Hum Genet. 2006; 119: 241-254Crossref PubMed Scopus (88) Google Scholar, 8Howell N. Elson J.L. Chinnery P.F. Turnbull D.M. mtDNA mutations and common neurodegenerative disorders.Trends Genet. 2005; 21: 583-586Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar).A high mutation load in sperm has been asserted by Thangaraj et al. (9Thangaraj K. Gupta N.J. Pavani K. Reddy A.G. Subramainan S. Rani D.S. et al.Y chromosome deletions in azoospermic men in India.J Androl. 2003; 24: 588-597PubMed Google Scholar) in a single case of oligoasthenoteratozoospermia (OAT): "The mitochondrial genes COI, COII, ATPase6, ATPase8, tRNA serine I, tRNA lysine, and ND3 were amplified and sequenced from sperm and blood cells of the man with OAT. A comparison of the sequences of the above genes with a reference sequence revealed a total of 36 nucleotide substitutions in the sperm mtDNA but not in the DNA from the blood cells." Reports of such exorbitant numbers of somatic mutations can, as a rule, always be ascribed to artefacts, such as sample mix-up (10Salas A. Yao Y.-G. Macaulay V. Vega A. Carracedo Á Bandelt H.-J. A critical reassessment of the role of mitochondria in tumorigenesis.PLoS Med. 2005; 2: e296Crossref PubMed Scopus (183) Google Scholar). In that case, however, not unexpectedly, a specific nuclear insert of mitochondrial DNA (numt) had been targeted instead, as was clearly demonstrated by Bravi et al. (11Bravi C.M. Parson W. Bandelt H.-J. Numts revisited.in: Bandelt H.-J. Macaulay V. Richards M. Human mitochondrial DNA and the evolution of Homo sapiens. Springer-Verlag, Berlin-Heidelberg2006: 31-46Google Scholar). An inadvertent base shift (by +1) may have obscured the identity of that "classic" numt, which had already played a role in an earlier case (12Davis R.E. Miller S. Herrnstadt C. Ghosh S.S. Fahy E. Shinobu L.A. et al.Mutations in mitochondrial cytochrome c oxidase genes segregate with late-onset Alzheimer disease.Proc Natl Acad Sci U S A. 1997; 94: 4526-4531Crossref PubMed Scopus (316) Google Scholar).Summarizing, from the publication record up to 2005, there has been no convincing evidence for the action of mtDNA substitutions, either directly in the sperm (as somatic mutations other than deletions) nor indirectly through haplogroup background by predisposing to reduced motility. A most remarkable finding was recently announced by Selvi Rani et al. (13Selvi Rani D. Vanniarajan A. Gupta N.J. Chakravarty B. Singh L. Thangaraj K. A novel missense mutation C11994T in the mitochondrial ND4 gene as a cause of low sperm motility in the Indian subcontinent.Fertil Steril. 2006; 86: 1783-1785Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar): Among men (from India) with fertility problems, all 34 individuals with oligoasthenozoospermia (OA) showed the mutation C11994T in homoplasmic state. None of 150 normozoospermic controls showed this mutation.At face value, OA would thus look like a mitochondrial disorder par excellence, clear-cut, without etiologic or interpretative complications, such as in the case of LHON (4Carelli V. Achilli A. Valentino M.L. Rengo C. Semino O. Pala M. et al.Haplogroup effects and recombination of mitochondrial DNA: novel clues from the analysis of Leber hereditary optic neuropathy pedigrees.Am J Hum Genet. 2006; 78: 564-574Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar) or almost every other recognized mitochondrial disease. It is amazing that "this mutation appears not to affect systems other than the motility of the sperm" (13Selvi Rani D. Vanniarajan A. Gupta N.J. Chakravarty B. Singh L. Thangaraj K. A novel missense mutation C11994T in the mitochondrial ND4 gene as a cause of low sperm motility in the Indian subcontinent.Fertil Steril. 2006; 86: 1783-1785Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). There are, however, serious problems with this proposition. First, if all OA men from that study belonged to one and only one haplogroup characterized by C11994T, then it appears somewhat strange that: 1) this haplogroup was not yet discovered in systematic screening of complete mtDNA variation in India (14Palanichamy Mg. Sun C. Agrawal S. Bandelt H.-J. Kong Q.-P. Khan F. et al.Phylogeny of mitochondrial DNA macrohaplogroup N in India, based on complete sequencing: implications for the peopling of South Asia.Am J Hum Genet. 2004; 75: 966-978Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 15Sun C. Kong Q.-P. Palanichamy Mg Agrawal S. Bandelt H.-J. Yao Y.-G. et al.The dazzling array of basal branches in the mtDNA macrohaplogroup M from India as inferred from complete genomes.Mol Biol Evol. 2006; 23: 683-690Crossref PubMed Scopus (129) Google Scholar, 16Thangaraj K. Chaubey G. Singh V.K. Vanniarajan A. Thanseem I. Reddy A.G. Singh L. In situ origin of deep rooting lineages of mitochondrial macrohaplogroup M in India.BMC Genomics. 2006; 7: 151Crossref PubMed Scopus (80) Google Scholar); and 2) this would virtually constitute the only cause of OA in India. Second, if C11994T occurred repeatedly in different parts of the Indian mtDNA phylogeny, it is quite strange that this recurrent mutation has never been reported in more than 3,000 coding region sequences available from the worldwide database. Confronted with this conundrum, one could envision other causes for the C11994T phenomenon asserted by Selvi Rana et al. (13Selvi Rani D. Vanniarajan A. Gupta N.J. Chakravarty B. Singh L. Thangaraj K. A novel missense mutation C11994T in the mitochondrial ND4 gene as a cause of low sperm motility in the Indian subcontinent.Fertil Steril. 2006; 86: 1783-1785Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar):1.Some novel numt was amplified in all those OA samples for the amplified fragment containing position 11994 (17Parr R.L. Maki J. Reguly B. Dakubo G.D. Aguirre A. Wittock R. et al.The pseudo-mitochondrial genome influences mistakes in heteroplasmy interpretation.BMC Genomics. 2006; 7: 185Crossref PubMed Scopus (67) Google Scholar).2.Position 11944 was systematically hit by a phantom mutation in all those OA samples, just as the specific "Klinefelter mtDNA motif" arose in Oikawa et al. (18Oikawa H. Tun Z. Young D.R. Ozawa H. Yamazaki K. Tanaka E. Honda K. The specific mitochondrial DNA polymorphism found in Klinefelter's syndrome.Biochem Biophys Res Commun. 2002; 297: 341-345Crossref PubMed Scopus (13) Google Scholar, 19Bandelt H.-J. Kong Q.-P. Parson W. Salas A. More evidence for nonmaternal inheritance of mitochondrial DNA?.J Med Genet. 2005; 42: 957-960Crossref PubMed Scopus (60) Google Scholar).3.Some inadvertent alignment shift or misediting has produced a seeming 11994 mutation, similar to the case admitted by Hagelberg et al. (20Hagelberg E. Goldman N. Lió P. Whelan S. Schiefenhövel W. Clegg J.B. Bowden D.K. Evidence for mitochondrial DNA recombination in a human population of island Melanesia: correction.Proc R Soc London Ser B Biol Sci. 2000; 267: 1595-1596Crossref Scopus (28) Google Scholar).4.The OA samples were preselected according to a specific unknown (minor) haplogroup, in which C11994T thrives.5.One sample with 11994 has actually been found in one mtDNA, from which the fragment containing position 11994 was borrowed by all those 34 OA samples.Because the article by Selvi Rani et al. (13Selvi Rani D. Vanniarajan A. Gupta N.J. Chakravarty B. Singh L. Thangaraj K. A novel missense mutation C11994T in the mitochondrial ND4 gene as a cause of low sperm motility in the Indian subcontinent.Fertil Steril. 2006; 86: 1783-1785Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) presents no data—neither a report of the complete mtDNA variation observed in the 10 analyzed mtDNAs from men with OA, nor any pairs of electropherograms for the fragment containing C11994T—one cannot decide between the above five alternatives. Such information would be indispensable for identifying the potential cause of an artefact (21Bandelt H.-J. Achilli A. Kong Q.-P. Salas A. Lutz-Bonengel S. Sun C. et al.Low "penetrance" of phylogenetic knowledge in mitochondrial disease studies.Biochem Biophys Res Commun. 2005; 333: 122-130Crossref PubMed Scopus (64) Google Scholar, 22Bandelt H.-J. Yao Y.-G. Salas A. Kivisild T. Bravi C.M. High penetrance of sequencing errors and interpretative shortcomings in mtDNA sequence analysis of LHON patients.Biochem Biophys Res Commun. 2007; 352: 283-291Crossref PubMed Scopus (39) Google Scholar, 23Parson W. The art of reading sequence electropherograms.Ann Hum Genet. 2006; 71: 276-280Crossref PubMed Scopus (11) Google Scholar).Because the authors hypothesized that this novel mutation is a "widespread genetic cause of oligoasthenozoospermia" and went on to suggest that "the C11994T mutation in the mitochondrial ND4 gene should routinely be screened in individuals with moderate oligoasthenospermia," we decided to analyze the polymorphism C11994T by direct sequencing in the seven samples from a previous study (3Pereira L. Gonçalves J. Goios A. Rocha T. Amorim A. Human mtDNA haplogroups and reduced male fertility: real association or hidden population substructuring.Int J Androl. 2005; 28: 241-247Crossref PubMed Scopus (30) Google Scholar) classified as oligoasthenozoospermic (OA). In that study, both hypervariable regions (HVS-I and HVS-II) of the mtDNA control region were sequenced in a sample of 101 infertile males from southern Portugal. The infertile patients were selected after andrologic and hormonal evaluation. Only patients with idiopathic infertility, i.e., absence of history of hypospadias, urinary infection, epididymitis, orchitis, varicocele, prostatic abnormalities, testis tumors, and testicular maldescent, were included in the present study. All patients had a 46,XY normal karyotype, and all were previously screened for the detection of AZFa, AZFb, and AZFc microdeletions. Only patients without AZF microdeletions were included. Sperm analysis was performed according to World Health Organization guidelines (24World Health Organization WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction.3rd ed. Cambridge University Press, Cambridge, UK1992Google Scholar). Semen samples of all patients were classified into one of the following categories, briefly: oligozoospermia if sperm concentration was less than 20 million/mL but more than 0; asthenozoospermia if fewer than 50% spermatozoa with forward progression (categories A and B) or fewer than 25% spermatozoa with rapid linear progression; teratozoospermia if fewer than 30% spermatozoa with normal head morphology; oligoteratoasthenozoospermia if all three variables were disturbed; oligoasthenozoospermia if patients displayed both oligozoospermia and asthenozoospermia. All patients gave their written informed consent to all analyses, and the study was performed in compliance with the Helsinki Declaration and according to the guidelines of the Scientific and Ethics Committee of the Center of Human Genetics of the National Institute of Health, Lisbon.For checking the presence of the C11994T substitution, an mtDNA fragment was amplified by using the primers forward L11486 5′-AAA ACT AGG CGG CTA TGG TA-3′ and reverse H12076 5′-ATT TTC TGC TAG GGG GTG GA-3′. In this way, the sequence between positions 11531 and 12040 could be read in both directions. The temperature profile was 95°C for 10 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, for 35 cycles of amplification. The amplified samples were purified with Microspin S-300 HR columns (Amersham Biosciences, Uppsala, Sweden), according to the manufacturer's specifications. The sequence reactions were carried out using the kit Big-Dye Terminator Cycle Sequencing Ready Reaction (Applied Biosystems, Foster City, CA) with one of the above primers in both forward and reverse directions. A postpurification by Sephadex was used after the sequencing reaction, and samples were run in an automatic sequencer ABI 3100 (Applied Biosystems). The results are presented in Table 1. Electropherograms for inspection can be requested from the first author.Table 1mtDNA profileaAll mutations are transitions relative to rCRS (29) unless a suffix (A, C, +C, +CC) indicates a transversion (to A or C) or an insertion (of one or two cytosines); 0 signifies identity with rCRS. of seven oligoasthenozoospermic men from Portugal.Sample IDSperm/mLHVS-I (16024–16383) 16000+HVS-II (72–340)Fragment 11531–12040Haplogroup9900011 × 106126 248 29473 263 309+C 315+C11719 11812T29930346 × 106189 29872 263 315+C0HV09938430.22 × 106320A263 315+C0H0002177 × 106126 187 189 223 264 270 278 293 31173 152 182 185T 195 198 247 263 315+C 35711719L1b10218222 × 106304bRereading the electropherograms of this sample revealed that the originally reported transition at 16085 (3) seemed rather to be heteroplasmic in both strands, but subsequent resequencing of both strands for HVS-I confirmed the absence of this mutation.263 309+C 315+C0H0302421 × 106069 126 193 27873 150 152 263 295 315+C11719J2b9926663 × 106172 183C 189 219 239 27873 263 309+CC 315+C11719 11938U6a1a All mutations are transitions relative to rCRS 29Andrews R.M. Kubacka I. Chinnery P.F. Lightowlers R.N. Turnbull D.M. Howell N. Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA.Nat Genet. 1999; 23: 147Crossref PubMed Scopus (2519) Google Scholar unless a suffix (A, C, +C, +CC) indicates a transversion (to A or C) or an insertion (of one or two cytosines); 0 signifies identity with rCRS.b Rereading the electropherograms of this sample revealed that the originally reported transition at 16085 3Pereira L. Gonçalves J. Goios A. Rocha T. Amorim A. Human mtDNA haplogroups and reduced male fertility: real association or hidden population substructuring.Int J Androl. 2005; 28: 241-247Crossref PubMed Scopus (30) Google Scholar seemed rather to be heteroplasmic in both strands, but subsequent resequencing of both strands for HVS-I confirmed the absence of this mutation. Open table in a new tab The seven mtDNAs analyzed include at least one representative from each of the major West Eurasian branches (R0, JT, and U; 25Torroni A. Achilli A. Macaulay V. Richards M. Bandelt H.-J. Harvesting the fruit of the human mtDNA tree.Trends Genet. 2006; 22: 339-345Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar) of haplogroup R as well as one representative of an African haplogroup (L1b1). The mutations detected in region 11531–12040 are concordant with the haplogroup status, as originally inferred from HVS-I and HVS-II, i.e., the root of haplogroup R0 (embracing branches HV0 and H) is indistinguishable from rCRS within that fragment, haplogroup T2 bears the characteristic 11812 transition, and in haplogroup U6a1 the 16239 and 11938 transitions seem to be linked (26Maca-Meyer N. González A.M. Pestano J. Flores C. Larruga J.M. Cabrera V.M. Mitochondrial DNA transit between West Asia and North Africa inferred from U6 phylogeography.BMC Genetics. 2003; 4: 15Crossref PubMed Scopus (81) Google Scholar). Not a single occurrence of the 11994 transition was found here or in the mtDNAs of another 20 asthenozoospermic and 23 teratoasthenozoospermic men (27Pereira L. Gonçalves J. Franco-Duarte R. Silva J. Rocha T. Arnold C. et al.No evidence for an mtDNA role in sperm motility: data from complete sequencing of asthenozoospermic males.Mol Biol Evol. 2007; 24: 868-874Crossref PubMed Scopus (60) Google Scholar). Therefore C11994T cannot play a dominant role in oligoasthenozoopermy or other forms of asthenozoopermy, at least in Portugal, and it is therefore doubtful that this mutation should constitute the single cause in India.If the sequencing protocol of Selvi Rani et al. (13Selvi Rani D. Vanniarajan A. Gupta N.J. Chakravarty B. Singh L. Thangaraj K. A novel missense mutation C11994T in the mitochondrial ND4 gene as a cause of low sperm motility in the Indian subcontinent.Fertil Steril. 2006; 86: 1783-1785Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) followed Rieder et al. (28Rieder M.J. Taylor S.L. Tobe V.O. Nickerson D.A. Automating the identification of DNA variations using quality-based fluorescence re-sequencing: analysis of the human mitochondrial genome.Nucleic Acids Res. 1998; 26: 967-973Crossref PubMed Scopus (444) Google Scholar), then position 11994 was probably only well readable on the H strand of amplicon 17 (11314–12076) and on the L strand of amplicon 18 (11948–12772). It would be interesting to learn which further variation has been observed in the 10 mtDNAs that were completely sequenced by Selvi Rani et al. (13Selvi Rani D. Vanniarajan A. Gupta N.J. Chakravarty B. Singh L. Thangaraj K. A novel missense mutation C11994T in the mitochondrial ND4 gene as a cause of low sperm motility in the Indian subcontinent.Fertil Steril. 2006; 86: 1783-1785Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) and inspect the pairs of electropherograms supporting the findings. Otherwise, further mtDNA samples of oligoasthenozoopermic men from India would need investigation performed in other laboratories. "Defects of the mitochondrial genome are recognized as common causes of genetic disease. … However, establishing whether a detected sequence change is polymorphic or pathogenic is still a major difficulty owing to its highly polymorphic nature" (1Mitchell A.L. Elson J.L. Howell N. Taylor R.W. Turnbull D.M. Genes: mutation or polymorphism? Sequence variation in mitochondrial complex I.J Med Genet. 2006; 43: 175-179Crossref PubMed Scopus (100) Google Scholar). Homoplasmic substitutions in mitochondrial DNA may, in some cases, be responsible for a mitochondrial disorder. In other instances, a whole suite of mutations may be associated with a disorder or influence its phenotype. Many diseases involving impaired energy production of the cell have come into suspicion, at least temporarily, for having a mitochondrial DNA component. It therefore should not come as a surprise that subfertility of men has also become a target of mtDNA studies seeking pathogenic mutations. Ruiz-Pesini et al. (2Ruiz-Pesini E. Lapeña A.C. Díez-Sánchez C. Pérez-Martos A. Montoya J. Alvarez E. et al.Human mtDNA haplogroups associated with high or reduced spermatozoa motility.Am J Hum Genet. 2000; 67: 682-696Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar) from Spain have claimed that mtDNA "haplogroups H and T are significantly more abundant in nonasthenozoospermic and asthenozoospermic populations, respectively." It has, however, been demonstrated that such a seeming association can be spurious, even in a small country such as Portugal (3Pereira L. Gonçalves J. Goios A. Rocha T. Amorim A. Human mtDNA haplogroups and reduced male fertility: real association or hidden population substructuring.Int J Androl. 2005; 28: 241-247Crossref PubMed Scopus (30) Google Scholar). Indeed, as has been emphasized many times on similar occasions, "association studies can be confounded if patients and control subjects are not well matched" (4Carelli V. Achilli A. Valentino M.L. Rengo C. Semino O. Pala M. et al.Haplogroup effects and recombination of mitochondrial DNA: novel clues from the analysis of Leber hereditary optic neuropathy pedigrees.Am J Hum Genet. 2006; 78: 564-574Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Further, the claim by Holyoake et al. (5Holyoake J.A. McHugh P. Wu M. O'Carroll S. Benny P. Sin I.L. Sin F.Y.T. High incidence of single nucleotide substitutions in the mitochondrial genome is associated with poor semen parameters in men.Int J Androl. 2001; 24: 175-182Crossref PubMed Scopus (98) Google Scholar) that the mtDNA mutations G9055A (characteristic of haplogroup K) and G11719A (signifying nonR0 haplogroup status) would compromise the semen quality of those men who possess any of these mutations in their mtDNAs was evidently based on some single-stranded conformation polymorphism (SSCP) misanalysis (6Bandelt H.-J. Misanalysis gave false association of mtDNA mutations with infertility.Int J Androl. 2007; (in press)Google Scholar). Therefore one cannot yet posit that haplogroups play any role in low sperm motility. This may parallel the situation with Alzheimer's disease and Parkinson's disease, where claims of mtDNA association seem to have been premature (7Elson J.L. Herrnstadt C. Preston G. Thal L. Morris C.M. Edwardson J.A. et al.Does the mitochondrial genome play a role in the etiology of Alzheimer's disease?.Hum Genet. 2006; 119: 241-254Crossref PubMed Scopus (88) Google Scholar, 8Howell N. Elson J.L. Chinnery P.F. Turnbull D.M. mtDNA mutations and common neurodegenerative disorders.Trends Genet. 2005; 21: 583-586Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). A high mutation load in sperm has been asserted by Thangaraj et al. (9Thangaraj K. Gupta N.J. Pavani K. Reddy A.G. Subramainan S. Rani D.S. et al.Y chromosome deletions in azoospermic men in India.J Androl. 2003; 24: 588-597PubMed Google Scholar) in a single case of oligoasthenoteratozoospermia (OAT): "The mitochondrial genes COI, COII, ATPase6, ATPase8, tRNA serine I, tRNA lysine, and ND3 were amplified and sequenced from sperm and blood cells of the man with OAT. A comparison of the sequences of the above genes with a reference sequence revealed a total of 36 nucleotide substitutions in the sperm mtDNA but not in the DNA from the blood cells." Reports of such exorbitant numbers of somatic mutations can, as a rule, always be ascribed to artefacts, such as sample mix-up (10Salas A. Yao Y.-G. Macaulay V. Vega A. Carracedo Á Bandelt H.-J. A critical reassessment of the role of mitochondria in tumorigenesis.PLoS Med. 2005; 2: e296Crossref PubMed Scopus (183) Google Scholar). In that case, however, not unexpectedly, a specific nuclear insert of mitochondrial DNA (numt) had been targeted instead, as was clearly demonstrated by Bravi et al. (11Bravi C.M. Parson W. Bandelt H.-J. Numts revisited.in: Bandelt H.-J. Macaulay V. Richards M. Human mitochondrial DNA and the evolution of Homo sapiens. Springer-Verlag, Berlin-Heidelberg2006: 31-46Google Scholar). An inadvertent base shift (by +1) may have obscured the identity of that "classic" numt, which had already played a role in an earlier case (12Davis R.E. Miller S. Herrnstadt C. Ghosh S.S. Fahy E. Shinobu L.A. et al.Mutations in mitochondrial cytochrome c oxidase genes segregate with late-onset Alzheimer disease.Proc Natl Acad Sci U S A. 1997; 94: 4526-4531Crossref PubMed Scopus (316) Google Scholar). Summarizing, from the publication record up to 2005, there has been no convincing evidence for the action of mtDNA substitutions, either directly in the sperm (as somatic mutations other than deletions) nor indirectly through haplogroup background by predisposing to reduced motility. A most remarkable finding was recently announced by Selvi Rani et al. (13Selvi Rani D. Vanniarajan A. Gupta N.J. Chakravarty B. Singh L. Thangaraj K. A novel missense mutation C11994T in the mitochondrial ND4 gene as a cause of low sperm motility in the Indian subcontinent.Fertil Steril. 2006; 86: 1783-1785Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar): Among men (from India) with fertility problems, all 34 individuals with oligoasthenozoospermia (OA) showed the mutation C11994T in homoplasmic state. None of 150 normozoospermic controls showed this mutation. At face value, OA would thus look like a mitochondrial disorder par excellence, clear-cut, without etiologic or interpretative complications, such as in the case of LHON (4Carelli V. Achilli A. Valentino M.L. Rengo C. Semino O. Pala M. et al.Haplogroup effects and recombination of mitochondrial DNA: novel clues from the analysis of Leber hereditary optic neuropathy pedigrees.Am J Hum Genet. 2006; 78: 564-574Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar) or almost every other recognized mitochondrial disease. It is amazing that "this mutation appears not to affect systems other than the motility of the sperm" (13Selvi Rani D. Vanniarajan A. Gupta N.J. Chakravarty B. Singh L. Thangaraj K. A novel missense mutation C11994T in the mitochondrial ND4 gene as a cause of low sperm motility in the Indian subcontinent.Fertil Steril. 2006; 86: 1783-1785Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). There are, however, serious problems with this proposition. First, if all OA men from that study belonged to one and only one haplogroup characterized by C11994T, then it appears somewhat strange that: 1) this haplogroup was not yet discovered in systematic screening of complete mtDNA variation in India (14Palanichamy Mg. Sun C. Agrawal S. Bandelt H.-J. Kong Q.-P. Khan F. et al.Phylogeny of mitochondrial DNA macrohaplogroup N in India, based on complete sequencing: implications for the peopling of South Asia.Am J Hum Genet. 2004; 75: 966-978Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 15Sun C. Kong Q.-P. Palanichamy Mg Agrawal S. Bandelt H.-J. Yao Y.-G. et al.The dazzling array of basal branches in the mtDNA macrohaplogroup M from India as inferred from complete genomes.Mol Biol Evol. 2006; 23: 683-690Crossref PubMed Scopus (129) Google Scholar, 16Thangaraj K. Chaubey G. Singh V.K. Vanniarajan A. Thanseem I. Reddy A.G. Singh L. In situ origin of deep rooting lineages of mitochondrial macrohaplogroup M in India.BMC Genomics. 2006; 7: 151Crossref PubMed Scopus (80) Google Scholar); and 2) this would virtually constitute the only cause of OA in India. Second, if C11994T occurred repeatedly in different parts of the Indian mtDNA phylogeny, it is quite strange that this recurrent mutation has never been reported in more than 3,000 coding region sequences available from the worldwide database. Confronted with this conundrum, one could envision other causes for the C11994T phenomenon asserted by Selvi Rana et al. (13Selvi Rani D. Vanniarajan A. Gupta N.J. Chakravarty B. Singh L. Thangaraj K. A novel missense mutation C11994T in the mitochondrial ND4 gene as a cause of low sperm motility in the Indian subcontinent.Fertil Steril. 2006; 86: 1783-1785Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar):1.Some novel numt was amplified in all those OA samples for the amplified fragment containing position 11994 (17Parr R.L. Maki J. Reguly B. Dakubo G.D. Aguirre A. Wittock R. et al.The pseudo-mitochondrial genome influences mistakes in heteroplasmy interpretation.BMC Genomics. 2006; 7: 185Crossref PubMed Scopus (67) Google Scholar).2.Position 11944 was systematically hit by a phantom mutation in all those OA samples, just as the specific "Klinefelter mtDNA motif" arose in Oikawa et al. (18Oikawa H. Tun Z. Young D.R. Ozawa H. Yamazaki K. Tanaka E. Honda K. The specific mitochondrial DNA polymorphism found in Klinefelter's syndrome.Biochem Biophys Res Commun. 2002; 297: 341-345Crossref PubMed Scopus (13) Google Scholar, 19Bandelt H.-J. Kong Q.-P. Parson W. Salas A. More evidence for nonmaternal inheritance of mitochondrial DNA?.J Med Genet. 2005; 42: 957-960Crossref PubMed Scopus (60) Google Scholar).3.Some inadvertent alignment shift or misediting has produced a seeming 11994 mutation, similar to the case admitted by Hagelberg et al. (20Hagelberg E. Goldman N. Lió P. Whelan S. Schiefenhövel W. Clegg J.B. Bowden D.K. Evidence for mitochondrial DNA recombination in a human population of island Melanesia: correction.Proc R Soc London Ser B Biol Sci. 2000; 267: 1595-1596Crossref Scopus (28) Google Scholar).4.The OA samples were preselected according to a specific unknown (minor) haplogroup, in which C11994T thrives.5.One sample with 11994 has actually been found in one mtDNA, from which the fragment containing position 11994 was borrowed by all those 34 OA samples. Because the article by Selvi Rani et al. (13Selvi Rani D. Vanniarajan A. Gupta N.J. Chakravarty B. Singh L. Thangaraj K. A novel missense mutation C11994T in the mitochondrial ND4 gene as a cause of low sperm motility in the Indian subcontinent.Fertil Steril. 2006; 86: 1783-1785Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) presents no data—neither a report of the complete mtDNA variation observed in the 10 analyzed mtDNAs from men with OA, nor any pairs of electropherograms for the fragment containing C11994T—one cannot decide between the above five alternatives. Such information would be indispensable for identifying the potential cause of an artefact (21Bandelt H.-J. Achilli A. Kong Q.-P. Salas A. Lutz-Bonengel S. Sun C. et al.Low "penetrance" of phylogenetic knowledge in mitochondrial disease studies.Biochem Biophys Res Commun. 2005; 333: 122-130Crossref PubMed Scopus (64) Google Scholar, 22Bandelt H.-J. Yao Y.-G. Salas A. Kivisild T. Bravi C.M. High penetrance of sequencing errors and interpretative shortcomings in mtDNA sequence analysis of LHON patients.Biochem Biophys Res Commun. 2007; 352: 283-291Crossref PubMed Scopus (39) Google Scholar, 23Parson W. The art of reading sequence electropherograms.Ann Hum Genet. 2006; 71: 276-280Crossref PubMed Scopus (11) Google Scholar). Because the authors hypothesized that this novel mutation is a "widespread genetic cause of oligoasthenozoospermia" and went on to suggest that "the C11994T mutation in the mitochondrial ND4 gene should routinely be screened in individuals with moderate oligoasthenospermia," we decided to analyze the polymorphism C11994T by direct sequencing in the seven samples from a previous study (3Pereira L. Gonçalves J. Goios A. Rocha T. Amorim A. Human mtDNA haplogroups and reduced male fertility: real association or hidden population substructuring.Int J Androl. 2005; 28: 241-247Crossref PubMed Scopus (30) Google Scholar) classified as oligoasthenozoospermic (OA). In that study, both hypervariable regions (HVS-I and HVS-II) of the mtDNA control region were sequenced in a sample of 101 infertile males from southern Portugal. The infertile patients were selected after andrologic and hormonal evaluation. Only patients with idiopathic infertility, i.e., absence of history of hypospadias, urinary infection, epididymitis, orchitis, varicocele, prostatic abnormalities, testis tumors, and testicular maldescent, were included in the present study. All patients had a 46,XY normal karyotype, and all were previously screened for the detection of AZFa, AZFb, and AZFc microdeletions. Only patients without AZF microdeletions were included. Sperm analysis was performed according to World Health Organization guidelines (24World Health Organization WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction.3rd ed. Cambridge University Press, Cambridge, UK1992Google Scholar). Semen samples of all patients were classified into one of the following categories, briefly: oligozoospermia if sperm concentration was less than 20 million/mL but more than 0; asthenozoospermia if fewer than 50% spermatozoa with forward progression (categories A and B) or fewer than 25% spermatozoa with rapid linear progression; teratozoospermia if fewer than 30% spermatozoa with normal head morphology; oligoteratoasthenozoospermia if all three variables were disturbed; oligoasthenozoospermia if patients displayed both oligozoospermia and asthenozoospermia. All patients gave their written informed consent to all analyses, and the study was performed in compliance with the Helsinki Declaration and according to the guidelines of the Scientific and Ethics Committee of the Center of Human Genetics of the National Institute of Health, Lisbon. For checking the presence of the C11994T substitution, an mtDNA fragment was amplified by using the primers forward L11486 5′-AAA ACT AGG CGG CTA TGG TA-3′ and reverse H12076 5′-ATT TTC TGC TAG GGG GTG GA-3′. In this way, the sequence between positions 11531 and 12040 could be read in both directions. The temperature profile was 95°C for 10 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, for 35 cycles of amplification. The amplified samples were purified with Microspin S-300 HR columns (Amersham Biosciences, Uppsala, Sweden), according to the manufacturer's specifications. The sequence reactions were carried out using the kit Big-Dye Terminator Cycle Sequencing Ready Reaction (Applied Biosystems, Foster City, CA) with one of the above primers in both forward and reverse directions. A postpurification by Sephadex was used after the sequencing reaction, and samples were run in an automatic sequencer ABI 3100 (Applied Biosystems). The results are presented in Table 1. Electropherograms for inspection can be requested from the first author. The seven mtDNAs analyzed include at least one representative from each of the major West Eurasian branches (R0, JT, and U; 25Torroni A. Achilli A. Macaulay V. Richards M. Bandelt H.-J. Harvesting the fruit of the human mtDNA tree.Trends Genet. 2006; 22: 339-345Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar) of haplogroup R as well as one representative of an African haplogroup (L1b1). The mutations detected in region 11531–12040 are concordant with the haplogroup status, as originally inferred from HVS-I and HVS-II, i.e., the root of haplogroup R0 (embracing branches HV0 and H) is indistinguishable from rCRS within that fragment, haplogroup T2 bears the characteristic 11812 transition, and in haplogroup U6a1 the 16239 and 11938 transitions seem to be linked (26Maca-Meyer N. González A.M. Pestano J. Flores C. Larruga J.M. Cabrera V.M. Mitochondrial DNA transit between West Asia and North Africa inferred from U6 phylogeography.BMC Genetics. 2003; 4: 15Crossref PubMed Scopus (81) Google Scholar). Not a single occurrence of the 11994 transition was found here or in the mtDNAs of another 20 asthenozoospermic and 23 teratoasthenozoospermic men (27Pereira L. Gonçalves J. Franco-Duarte R. Silva J. Rocha T. Arnold C. et al.No evidence for an mtDNA role in sperm motility: data from complete sequencing of asthenozoospermic males.Mol Biol Evol. 2007; 24: 868-874Crossref PubMed Scopus (60) Google Scholar). Therefore C11994T cannot play a dominant role in oligoasthenozoopermy or other forms of asthenozoopermy, at least in Portugal, and it is therefore doubtful that this mutation should constitute the single cause in India. If the sequencing protocol of Selvi Rani et al. (13Selvi Rani D. Vanniarajan A. Gupta N.J. Chakravarty B. Singh L. Thangaraj K. A novel missense mutation C11994T in the mitochondrial ND4 gene as a cause of low sperm motility in the Indian subcontinent.Fertil Steril. 2006; 86: 1783-1785Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) followed Rieder et al. (28Rieder M.J. Taylor S.L. Tobe V.O. Nickerson D.A. Automating the identification of DNA variations using quality-based fluorescence re-sequencing: analysis of the human mitochondrial genome.Nucleic Acids Res. 1998; 26: 967-973Crossref PubMed Scopus (444) Google Scholar), then position 11994 was probably only well readable on the H strand of amplicon 17 (11314–12076) and on the L strand of amplicon 18 (11948–12772). It would be interesting to learn which further variation has been observed in the 10 mtDNAs that were completely sequenced by Selvi Rani et al. (13Selvi Rani D. Vanniarajan A. Gupta N.J. Chakravarty B. Singh L. Thangaraj K. A novel missense mutation C11994T in the mitochondrial ND4 gene as a cause of low sperm motility in the Indian subcontinent.Fertil Steril. 2006; 86: 1783-1785Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) and inspect the pairs of electropherograms supporting the findings. Otherwise, further mtDNA samples of oligoasthenozoopermic men from India would need investigation performed in other laboratories. The authors thank Yong-Gang Yao for bringing the Selvi Rani et al. article to their attention.
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