Krausz, Csilla Gabriella

Tyler Smith, C

English

Florence Research

editorialsn engl j med 360;9 nejm.org february 26, 2009 925of the dietary fat to carbohydrate ratio of isoenergetically dense diets: effect on food intake in feeding men ad libitum. Int J Obes Relat Metab Disord 1996;20:651-60.Davis EM, Clark JM, Carrese JA, Gary TL, Cooper LA. Racial 9. and socioeconomic differences in the weight-loss experiences of obese women. Am J Public Health 2005;95:1539-43.Franz MJ, VanWormer JJ, Crain AL, et al. Weight-loss out-10. comes: a systematic review and meta-analysis of weight-loss clinical trials with a minimum 1-year follow-up. J Am Diet Assoc 2007;107:1755-67.Romon M, Lommez A, Tafflet M, et al. Downward trends in 11. the prevalence of childhood overweight in the setting of 12-year school- and community-based programmes. Public Health Nutr 2008 December 23 (Epub ahead of print).Westley H. Thin living. BMJ 2007;335:1236-7.12. Copyright © 2009 Massachusetts Medical Society.The Will-o’-the-Wisp of Genetics — Hunting  for the Azoospermia Factor GeneChris Tyler-Smith, Ph.D., and Csilla Krausz, M.D., Ph.D.It has been known for decades that the Y chro-mosome carries genes required for spermatogen-esis and male fertility. But it has been frustrat-ingly difficult to pin down the relevant genes, to such an extent that a finding reported by Luddi et al.1 in this issue of the Journal — that the ubiq-uitin-specific peptidase 9, Y-linked gene (USP9Y) is not essential for normal spermatogenesis — represents a noteworthy advance in the field.Approximately 15% of couples have difficulty conceiving; the causes are many and include ge-netic factors. The most common known genetic causes of spermatogenic failure are karyotype anomalies and Y-chromosome microdeletions. Karyotype analysis and screening for Yq deletions are now routine diagnostic tests that are able to help elucidate both the cause of spermatogenic perturbations and their clinical consequences. It is therefore of some interest to know which genes are essential to spermatogenesis; with this infor-mation, a more specific diagnosis might be pos-sible, and the clinical management — including genetic counseling — of infertile couples would be greatly improved.The search for these genes began in 1976, when Tiepolo and Zuffardi2 reported cytogenetically vis-ible microdeletions in Yq in six azoospermic men. Such deletions must be very large to be visible and are therefore likely to involve the absence of many genes (and unlikely to reveal the causal gene), but this observation provided a starting point for a classic approach to identifying the gene or genes responsible for this phenotype. Smaller deletions within the Yq interval would be sought using mo-lecular methods, leading to the identification of one or a few candidate genes, and inactivating mutations would then be used to confirm which of the candidates was responsible. This strategy has proved successful in the identification of the causal genes underlying countless single-gene dis-orders since its initial application to chronic gran-ulomatous disease in 1986.3At first, the application of this approach for Y-chromosomal spermatogenic abnormalities seemed promising. Molecular analysis did indeed identify smaller deletions within Yq that were spe-cific to men with spermatogenic failure. These were classified, on the basis of chromosomal lo-cation, as three azoospermia factor (AZF) regions: AZFa, AZFb, and AZFc.4 AZFb and AZFc were later shown to contain large segments of duplicated sequence and, in fact, to overlap each other. Given the challenge of tracking down specific loci in chromosomal regions enriched in sequence dupli-cations, it is perhaps understandable that the gene or genes responsible for the AZFb–AZFc spermato-genesis phenotype have still not been identified.In contrast, the AZFa interval consists of reg-ular single-copy DNA (i.e., DNA that does not contain a supranormal level of repeated sequence) that should not present unusual complications for homing in on the key gene. And in 1999, Sun et al.5 described what seemed to be a definitive re-sult. They sequenced the two genes in the AZFa region (USP9Y and the DEAD [Asp–Glu–Ala–Asp] box polypeptide 3, Y-linked gene, DDX3Y) in 576 infertile men and discovered exactly the kind of mutation they were looking for, in a man with spermatogenic failure: a de novo deletion of 4 bp within the USP9Y gene that destroyed a splice-donor site, resulting in the skipping of the next exon and truncation of approximately 90% of the peptide sequence (Fig. 1). Thus, it appeared that the loss of USP9Y was responsible for the observed azoospermia and that one gene underlying sper-matogenic failure had been successfully found.T h e  n e w  e ngl a nd  j o u r na l  o f  m e dic i n en engl j med 360;9 nejm.org february 26, 2009926But subsequent findings complicated this sim-ple conclusion. The phenotype associated with the loss of USP9Y turned out to be variable, ranging from normospermia in chimpanzees and bono-bos, in which the gene became inactivated mil-lions of years ago,8 to moderate oligoasthenotera-tozoospermia in humans,7 to azoospermia.5 The report by Luddi et al. now adds normospermia in humans to this list of phenotypes. These obser-vations have bearing on two related questions: Is the inactivation of USP9Y or DDX3Y responsible for the AZFa azoospermic phenotype? and What con-clusions should we draw from the variation in the USP9Y-associated phenotype?There are a number of reasons why the first, apparently simple, question has been so difficult to resolve. First, AZFa deletions are themselves rare. In literature reports, they represent less than 5% of all AZF deletions.9 Second, AZFa deletions leading to azoospermia obviously cannot be in-herited, and so all are de novo mutations. They usually arise by the same mechanism: homologous recombination between two misaligned human endogenous retrovirus sequences (HERVs, ele-ments of viral origin inserted into the genome and inherited as part of the chromosome) spaced about 790 kb apart (Fig. 1), encompassing both genes.10 Mutations due to HERV misalignment are therefore not informative for distinguishing between the contributions of the two genes. Years of study have identified just a handful of patients who carry nonstandard deletions that affect only one of the genes and are thus informative (Fig. 1). Third, all of the confirmed single-gene mutations affect USP9Y; we have no information about the phenotype associated with the loss of DDX3Y only.The study by Luddi et al. clearly shows that complete USP9Y deletion is compatible with nor-mal spermatogenesis and fertility. But previous studies5-7 show, equally clearly, that its loss can disrupt spermatogenesis to varying degrees and that natural transmission (fertility) is possible when the phenotype is mild.7 So we have to con-clude that the phenotype associated with the loss of USP9Y varies according to the genetic or other background of the carrier, although the relevant modifying factors remain unknown. This varia-tion in phenotype contrasts sharply with the effect of losing both genes, which invariably results in the Sertoli-cell–only syndrome (characterized by the lack of germ cells) and, consequently, azoo-spermia. This consistent effect strongly suggests a key role of DDX3Y in spermatogenesis, a con-clusion supported by expression of the gene in 33p912.8 12.9 13.0 13.1 13.2 13.3 13.4 13.5 13.6 13.7 13.8Reference Sequence MbAUTHOR:FIGURE:JOB:4-CH/TRETAKESIZEICMCASEEMail LineH/TComboRevisedAUTHOR, PLEASE NOTE: Figure has been redrawn and type has been reset.Please check carefully.REG FEnon1st2nd3rdTyler-Smith1 of 102-26-09ARTIST: ts36009 ISSUE:Mutation or PatientStandard AZFa deletionSAYERWHT2780  1115/0 (transmitted deletion)A333AZFa deletion (transmitted deletion) described by Luddi et al. GenesUSP9Y DDX3YHERV HERVSperm PhenotypeAzoospermiaNormozoospermiaAzoospermiaSevere OATMild OATModerate OATFigure 1. Known Deletions of the Azoospermia Factor Region a (AZFa) of the Human Y Chromosome and Their As-sociated Spermatogenic Phenotypes.Part of the reference sequence, 1 Mb in length, of the Y chromosome is shown (top), highlighting ubiquitin-specific peptidase 9, Y-linked gene (USP9Y) and the DEAD (Asp–Glu–Ala–Asp) box polypeptide 3, Y-linked gene (DDX3Y)  (red arrows) and the two human endogenous retrovirus sequences (HERVs) (orange boxes). Along the same se-quence, regions are shown (bottom) as retained (black boxes) or missing (thin dotted line) in patients, including the standard AZFa deletion resulting from nonallelic homologous recombination between the two HERV sequences, as well as confirmed gene-specific deletions. Note the variability in phenotype associated with USP9Y-only deletions and the lack of known DDX3Y-only deletions. The white boxes in SAYER (described by Brown et al.6) indicate the ex-tent of uncertainty in the location of the breakpoint in this patient, and the size of the 4-bp deletion in WHT2780 (described by Sun et al.5) has been exaggerated to make it visible at this scale. The 1115/0 and A333 deletions are described by Krausz et al.7 “Transmitted” indicates a deletion that has been passed naturally from father to son on at least one occasion. OAT denotes oligoasthenoteratozoospermia.editorialsn engl j med 360;9 nejm.org february 26, 2009 927premeiotic spermatogonia (USP9Y is expressed in postmeiotic spermatids).11 We predict that the loss of DDX3Y alone is associated with a more severe testicular phenotype, most likely azoo-spermia.What are the clinical implications of these con-clusions? Screening for USP9Y deletion should not be performed routinely until there is a better un-derstanding of the factors that modify the phe-notype. Luddi et al. suggest that revision of the European Academy of Andrology–European Mo-lecular Genetics Quality Network screening guide-lines may be warranted to ensure that DDX3Y-only deletions are detected. The lack of confirmed de-letions of this type after many years of world-wide screening in research laboratories9 means that the cost of such a step may currently out-weigh the clinical benefit, but we can look for-ward to a time when the thousand-dollar genome will make such information, and much more, readily available. In the meantime, the article by Luddi et al. focuses our attention even more strongly on understanding the role of DDX3Y.No potential conflict of interest relevant to this article was re-ported.From the Wellcome Trust Sanger Institute, Hinxton, United King-dom (C.T.-S.); and the Andrology Unit, Department of Clinical Physiopathology, University of Florence, Florence, Italy (C.K.).Luddi A, Margollicci M, Gambera L, et al. Spermatogenesis 1. in a man with complete deletion of USP9Y. N Engl J Med 2009; 360:881-5.Tiepolo L, Zuffardi O. Localization of factors controlling 2. spermatogenesis in the nonfluorescent portion of the human Y chromosome long arm. Hum Genet 1976;34:119-24.Royer-Pokora B, Kunkel LM, Monaco AP, et al. Cloning the 3. gene for an inherited human disorder — chronic granulomatous disease — on the basis of its chromosomal location. Nature 1986;322:32-8.Vogt PH, Edelmann A, Kirsch S, et al. Human Y chromo-4. some azoospermia factors (AZF) mapped to different subregions in Yq11. Hum Mol Genet 1996;5:933-43.Sun C, Skaletsky H, Birren B, et al. An azoospermic man 5. with a de novo point mutation in the Y-chromosomal gene USP9Y. Nat Genet 1999;23:429-32.Brown GM, Furlong RA, Sargent CA, et al. Characterisation 6. of the coding sequence and fine mapping of the human DFFRY gene and comparative expression analysis and mapping to the Sxrb interval of the mouse Y chromosome of the Dffry gene. Hum Mol Genet 1998;7:97-107.Krausz C, Degl’Innocenti S, Nuti F, et al. Natural transmis-7. sion of USP9Y gene mutations: a new perspective on the role of AZFa genes in male fertility. Hum Mol Genet 2006;15:2673-81.Perry GH, Tito RY, Verrelli BC. The evolutionary history of 8. human and chimpanzee Y-chromosome gene loss. Mol Biol Evol 2007;24:853-9.Krausz C, Degl’Innocenti S. Y chromosome and male infer-9. tility: update, 2006. Front Biosci 2006;11:3049-61.Kamp C, Hirschmann P, Voss H, Huellen K, Vogt PH. Two 10. long homologous retroviral sequence blocks in proximal Yq11 cause AZFa microdeletions as a result of intrachromosomal re-combination events. Hum Mol Genet 2000;9:2563-72.Vogt PH, Falcao CL, Hanstein R, Zimmer J. The AZF pro-11. teins. Int J Androl 2008;31:383-94.Copyright © 2009 Massachusetts Medical Society.

The will-o&apos;-the-wisp of genetics--hunting for theazoospermia factor gene.

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The will-o'-the-wisp of genetics--hunting for theazoospermia factor gene.

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The will-o&apos;-the-wisp of genetics--hunting for theazoospermia factor gene.

Abstract

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Florence Research

The will-o'-the-wisp of genetics--hunting for theazoospermia factor gene.