Whole-exome sequencing in patients with maturation arrest: a potential additional diagnostic tool for prevention of recurrent negative testicular sperm extraction outcomes.

STUDY QUESTION: Could whole-exome sequencing (WES) be useful in clinical practice for men with maturation arrest (MA) after a first testicular sperm extraction (TESE)? SUMMARY ANSWER: WES in combination with TESE yields substantial additional information and may potentially be added as a test to predict a negative outcome of a recurrent TESE in patients with MA. WHAT IS KNOWN ALREADY: At present, the only definitive contraindications for TESE in men with non-obstructive azoospermia (NOA) are a 46,XX karyotype and microdeletions in the azoospermia factor a (AZFa) and/or AZFb regions. After a first negative TESE with MA, no test currently exists to predict a negative outcome of a recurrent TESE. STUDY DESIGN, SIZE, DURATION: In a cohort study, we retrospectively included 26 patients with idiopathic NOA caused by complete MA diagnosed after a first TESE. PARTICIPANTS/MATERIALS, SETTING, METHODS: Twenty-six men with MA at the spermatocyte stage in all seminiferous tubules, according to a histopathological analysis performed independently by two expert histologists, and a normal karyotype (i.e. no AZF gene microdeletions on the Y chromosome) were included. Single-nucleotide polymorphism comparative genomic hybridization array and WES were carried out. The results were validated with Sanger sequencing. For all the variants thought to influence spermatogenesis, we used immunohistochemical techniques to analyse the level of the altered protein. MAIN RESULTS AND THE ROLE OF CHANCE: Deleterious homozygous variants were identified in all seven consanguineous patients and in three of the 19 non-consanguineous patients. Compound heterozygous variants were identified in another 5 of the 19 non-consanguineous patients. No recurrent variants were identified. We found new variants in genes known to be involved in azoospermia or MA [including testis expressed 11 (TEX11), meiotic double-stranded break formation protein 1 (MEI1), proteasome 26s subunit, ATPase 3 interacting protein (PSMC3IP), synaptonemal complex central element protein 1 (SYCE1) and Fanconi anaemia complementation group M (FANCM) and variants in genes not previously linked to human MA (including CCCTC-binding factor like (CTCFL), Mov10 like RISC complex RNA helicase 1 (MOV10L1), chromosome 11 open reading frame 80 (C11ORF80) and exonuclease 1 (EXO1)]. LARGE SCALE DATA: Data available on request. LIMITATIONS, REASONS FOR CAUTION: More data are required before WES screening can be used to avoid recurrent TESE, although screening should be recommended for men with a consanguineous family background. WES is still a complex technology and can generate incidental findings. WIDER IMPLICATIONS OF THE FINDINGS: Our results confirmed the genetic aetiology of MA in most patients: the proportion of individuals with at least one pathologic variant was 50% in the overall study population and 100% in the consanguineous patients. With the exception of MEI1 (compound heterozygous variants of which were identified in two cases), each variant corresponded to a specific gene-confirming the high degree of genetic heterogeneity in men with MA. Our results suggest that WES screening could help to avoid recurrent, futile TESE in men with MA in general and in consanguineous individuals in particular, but these results need to be confirmed in future studies before clinical implementation. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by the Fondation Maladies Rares (Paris, France), Merck (Kenilworth, NJ, USA), IRSF (Montigny le Bretonneux, France) and Agence de la Biomédecine (Saint Denis, France). There are no competing interests. TRIAL REGISTRATION NUMBER: N/A.

A MEI1 homozygous missense mutation associated with meiotic arrest in a consanguineous family.

Although meiotic arrest in males is observed in about 25% of azoospermic patients, pure homogeneous arrest in all seminiferous tubules is less frequent, and may be due to mutation of a single gene. However, given the large number of genes involved in meiosis, this gives rises to extensive genetic heterogeneity. Only two genetic abnormalities have been reported on a regular basis: the X-linked exonic TEX11 deletion, and the AZFb microdeletion on the Y chromosome. Other single gene defects were private and found in consanguineous families. Here, we report on a homozygous missense mutation in the gene coding for meiotic double-stranded break formation protein 1 (MEI1; c.C3307T:p.R1103W) observed in two brothers (from a consanguineous Tunisian family) with non-obstructive azoospermia and meiotic arrest. A fertile brother was heterozygous for the mutation. All the queried databases predicted that this mutation is damaging, and it has previously been reported that Mei1 knock-out is associated with meiotic arrest in a murine model. Hence, meiotic arrest in the two brothers was probably caused by an alteration in a gene known to be fundamental for chromosome synapsis.

CYP1B1 mutations in French patients with early-onset primary open-angle glaucoma.

INTRODUCTION: Primary open-angle glaucoma (POAG) is a leading cause of visual impairment worldwide and a complex genetic disorder that affects mostly adults. Mutations in the MYOCILIN (MYOC) and OPTINEURIN genes account for rare forms with a Mendelian inheritance and for <5% of all POAG cases. The CYP1B1 gene, a member of the cytochrome P450 gene family, is a major cause of primary congenital glaucoma (PCG), a rare and severely blinding disease with recessive inheritance. However, CYP1B1 mutations have also been associated with cases of juvenile-onset glaucoma in some PCG families or shown to modify the age of onset of glaucoma linked to a MYOC mutation in a large family. OBJECTIVE: To investigate the role of CYP1B1 mutations in POAG predisposition, irrespective of the presence of a MYOC mutation. METHODS AND SUBJECTS: CYP1B1 coding region variation was characterised by denaturing high performance liquid chromatography (DHPLC) and sequencing in 236 unrelated French Caucasian POAG patients and 47 population-matched controls. RESULTS: Eleven (4.6%) patients carried one or two mutated CYP1B1 gene(s) and no MYOC mutation. They showed juvenile or middle-age onset of disease (median age at diagnosis, 40 years, range 13-52), significantly earlier than in non-carrier patients. Apart from one, all mutations detected in POAG patients were previously associated with PCG. CONCLUSION: CYP1B1 mutations might pose a significant risk for early-onset POAG and might also modify glaucoma phenotype in patients who do not carry a MYOC mutation.

Genetic control of autoantibody expression in autoimmune myasthenia gravis: role of the self-antigen and of HLA-linked loci.

Autoantibodies against the muscle acetylcholine receptor (AChR) play an essential role in the pathophysiology of autoimmune myasthenia gravis (MG). Their serum titers, however, vary considerably among patients. Our aim was to investigate whether their variation might be explained by genetic factors. Using different methods, we have obtained strong evidence for a three-locus association influencing autoantibody titers in MG patients with thymus hyperplasia or with a normal thymus. Two of the loci, one encoding the AChR alpha-subunit, the other encoding the alpha-chain of the class II antigen-presentation molecule, HLA-DQ, demonstrated interaction to determine high autoantibody titers. The third locus was associated with the 8.1 ancestral HLA haplotype. It exerted an additive effect and it is postulated to have a nonantigen specific immunoregulatory function. Our study demonstrates for the first time that polymorphism of an autoantigen gene may quantitatively modify the immune response against it. Altogether, the data lend support to a three-gene model to explain autoantibody expression in a subset of MG patients.

Association of the gene encoding the delta-subunit of the muscle acetylcholine receptor (CHRND) with acquired autoimmune myasthenia gravis.

The muscle acetylcholine receptor (AChR) is the main target self-antigen in acquired autoimmune myasthenia gravis (MG). Here, we investigated an association of MG with the CHRND gene encoding the delta-subunit of the AChR. Using a microsatellite repeat located in the second intron of the gene, we observed a preferential transmission of the allele 268 in 114 one-generation families with one myasthenic child (Pc=0.0154). This allele was also over-represented in a group of 350 unrelated nonthymoma MG patients (OR=1.78, P=0.038), but not in 84 thymoma patients, compared to 168 healthy controls. Moreover, among nonthymoma patients, those lacking serum anti-titin antibodies appeared to be best associated (OR=2.07, P=0.017). In contrast, there was no distortion in the transmission of a single-nucleotide substitution polymorphisms (SNPs) in the 3' untranslated region of CHRND nor in that of two SNPs located in the closely linked CHRNG gene, 4.5 kb telomeric to CHRND. The data warrant a detailed investigation of CHRND polymorphism in MG patients.

TNFA-TNFB haplotypes modify susceptibility to type I diabetes mellitus independently of HLA class II in a Moroccan population.

The contribution of single nucleotide polymorphisms in tumor necrosis factors (TNF) alpha and beta to autoimmune diseases, and to type 1 diabetes mellitus (T1DM) in particular, is not well established, and may be confounded by linkage disequilibrium to class II HLA genes. At least two polymorphisms seem to have functional relevance in the respective genes: TNFA-307 and TNFB+252. We have typed these two polymporphisms in samples of Moroccan T1DM patients and controls for which class II HLA genes had already been typed. Tumor necrosis factors and compound TNF-class II HLA haplotypes were inferred; it was the first time that such a design had been implemented. Independent of linkage disequilibrium with class II HLA, TNF haplotype TNFA-307*2 - TNFB+252*2 showed a significant protective effect (OR = 0.031), partly exacerbated by partial linkage to protective class II haplotypes. Such effect could be detected because Morocco shows the highest frequency of the TNFA-307*2 allele yet reported. This highlights the possible population differences in alleles contributing to autoimmune diseases.

A novel frameshift founder mutation in the cytochrome P450 1B1 (CYP1B1) gene is associated with primary congenital glaucoma in Morocco.

Primary congenital glaucoma (PCG) is a heterogeneous autosomal recessive disorder caused by unknown developmental defect(s) of the anterior chamber of the eye. A member of the cytochrome P450 gene family, CYP1B1, was found to be mutated in PCG patients in different populations, albeit to a variable extent. In this study, CYP1B1 mutations were searched for in 32 unrelated PCG patients from Morocco. Two mutations were detected in 11 (34%) patients. One, 4339delG, is novel and causes a frameshift at residue 179. The other, G61E, was previously found in patients from Turkey and Saudi Arabia. Seven patients were homozygous for 4339delG and two other patients for G61E, whereas the two remaining patients were compound heterozygotes. The close association of 4339delG with a rare allele of D2S177, a microsatellite marker located 270 kb upstream of CYP1B1, strongly suggested a founder effect for 4339delG. The occurrence of this mutation was tentatively dated at between 900 and 1700 years ago. Typing 4339delG and G61E mutations should help to prevent blindness resulting from a delayed diagnosis of PCG in Morocco.

Further mapping of the Idd5.1 locus for autoimmune diabetes in NOD mice.

The Idd5 locus for autoimmune diabetes in nonobese diabetic (NOD) mice has been mapped to the proximal half of chromosome 1 and appears to include two loci, Idd5.1 and Idd5.2, Idd5.1 being a candidate homolog of the human IDDM12 locus. Using new recombinant congenic lines, we have reduced the Idd5.1 interval to 5 cM at most, between D1Mit279 and D1Mit19 (not included). This interval now excludes the Casp8 and Cflar (Flip) candidate genes. It still retains Cd28 and Ctla4 and also includes Icos (inducible costimulator). The previously reported differential expression of Ctla4, which is induced at a lower level in NOD than in B6-activated T-cells, was found independent of Idd5.1 itself because Ctla4 expression was induced at a low level in T-cells from Idd5.1-congenic mice. The Idd5.1 locus protected against both spontaneous and cyclophosphamide-induced diabetes, but it did not prevent inflammatory infiltration of the islets of Langerhans. Furthermore, diabetogenic precursor spleen cells from prediabetic NOD and Idd5.1-congenic mice were equally capable of transferring diabetes to immunodeficient NOD.scid/scid recipient mice. The Idd5.1 locus might affect a late event of disease development, subsequent to the onset of insulitis and possibly taking place in the islets of Langerhans.

Linkage of HLA to myasthenia gravis and genetic heterogeneity depending on anti-titin antibodies.

BACKGROUND: MG is an autoimmune disease of the neuromuscular junction. MG with thymus hyperplasia has been associated with, but not genetically linked to, the HLA-DR3 haplotype. OBJECTIVE: To re-evaluate the association of HLA with MG in 656 patients with generalized disease and to test linkage of HLA to MG with thymus hyperplasia. METHOD: Patients were genotyped for HLA-DRB1. Data analysis included case-control comparisons after subgrouping patients by thymus histopathology. The transmission of parental alleles to MG offspring with thymus hyperplasia was studied in simplex families using the transmission/disequilibrium test (TDT) as a test of linkage. RESULTS: MG with thymus hyperplasia was positively associated with DR3 (OR = 4.5, p = 1 x 10(-6)) and negatively associated with DR7 (OR = 0.28, p = 1 x 10(-6)), based on both case-control comparisons and TDT. No association was detected with thymomas. Conversely, patients who lacked thymus anomalies but expressed anti-titin antibodies (ATA) had an increase of DR7 (OR = 2.08, p = 4 x 10(-3)) and a decrease of DR3 (OR = 0.33, p = 9 x 10(-3)). CONCLUSIONS: The authors established linkage of HLA to MG and thymus hyperplasia, defining the MYAS1 locus. Moreover, DR3 and DR7, or closely linked genes, have opposing effects on MG phenotypes. Nonthymomatous patients with ATA may be a pathogenetically distinct subset of MG patients.

Association of a single nucleotide polymorphism in the TIGR/MYOCILIN gene promoter with the severity of primary open-angle glaucoma.

Primary open-angle glaucoma (POAG) is a highly prevalent optic neuropathy and a major cause of irreversible blindness, with elevation of intraocular pressure (IOP) being a primary risk factor. The trabecular meshwork-inducible glucocorticoid response (TIGR)/MYOCILIN (MYOC) gene coding region is mutated in 3-4% of POAG patients. Here, in a retrospective study of 142 POAG patients, we evaluated the influence on glaucoma phenotype of a novel biallelic polymorphism (-1000C/G) located in the upstream region of the MYOC gene. Allele frequencies were similar among patients and controls. However, the G allele (frequency 17.6%), also designated as MYOC.mt1, was associated with an increased IOP (+4.9 mmHg, p=0.0004) and a more damaged visual field (p=0.02). Both effects were predominant in females. Moreover, whereas IOP in MYOC.mt1 noncarriers decreased very markedly to the normal range between diagnosis and inclusion in the study (p=3 x 10(-5) in both males and females), reflecting successful therapy, it decreased less noticeably in MYOC.mt1+ male patients (p=0.005) and not at all in MYOC.mt1+ female patients. MYOC.mt1 appears therefore to be an indicator of poor IOP control and greater visual field damage in diagnosed POAG patients, potentially due to a lack of response to therapeutic intervention. Its typing might help in the selection of treatment paradigms for the management of POAG patients.

Genetic influences on the end-stage effector phase of arthritis.

K/BxN T cell receptor transgenic mice are a model of inflammatory arthritis, most similar to rheumatoid arthritis, that is critically dependent on both T and B lymphocytes. Transfer of serum, or just immunoglobulins, from arthritic K/BxN animals into healthy recipients provokes arthritis efficiently, rapidly, and with high penetrance. We have explored the genetic heterogeneity in the response to serum transfer, thereby focussing on the end-stage effector phase of arthritis, leap-frogging the initiating events. Inbred mouse strains showed clear variability in their responses. A few were entirely refractory to disease induction, and those which did develop disease exhibited a range of severities. F1 analyses suggested that in most cases susceptibility was controlled in a polygenic additive fashion. One responder/nonresponder pair (C57Bl/6 x NOD) was studied in detail via a genome scan of F2 mice; supplementary information was provided by the examination of knock-out and congenic strains. Two genomic regions that are major, additive determinants of the rapidity and severity of K/BxN serum-transferred arthritis were highlighted. Concerning the first region, on proximal chromosome (chr)2, candidate assignment to the complement gene C5 was confirmed by both strain segregation analysis and functional data. Concerning the second, on distal chr1, coinciding with the Sle1 locus implicated in susceptibility to lupus-like autoimmune disease, a contribution by the fcgr2 candidate gene was excluded. Two other regions, on chr12 and chr18 may also contribute to susceptibility to serum-transferred arthritis, albeit to a more limited degree. The contributions of these loci are additive, but gene dosage effects at the C5 locus are such that it largely dominates. The clarity of these results argues that our focus on the terminal effector phase of arthritis in the K/BxN model will bear fruit.

Anti-titin antibodies in myasthenia gravis: tight association with thymoma and heterogeneity of nonthymoma patients.

BACKGROUND: Titin is the major autoantigen recognized by anti-striated muscle antibodies, which are characteristic of generalized myasthenia gravis (MG). OBJECTIVE: To seek a correlation between anti-titin antibodies and other features of MG patients, including histopathology, age at diagnosis, anti-acetylcholine receptor (anti-AChR), autoantibody titers, and clinical severity. METHODS: A novel, highly specific radioligand assay was performed on a large group of 398 patients with generalized MG. RESULTS: Among thymectomized patients, anti-titin antibodies were present in most patients with thymoma (56/70 [80%]), contrasting with only a minority of patients with thymus atrophy or hyperplasia (17/165 [10%]). They were also present in 64 (41%) of 155 nonthymectomized patients who had a radiologically normal thymus. In these patients and in those who had a histologically normal thymus, anti-titin antibodies were associated with a later age at onset of disease and with intermediate titers of anti-AChR antibodies. After controlling for these 2 variables, disease severity was not significantly influenced by anti-titin antibodies. CONCLUSIONS: Anti-titin antibodies are a sensitive marker of thymoma associated with MG in patients 60 years and younger, justifying the insistent search for a thymoma in MG patients of this age group who have these antibodies. In nonthymoma patients, anti-titin antibodies represent an interesting marker complementary to the anti-AChR antibody titer, identifying a restricted subset of patients. These clinical correlations should prompt further studies to examine the mechanisms leading to the production of anti-titin antibodies.

CTLA-4-/- mice display T cell-apoptosis resistance resembling that ascribed to autoimmune-prone non-obese diabetic (NOD) mice.

The genes conferring susceptibility to autoimmune (insulin-dependent) diabetes mellitus (IDDM) are, in most cases, not defined. Among the loci so far identified as associated with murine IDDM (Idd1-19), only the nature of Idd1 has been assessed. Here we show that thymocytes and peripheral lymphocytes of the non-obese diabetic (NOD) mouse are relatively resistant to apoptosis induced by gamma-irradiation. By linkage analysis of F2 progeny mice, we map this trait to a locus on chromosome 1 containing the Idd5 diabetes susceptibility region. By the use of congenic mice, we confirm the linkage data and map this locus to a 6 cM region on proximal chromosome 1. Ctla4, being localized in this chromosomal region and mediating crucial functions in T cell biology, is a logical candidate gene in the Idd5 susceptibility region. In line with this, we demonstrate that T cells from Ctla4(-/-)deficient mice show a similar resistance to gamma-irradiation-induced apoptosis as observed in the NOD mice. This reinforces the notion that CTLA-4 contributes to the pathogenesis of autoimmune diabetes.

XMR is associated with the asynapsed segments of sex chromosomes in the XY body of mouse primary spermatocytes.

The XMR (Xlr-related, meiosis-regulated) protein is an M(r) 30,000 nuclear protein closely associated with the XY body in mouse primary spermatocytes. It shows sequence similarity with several other meiosis-specific proteins. In the present study, we investigated the fine immunolocalization of XMR in the XY body by laser confocal and electron microscopy. It was found that XMR was associated with the asynapsed segments of sex chromosomes, including their axes and the surrounding chromatin loops. In contrast, the pseudoautosomal region and the opposite free end of the X were unlabeled for XMR. In mice with the reciprocal T(X;16)16H translocation, XMR was also associated with the heterochromatic translocation product that emerges from the XY body. These findings at the subchromosomal level point to a role for XMR in chromatin condensation and transcriptional inactivation. XMR is unique among proteins in being capable of association with the XY body. It could play a specific role in a mechanism of male X-chromosome inactivation in mammals.

Genetic and physical mapping of a type 1 diabetes susceptibility gene (IDDM12) to a 100-kb phagemid artificial chromosome clone containing D2S72-CTLA4-D2S105 on chromosome 2q33.

Polymorphic markers within the CTLA4 gene on chromosome 2q33 have been shown to be associated with type 1 diabetes. Therefore, a gene responsible for the disease (IDDM12) most likely lies within a region of <1-2 cM of CTLA4. To define more precisely the IDDM12 interval, we genotyped a multiethnic (U.S. Caucasian, Mexican-American, French, Spanish, Korean, and Chinese) collection of 178 simplex and 350 multiplex families for 10 polymorphic markers within a genomic interval of approximately 300 kb, which contains the candidate genes CTLA4 and CD28. The order of these markers (D2S346, CD28, GGAA19E07, D2S307, D2S72, CTLA4, D2S105, and GATA52A04) was determined by sequence tagged site content mapping of bacterial artificial chromosome (BAC) and yeast artificial chromosome (YAC) clones. The transmission disequilibrium test (TDT) analyses of our data revealed significant association/linkage with three markers within CTLA4 and two immediate flanking markers (D2S72 and D2S105) on each side of CTLA4 but not with more distant markers including the candidate gene CD28. Tsp analyses revealed significant association only with the three polymorphic markers within the CTLA4 gene. The markers linked and associated with type 1 diabetes are contained within a phagemid artificial chromosome clone of 100 kb, suggesting that the IDDM12 locus is either CTLA4 or an unknown gene in very close proximity.

High level expression of the Xlr nuclear protein in immature thymocytes and colocalization with the matrix-associated region-binding SATB1 protein.

The X-linked lymphocyte-regulated (Xlr) protein is a 30,000 Mr nuclear protein bearing homology with meiosis-specific proteins and expressed in late stage B lymphoid cell lines. In the present study we investigated its expression in the T lymphoid lineage. In adults, a high level of expression was detected in CD4-CD8- thymocytes. Most remarkably, the peak of Xlr expression occurred early during thymus cell ontogeny, precisely on days 14-15 of gestation, and was associated with the first wave of pre-T cell differentiation. Its onset preceded the rearrangement of TCR genes, as Xlr expression was conserved in thymus cells from RAG1(0/0) mice. The lower expression of Xlr on day 13 of fetal development, the bright Thy1+ phenotype of Xlr-positive cells, their large size, and their absence from subcapsular areas suggest that Xlr expression must be turned on within the thymus and not in prethymic precursors. From day 16 of gestation, Xlr expression decreased markedly. At birth and later, Xlr(high) cells were mostly large cells scattered throughout the cortical area. As shown by confocal microscopy, expression of Xlr closely overlapped that of SATB1, which binds special AT-rich DNA sequences associated with the nuclear matrix and plays an important regulatory role for many genes. The remarkably regulated expression of Xlr in the lymphoid cell lineage and of its homologue Xmr in the germ cell lineage suggests that they might play an important role in chromatin metabolism at critical stages of differentiation during which the genome undergoes irreversible rearrangements.

Novel TIGR/MYOC mutations in families with juvenile onset primary open angle glaucoma.

Mutations in the trabecular meshwork induced glucocorticoid response protein (TIGR) or myocilin (MYOC) has recently been shown to cause juvenile onset primary open angle glaucoma (JOAG). In this study, we identified two new mutations (Asp380Ala and Ser502Pro) in two British families and another (Pro370Leu) in a French-Canadian family. These mutations were not present in a total of 106 normal chromosomes. In another Turkish family with JOAG, we also detected a sequence variant that was proven to be an amino acid polymorphism (Arg76Lys). No other sequence changes were found in the entire coding region and splice junctions of the TIGR/MYOC gene in this family. However, it is still possible that mutations either in the TIGR promoter or in another neighbouring gene could cause glaucoma in this JOAG family. Our results confirm the role of the TIGR/MYOC gene in the aetiology of the JOAG phenotype.