Fertility defects revealing germline biallelic nonsense NBN mutations

Biallelic mutations in the NBN/NBS1 gene are the cause of Nijmegen breakage syndrome (NBS), a severe pediatric disease characterized by dysmorphy with a bird‐like face, microcephaly, growth retardation, immune deficiency, and proneness to cancer. We here report two adult siblings that are compound heterozygotes for two previously unreported NBN nonsense mutations. These patients presented with the unique clinical symptom of fertility defects. Contrasting with the absence of any developmental abnormality, biological analyses revealed defects similar to those observed in NBS patients, including chromosomal instability, cellular hyperradiosensitivity and checkpoint defects as measured by radioresistant DNA synthesis (RDS). NBN mutations should thus be considered a new cause of infertility, and should be searched for if associated with the biological abnormalities of NBS. Hum Mutat 0, 1–7, 2008. © 2008 Wiley‐Liss, Inc.     

Human RAD50 deficiency: Confirmation of a distinctive phenotype

DNA double‐strand breaks (DSBs) are highly toxic DNA lesions that can lead to chromosomal instability, loss of genes and cancer. The MRE11/RAD50/NBN (MRN) complex is keystone involved in signaling processes inducing the repair of DSB by, for example, in activating pathways leading to homologous recombination repair and nonhomologous end joining. Additionally, the MRN complex also plays an important role in the maintenance of telomeres and can act as a stabilizer at replication forks. Mutations in NBN and MRE11 are associated with Nijmegen breakage syndrome (NBS) and ataxia telangiectasia (AT)‐like disorder, respectively. So far, only one single patient with biallelic loss of function variants in RAD50 has been reported presenting with features classified as NBS‐like disorder. Here, we report a long‐term follow‐up of an unrelated patient with facial dysmorphisms, microcephaly, skeletal features, and short stature who is homozygous for a novel variant in RAD50. We could show that this variant, c.2524G > A in exon 15 of the RAD50 gene, induces aberrant splicing of RAD50 mRNA mainly leading to premature protein truncation and thereby, most likely, to loss of RAD50 function. Using patient‐derived primary fibroblasts, we could show abnormal radioresistant DNA synthesis confirming pathogenicity of the identified variant. Immunoblotting experiments showed strongly reduced protein levels of RAD50 in the patient‐derived fibroblasts and provided evidence for a markedly reduced radiation‐induced AT‐mutated signaling. Comparison with the previously reported case and with patients presenting with NBS confirms that RAD50 mutations lead to a similar, but distinctive phenotype.     

Mutation analysis of tubulin beta 8 class VIII in infertile females with oocyte or embryonic defects

Variants of tubulin beta 8 class VIII (TUBB8) have been shown to be associated with female infertility characterized by oocyte or embryonic defects. To further investigate the mutational spectrum of TUBB8 and the prevalence of variants, we performed Sanger sequencing of TUBB8 on a total of 115 infertile females who had undergone repeated in vitro fertilization cycles with oocyte or embryonic defects and 200 healthy controls. A total of 31 variants which were absent from the controls were identified in 36 unrelated individuals, accounting for a large proportion of this cohort (31.3%). All of the variants including heterozygous/homozygous missense variants and a heterozygous frameshift insertion variant were at conserved sites and predicted to be deleterious. Besides, these variants had diverse phenotypic effects, including not only oocyte maturation arrest, fertilization failure, and early embryonic arrest, but also multi‐pronuclei (MPN) formation, which is a new phenotype associated with TUBB8 variants. Overall, this study reveals a large number of variants of the TUBB8 gene in infertile females with oocyte or embryonic defects. Our results not only broaden the mutational and phenotypic spectra of TUBB8 variants, but also further confirm the critical role of TUBB8 in oocyte maturation, fertilization, and early embryonic development.     

Novel homozygous truncating variants in ZMYND15 causing severe oligozoospermia and their implications for male infertility

Sequence variants of ZMYND15 cause azoospermia in humans, but they have not yet been reported in infertile men with severe oligozoospermia (SO). We performed whole‐exome and Sanger sequencing to identify suspected causative variants in 414 idiopathic participating infertile men with SO or azoospermia. Three novel homozygous truncating variants in ZMYND15 were identified in three of the 219 (1.37%) unrelated patients with SO, including c.1209T>A(p.Tyr403*), c.1650delC (p.Glu551Lysfs*75), and c.1622_1636delinsCCAC (p.Leu541Profs*39). In silico bioinformatic analyses as well as in vivo and in vitro experiments showed that the ZMYND15 variants carried by the affected subjects might be the underlying cause for their infertility. One patient accepted intracytoplasmic sperm injection therapy, using his ejaculated sperm, and his wife successfully became pregnant. Our findings expand the disease phenotype spectrum by indicating that ZMYND15 variants cause SO and male infertility and suggest a possible correlation between the severity of male infertility caused by ZMYND15 variants and male age.     

Three new cases of ataxia‐telangiectasia‐like disorder: No impairment of the ATM pathway,but S‐phase checkpoint defect

Ataxia‐telangiectasia‐like disorder (ATLD) is a rare genomic instability syndrome caused by biallelic variants of MRE11 (meiotic recombination 11) characterized by progressive cerebellar ataxia and typical karyotype abnormalities. These symptoms are common to those of ataxia‐telangiectasia, which is consistent with the key role of MRE11 in ataxia‐telangiectasia mutated (ATM) activation after DNA double‐strand breaks. Three unrelated French patients were referred with ataxia. Only one had typical karyotype abnormalities. Unreported biallelic MRE11 variants were found in these three cases. Interestingly, one variant (c.424G>A) was present in two cases and haplotype analysis strongly suggested a French founder variant. Variants c.544G>A and c.314+4_314+7del lead to splice defects. The level of MRE11 in lymphoblastoid cell lines was consistently and dramatically reduced. Functional consequences were evaluated on activation of the ATM pathway via phosphorylation of ATM targets (KAP1 and CHK2), but no consistent defect was observed. However, an S‐phase checkpoint activation defect after camptothecin was observed in these patients with ATLD. In conclusion, we report the first three French ATLD patients and a French founder variant, and propose an S‐phase checkpoint activation study to evaluate the pathogenicity of MRE11 variants.     

Functional characterization of the first missense variant in CEP78, a founder allele associated with cone‐rod dystrophy,hearing loss,and reduced male fertility

Inactivating variants in the centrosomal CEP78 gene have been found in cone‐rod dystrophy with hearing loss (CRDHL), a particular phenotype distinct from Usher syndrome. Here, we identified and functionally characterized the first CEP78 missense variant c.449T>C, p.(Leu150Ser) in three CRDHL families. The variant was found in a biallelic state in two Belgian families and in a compound heterozygous state—in trans with c.1462‐1G>T—in a third German family. Haplotype reconstruction showed a founder effect. Homology modeling revealed a detrimental effect of p.(Leu150Ser) on protein stability, which was corroborated in patients' fibroblasts. Elongated primary cilia without clear ultrastructural abnormalities in sperm or nasal brushes suggest impaired cilia assembly. Two affected males from different families displayed sperm abnormalities causing infertility. One of these is a heterozygous carrier of a complex allele in SPAG17, a ciliary gene previously associated with autosomal recessive male infertility. Taken together, our data indicate that a missense founder allele in CEP78 underlies the same sensorineural CRDHL phenotype previously associated with inactivating variants. Interestingly, the CEP78 phenotype has been possibly expanded with male infertility. Finally, CEP78 loss‐of‐function variants may have an underestimated role in misdiagnosed Usher syndrome, with or without sperm abnormalities.     

Type I Procollagen C‐Propeptide Defects: Study of Genotype–Phenotype Correlation and Predictive Role of Crystal Structure

The type I procollagen carboxyterminal(C‐)propeptides are crucial in directing correct assembly of the procollagen heterotrimers. Defects in these domains have anecdotally been reported in patients with Osteogenesis Imperfecta (OI) and few genotype–phenotype correlations have been described. To gain insight in the functional consequences of C‐propeptide defects, we performed a systematic review of clinical, molecular, and biochemical findings in all patients in whom we identified a type I procollagen C‐propeptide defect, and compared this with literature data. We report 30 unique type I procollagen C‐propeptide variants, 24 of which are novel. The outcome of COL1A1 nonsense and frameshift variants depends on the location of the premature termination codon. Those located prior to 50–55 nucleotides upstream of the most 3’ exon–exon junction lead to nonsense‐mediated mRNA decay (NMD) and cause mild OI. Those located beyond this boundary escape NMD, generally lead to production of stable, overmodified procollagen chains, which may partly be retained intracellularly, and are usually associated with severe‐to‐lethal OI. Proα1(I)‐C‐propeptide defects that permit chain association result in more severe phenotypes than those inhibiting chain association. We demonstrate that the crystal structure of the proα1(III)‐C‐propeptide is a reliable tool to predict phenotypic severity for most COL1A1‐C‐propeptide missense variants, whereas for COL1A2‐C‐propeptide variants, the phenotypic outcome is milder than predicted.     

NBS1 directly activates ATR independently of MRE11 and TOPBP1

NBS1 plays unique and essential roles in ATM activation in response to DNA double‐strand breaks. We found that CHK1 phosphorylation and FANCD2 ubiquitination induced by various DNA replication‐stalling agents were abrogated in Nbs1 knockout DT40 cells but not in conditional Mre11 knockout cells, indicating an MRE11‐independent role for NBS1 in ATR activation. The results of in vitro ATR kinase assay indicated that the N‐terminal region of NBS1 directly activates ATR independently of TOPBP1, consistent with the findings that this region of NBS1 directly interacts with ATR. This conclusion was furthermore supported by the results of in vivo experiments; the expression of the N‐terminal region of NBS1 fused to PCNA induces ATR activation in Rad17 knockout cells, and the expression of the ATR activation domain of TOPBP1 fused to PCNA induces ATR activation in Nbs1 knockout cells. These results therefore indicate that NBS1 and TOPBP1 have the potential to activate ATR independently, although both are required for functional activation of ATR in vivo.     

A novel phenotypic marker for ATM‐deficient 129S6/SvEvTac‐ATMtm1Awb/J mice

Ataxia‐telangiectasia (A‐T) is a human autosomal recessive disorder characterized by neuronal degeneration as well as many other physiological and somatic defects. ATM (A‐T, mutated), the gene mutated in A‐T, encodes a 370 kDa protein kinase. ATM knockout mouse models (ATM^?/?) show growth retardation, infertility, neurological dysfunction, defects in T‐lymphocytes, and extreme sensitivity to ionizing radiation. We have recently established multiple ATM^+/? breeding pairs and discovered that all ATM^?/? offspring exhibit a nonpigmented section of tail, usually at or near the tip. To our knowledge, this is the first time that a phenotype of nonpigmented tail has been reported in ATM^?/? knockout mice. We believe that the sections of nonpigmented tail of 129S6/SvEvTac‐ATM^tm1Awb/J mice provide a novel phenotypic marker for research using this ATM knockout mouse model. Results from histochemistry and immunoblotting analysis further demonstrate that while melanocyte precursors or melanoblasts are present in the nonpigmented tail tissue of ATM^?/? mice, they fail to differentiate fully into mature melanocytes. The potential connection between this phenotype and other clinical symptoms caused by ATM deficiency, such as progressive neuronal degeneration, is discussed in this article. Anat Rec, 2007. © 2007 Wiley‐Liss, Inc.     

Genotype–phenotype correlations in individuals with pathogenic RERE variants

Heterozygous variants in the arginine‐glutamic acid dipeptide repeats gene (RERE) have been shown to cause neurodevelopmental disorder with or without anomalies of the brain, eye, or heart (NEDBEH). Here, we report nine individuals with NEDBEH who carry partial deletions or deleterious sequence variants in RERE. These variants were found to be de novo in all cases in which parental samples were available. An analysis of data from individuals with NEDBEH suggests that point mutations affecting the Atrophin‐1 domain of RERE are associated with an increased risk of structural eye defects, congenital heart defects, renal anomalies, and sensorineural hearing loss when compared with loss‐of‐function variants that are likely to lead to haploinsufficiency. A high percentage of RERE pathogenic variants affect a histidine‐rich region in the Atrophin‐1 domain. We have also identified a recurrent two‐amino‐acid duplication in this region that is associated with the development of a CHARGE syndrome‐like phenotype. We conclude that mutations affecting RERE result in a spectrum of clinical phenotypes. Genotype–phenotype correlations exist and can be used to guide medical decision making. Consideration should also be given to screening for RERE variants in individuals who fulfill diagnostic criteria for CHARGE syndrome but do not carry pathogenic variants in CHD7.     

Nijmegen breakage syndrome (NBS) with neurological abnormalities and without chromosomal instability

Background

Nijmegen breakage syndrome (NBS) is an autosomal recessive chromosomal instability disorder with hypersensitivity to ionising radiation. The clinical phenotype is characterised by congenital microcephaly, mild dysmorphic facial appearance, growth retardation, immunodeficiency, and greatly increased risk for lymphoreticular malignancy. Most NBS patients are of Slavic origin and homozygous for the founder mutation 657del5. The frequency of 657del5 heterozygotes in the Czech population is 1:150. Recently, another NBS1 mutation, 643C>T(R215W), with uncertain pathogenicity was found to have higher frequency among tumour patients of Slavic origin than in controls. This alteration results in the substitution of the basic amino acid arginine with the non‐polar tryptophan and thus could potentially interfere with the function of the NBS1 protein, nibrin.

Methods and Results

Children with congenital microcephaly are routinely tested for the 657del5 mutation in the Czech and Slovak Republics. Here, we describe for the first time a severe form of NBS without chromosomal instability in monozygotic twin brothers with profound congenital microcephaly and developmental delay who are compound heterozygotes for the 657del5 and 643C>T(R215W) NBS1 mutations. Both children showed reduced expression of full length nibrin when compared with a control and a heterozygote for the 657del5 mutation. Radiation response processes such as phosphorylation of ATM and phosphorylation/stabilisation of p53, which are promoted by NBS1, are strongly reduced in cells from these patients.

Conclusions

Interestingly, the patients are more severely affected than classical NBS patients. Consequently, we postulate that homozygosity for the 643C>T(R215W) mutation will also lead to a, possibly very, severe disease phenotype.     

Null variants and deletions in BRWD3 cause an X‐linked syndrome of mild–moderate intellectual disability,macrocephaly, and obesity: A series of 17 patients

BRWD3 has been described as a cause of X‐linked intellectual disability, but relatively little is known about the specific phenotype. We report the largest BRWD3 patient series to date, comprising 17 males with 12 distinct null variants and 2 partial gene deletions. All patients presented with intellectual disability, which was classified as moderate (65%) or mild (35%). Behavioral issues were present in 75% of patients, including aggressive behavior, attention deficit/hyperactivity and/or autistic spectrum disorders. Mean head circumference was +2.8 SD (2.8 standard deviations above the mean), and mean BMI was +2.0 SD (in the context of a mean height of +1.3 SD), indicating a predominant macrocephaly/obesity phenotype. Shared facial features included a tall chin, prognathism, broad forehead, and prominent supraorbital ridge. Additional features, reported in a minority (<30%) of patients included cryptorchidism, neonatal hypotonia, and small joint hypermobility. This study delineates the clinical features associated with BRWD3 null variants and partial gene deletions, and suggests that BRWD3 should be included in the differential diagnosis of patients with an overgrowth‐intellectual disability (OGID) phenotype, particularly in male patients with a mild or moderate intellectual disability associated with macrocephaly and/or obesity.     

Deletions Overlapping VCAN Exon 8 Are New Molecular Defects for Wagner Disease

Wagner disease is a rare nonsyndromic autosomal‐dominant vitreoretinopathy, associated with splice mutations specifically targeting VCAN exon 8. We report the extensive genetic analysis of two Wagner probands, previously found negative for disease‐associated splice mutations. Next‐generation sequencing (NGS), quantitative real‐time PCR, and long‐range PCR identified two deletions (3.4 and 10.5 kb) removing at least one exon–intron boundary of exon 8, and both correlating with an imbalance of VCAN mRNA isoforms. We showed that the 10.5‐kb deletion occurred de novo, causing somatic mosaicism in the proband's mother who had an unusually mild asymmetrical phenotype. Therefore, exon 8 deletions are novel VCAN genetic defects responsible for Wagner disease, and VCAN mosaic mutations may be involved in the pathogenesis of Wagner disease with attenuated phenotype. NGS is then an effective screening tool for genetic diagnosis of Wagner disease, improving the chance of identifying all disease‐causative variants as well as mosaic mutations in VCAN.     

Transferrin Receptor (CD71) Expression in Peritoneal Macrophages from Fertile and Infertile Women With and Without Endometriosis

PROBLEM: Hyperactivated macrophages are implicated in the pathophysiology of endometriosis-associated infertility. This study investigates transferrin receptor expression (CD71) as a marker of hyperactivity in peritoneal macrophages of infertile patients with minimal to mild endometriosis (group 1, n = 25). METHOD OF STUDY: Expression of the activation antigen CD71 on peritoneal fluid macrophages was determined by a specific monoclonal anti-CD71 antibody using indirect immunofluorescence technique and was analyzed by flow cytometry. Three different control groups of women were used: women with unexplained infertility (group 2, n = 25), fertile women with endometriosis (group 3, n =10), and fertile women without endometriosis (group 4, n = 25). RESULTS: The percentage of CD71 positive cells was significantly increased in infertile women with endometriosis as compared with the three control groups. There were no differences among groups 2, 3, and 4 with respect to the percentage of CD71 positive macrophages. CONCLUSIONS: Our results favor the concept that hyperactivated macrophages play a role in the pathophysiology of endometriosis-associated subfertility, a feature which is lacking in patients with unexplained infertility.     

CSGALNACT1‐congenital disorder of glycosylation: A mild skeletal dysplasia with advanced bone age

Congenital disorders of glycosylation (CDGs) comprise a large number of inherited metabolic defects that affect the biosynthesis and attachment of glycans. CDGs manifest as a broad spectrum of disease, most often including neurodevelopmental and skeletal abnormalities and skin laxity. Two patients with biallelic CSGALNACT1 variants and a mild skeletal dysplasia have been described previously. We investigated two unrelated patients presenting with short stature with advanced bone age, facial dysmorphism, and mild language delay, in whom trio‐exome sequencing identified novel biallelic CSGALNACT1 variants: compound heterozygosity for c.1294G>T (p.Asp432Tyr) and the deletion of exon 4 that includes the start codon in one patient, and homozygosity for c.791A>G (p.Asn264Ser) in the other patient. CSGALNACT1 encodes CSGalNAcT‐1, a key enzyme in the biosynthesis of sulfated glycosaminoglycans chondroitin and dermatan sulfate. Biochemical studies demonstrated significantly reduced CSGalNAcT‐1 activity of the novel missense variants, as reported previously for the p.Pro384Arg variant. Altered levels of chondroitin, dermatan, and heparan sulfate moieties were observed in patients’ fibroblasts compared to controls. Our data indicate that biallelic loss‐of‐function mutations in CSGALNACT1 disturb glycosaminoglycan synthesis and cause a mild skeletal dysplasia with advanced bone age, CSGALNACT1‐CDG.     

A possible mechanism of autoimmune-mediated infertility in women with endometriosis

Citation Inagaki J, Hao L, Nakatsuka M, Yasuda T, Hiramatsu Y, Shoenfeld Y, Matsuura E. A possible mechanism of autoimmune‐mediated infertility in women with endometriosis. Am J Reprod Immunol 2011; 66: 90–99 Problem Endometriosis has been proposed to be an autoimmune disease because of the presence of a variety of autoantibodies specific for endometrial or ovarian antigens. The object of the present study is to characterize binding specificity of anti‐laminin‐111 autoantibodies in infertile patients with endometriosis and to investigate whether these autoantibodies affect the in vitro embryo development. Method of study An ELISA analysis using overlapping synthesized peptides that covered the entire G domain of laminin‐α1 chain was performed in infertile patients with endometriosis (n = 45). Mouse blastocysts were cultured in media containing the purified IgG from one antibody‐positive serum on laminin‐111‐coated dishes. Results Anti‐laminin‐111 autoantibodies were directed to several particular biologically functional peptide sequences in laminin‐α 1 chain G domain. The tested IgG significantly inhibited the extent of in vitro trophoblast outgrowth. Conclusion Anti‐laminin‐111 autoantibodies may have major pathogenic roles on early reproductive failure including endometriosis‐associated infertility.