Aurora Sánchez

Prevalence of Y chromosome microdeletions in oligospermic and azoospermic candidates for intracytoplasmic sperm injection

OBJECTIVE To determine the prevalence and type of Y chromosome microdeletions in 136 consecutively seen intracytoplasmic sperm injection (ICSI) candidates and in 50 consecutively seen azoospermic men attending an infertility clinic. DESIGN Controlled clinical study. SETTING Genetics laboratory and infertility clinic at a University hospital. PATIENT(S) One hundred eighty-six men who were seen at an infertility clinic and who were referred to a genetics counseling service for genetic assessment before ICSI. INTERVENTION(S) Collection of semen and blood samples. MAIN OUTCOME MEASURE(S) Semen analysis; serum FSH, LH, and T levels; karyotype analysis; and presence or absence of several single tagged site markers along the Y chromosome (sY274, sY238, sY276, sY84, sY102, sY143, sY153, sY254, sY269, sY202, sY158, sY160). RESULT(S) Yq chromosome microdeletions were detected in 10 (5.4%) of 186 consecutively seen ICSI candidates. The number of microdeletions was much higher in azoospermic patients (16%; 8 of 50) than in oligospermic patients (1.5%; 2 of 136). Two of the azoospermic patients with a Yq microdeletion also had sex chromosome aneuploidy mosaicism. No microdeletions were detected in 100 consecutively seen fathers who were included as controls. CONCLUSION(S) The prevalence of Yq microdeletions in the azoospermic group was much higher than in the oligospermic group and was consistent with the prevalence of Yq microdeletions detected in other series of azoospermic men in different geographic areas. All Yq microdeletions found in our patients belong to the AZFc region, indicating that microdeletions of the AZFa and AZFb regions are infrequent among oligospermic ICSI candidates or azoospermic males in our population.

Gene expression profiles for the human pancreas and purified islets in Type 1 diabetes: new findings at clinical onset and in long-standing diabetes

Type 1 diabetes (T1D) is caused by the selective destruction of the insulin‐producing β cells of the pancreas by an autoimmune response. Due to ethical and practical difficulties, the features of the destructive process are known from a small number of observations, and transcriptomic data are remarkably missing. Here we report whole genome transcript analysis validated by quantitative reverse transcription–polymerase chain reaction (qRT–PCR) and correlated with immunohistological observations for four T1D pancreases (collected 5 days, 9 months, 8 and 10 years after diagnosis) and for purified islets from two of them. Collectively, the expression profile of immune response and inflammatory genes confirmed the current views on the immunopathogenesis of diabetes and showed similarities with other autoimmune diseases; for example, an interferon signature was detected. The data also supported the concept that the autoimmune process is maintained and balanced partially by regeneration and regulatory pathway activation, e.g. non‐classical class I human leucocyte antigen and leucocyte immunoglobulin‐like receptor, subfamily B1 (LILRB1). Changes in gene expression in islets were confined mainly to endocrine and neural genes, some of which are T1D autoantigens. By contrast, these islets showed only a few overexpressed immune system genes, among which bioinformatic analysis pointed to chemokine (C‐C motif) receptor 5 (CCR5) and chemokine (CXC motif) receptor 4) (CXCR4) chemokine pathway activation. Remarkably, the expression of genes of innate immunity, complement, chemokines, immunoglobulin and regeneration genes was maintained or even increased in the long‐standing cases. Transcriptomic data favour the view that T1D is caused by a chronic inflammatory process with a strong participation of innate immunity that progresses in spite of the regulatory and regenerative mechanisms.

Cytogenetic study of spontaneous abortions using semi‐direct analysis of chorionic villi samples detects the broadest spectrum of chromosome abnormalities

Conventional tissue culturing and karyotyping of spontaneous abortions has limitations such as culture failure, external contamination and selective growth of maternal cells. Molecular cytogenetic techniques such as FISH, QF‐PCR, and CGH allow diagnosis on uncultured cells but are also limited as to the spectrum of cytogenetic abnormalities detected. We describe the cytogenetic findings in a series of 116 first trimester arrested pregnancies, obtained through chorionic villi sampling (CVS) and semi‐direct analysis that avoids some of the long‐culture pitfalls such as maternal contamination, and compare our results with those that would have been obtained theoretically using molecular cytogenetic techniques. Samples were obtained by transcervical CVS from women with a diagnosis of missed abortion, most of them referred for cytogenetic prenatal diagnosis. Cytogenetic analysis was performed using semi‐direct technique. A karyotype was obtained in 103 cases. Eighty‐two abnormal karyotypes were found (80%), including 12 triploidies, 10 monosomies, 61 trisomies, and 9 structural abnormalities; a double abnormality being present in 10 cases. Between 10% and 50% of our abnormal results would have been missed using the most common molecular cytogenetic techniques. Semi‐direct analysis of CVS may still be considered as a comprehensive, reasonably rapid, cost‐effective and reliable method for detecting the broadest spectrum of chromosome abnormalities in missed abortions.

X-chromosome tiling path array detection of copy number variants in patients with chromosome X-linked mental retardation

BackgroundAproximately 5–10% of cases of mental retardation in males are due to copy number variations (CNV) on the X chromosome. Novel technologies, such as array comparative genomic hybridization (aCGH), may help to uncover cryptic rearrangements in X-linked mental retardation (XLMR) patients. We have constructed an X-chromosome tiling path array using bacterial artificial chromosomes (BACs) and validated it using samples with cytogenetically defined copy number changes. We have studied 54 patients with idiopathic mental retardation and 20 controls subjects.ResultsKnown genomic aberrations were reliably detected on the array and eight novel submicroscopic imbalances, likely causative for the mental retardation (MR) phenotype, were detected. Putatively pathogenic rearrangements included three deletions and five duplications (ranging between 82 kb to one Mb), all but two affecting genes previously known to be responsible for XLMR. Additionally, we describe different CNV regions with significant different frequencies in XLMR and control subjects (44% vs. 20%).ConclusionThis tiling path array of the human X chromosome has proven successful for the detection and characterization of known rearrangements and novel CNVs in XLMR patients.

CDKN1C (p57Kip2) analysis in Beckwith–Wiedemann syndrome (BWS) patients: Genotype–phenotype correlations, novel mutations, and polymorphisms

Beckwith–Wiedemann syndrome (BWS) is an overgrowth syndrome characterized by macroglossia, macrosomia, and abdominal wall defects. It is a multigenic disorder caused in most patients by alterations in growth regulatory genes. A small number of individuals with BWS (5–10%) have mutations in CDKN1C, a cyclin‐dependent kinase inhibitor of G1 cyclin complexes that functions as a negative regulator of cellular growth and proliferation. Here, we report on eight patients with BWS and CDKN1C mutations and review previous reported cases. We analyzed 72 patients (50 BWS, 17 with isolated hemihyperplasia (IH), three with omphalocele, and two with macroglossia) for CDKN1C defects with the aim to search for new mutations and to define genotype–phenotype correlations. Our findings suggest that BWS patients with CDKN1C mutations have a different pattern of clinical malformations than those with other molecular defects. Polydactyly, genital abnormalities, extra nipple, and cleft palate are more frequently observed in BWS with mutations in CDKN1C. The clinical observation of these malformations may help to decide which genetic characterization should be undertaken (i.e., CDKN1C screening), thus optimizing the laboratory evaluation for BWS.

Implications of the FMR1 gene in menopause: study of 147 Spanish women.

Objective To evaluate the relationship between the FMR1 premutation and premature ovarian failure (POF) in the Spanish population and the possible incorporation of this test in gynecological procedures for women with POF or early menopause (EM). Design Clinical and molecular genetic study. Ninety-eight premutated and six full-mutated carriers of fragile X syndrome and 43 women with POF were studied by polymerase chain reaction and Southern blot analysis for the CGG repeat expansion in the FMR1 gene. Results Among premutated carriers, 12.2% (12 of 98) presented with POF, and 15.3% (15 of 98) presented with EM. Neither POF nor EM was observed in any of the six full-mutated women. Two women of 43 from the POF population (4.65%) were carriers for the CGG premutation in the FMR1 gene. No correlation between CGG expansion size and age at menopause was found. A biased paternal origin of the premutation and a high twinning incidence was found in all premutated women, whether they had POF or not. Conclusions Our data support the hypothesis that the FMR1 gene is one of the genes associated with POF and EM. Analysis of the CGG expansion in the FMR1 gene may be justified in women with POF and EM until the real role of the FMR1 premutation is determined.

Antisense therapeutics for neurofibromatosis type 1 caused by deep intronic mutations

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder affecting 1:3,500 individuals. Disease expression is highly variable and complications are diverse. However, currently there is no specific treatment for the disease. NF1 is caused by mutations in the NF1 gene, approximately 2.1% of constitutional mutations identified in our population are deep intronic mutations producing the insertion of a cryptic exon into the mature mRNA. We used antisense morpholino oligomers (AMOs) to restore normal splicing in primary fibroblast and lymphocyte cell lines derived from six NF1 patients bearing three deep intronic mutations in the NF1 gene (c.288+2025T>G, c.5749+332A>G, and c.7908‐321C>G). AMOs were designed to target the newly created 5′ splice sites to prevent the incorporation of cryptic exons. Our results demonstrate that AMO treatment effectively restored normal NF1 splicing at the mRNA level for the three mutations studied in the different cell lines analyzed. We also found that AMOs had a rapid effect that lasted for several days, acting in a sequence‐specific manner and interfering with the splicing mechanism. Finally, to test whether the correction of aberrant NF1 splicing also restored neurofibromin function to wild‐type levels, we measured the amount of Ras‐GTP after AMO treatment in primary fibroblasts. The results clearly show an AMO‐dependent decrease in Ras‐GTP levels, which is consistent with the restoration of neurofibromin function. To our knowledge this is the first time that an antisense technique has been usedsuccessfully to correct NF1 mutations opening the possibility of a therapeutic strategy for this type of mutation not only for NF1 but for other genetic disorders. Hum Mutat 30, 454–462, 2009.

MLPA as first screening method for the detection of microduplications and microdeletions in patients with X-linked mental retardation

Purpose: Routine protocols for the study of mental retardation include karyotype, analysis for fragile X syndrome, and subtelomeric rearrangements. Nevertheless, detection of cryptic rearrangements requires more sensitive techniques. Mutation screening in all known genes responsible for X-linked mental retardation is not feasible, and linkage analysis is sometimes limited. Multiplex ligation probe amplification is a recently developed technique based on the amplification of specific probes that allows relative quantification of 40 to 46 different target DNA sequences in a single reaction.Methods: In the present study, we assessed multiplex ligation probe amplification for the detection of microduplications/microdeletions in 80 male patients with suspicion of X-linked mental retardation.Results: We detected four copy number aberrations (5%): three duplications (GDI1, RPS6KA3, and ARHGEF6) and one deletion (OPHN1). All these changes were confirmed by other molecular techniques, and patients were clinically re-evaluated.Conclusions: We strongly recommend the use of multiplex ligation probe amplification as a first screening method for the detection of copy number aberrations in patients with mental retardation because of its cost-effectiveness.

Screening for FMR1 and FMR2 mutations in 222 individuals from Spanish special schools: identification of a case of FRAXE-associated mental retardation

Abstract Fragile X syndrome is the most common inherited form of familial mental retardation. It results from a (CGG)n trinucleotide expansion in the FMR1 gene leading to the typical Martin-Bell phenotype. Clinical features vary depending on age and sex. Expansion of a (CCG)n repeat in the FMR2 gene corresponds to the FRAXE fragile site which lies distal to FRAXA and is also associated with mental retardation, but it is less frequent and lacks a consistent phenotype. Analysis of repeat expansions in these two genes allows the molecular diagnosis of these different entities. We report here the screening of the FRAXA and FRAXE mutations in 222 unrelated mentally retarded individuals attending Spanish special schools. PCR and/or Southern blotting methods were used. We detected full mutations in the FMR1 gene in 11 boys (4.9%) and 1 boy (0.5%) with a CCG repeat expansion in the FMR2 gene. The latter shows mild mental retardation with psychotic behaviour and no remarkable physical traits. Molecular studies revealed a mosaicism for methylation in the FMR2 gene. This case supports the observation that expansions greater than 100 repeats can be partially methylated and cause the phenotype.

Molecular analysis of the (CGG)n expansion in the FMR-1 gene in 59 Spanish fragile X syndrome families

The fragile X mental retardation syndrome is caused by an expansion of a trinucleotide repeat (CGG)n in the FMR-1 gene. Molecular genetic study of fragile X provides accurate diagnosis and facilitates genetic counseling in families with affected members. We present here the molecular study of 59 Spanish fragile X syndrome families using probe StB 12.3 and the polymerase chain reaction (PCR) of the (CGG)n repeat sequence of the FMR-1 gene. The results obtained have allowed us to characterize 455 individuals, including eight prenatal diagnoses. The clinical diagnosis of fragile X in 89 affected males was confirmed, 137 female carriers were identified (48 of whom were mentally retarded), 176 individuals “at risk” were found not to have the expansion, and 12 cases of normal transmitting males (NTM) were detected. In the sample studied, no de novo mutations were detected, nor any mutation different from that described for the (CGG)n expansion. One nonmentally retarded male was detected as having an unmethylated CpG island for the FMR-1 gene, but with more than 200 CGG repeats (high functioning male). The analysis of the (CGG)n repeat in 208 normal chromosomes gave an allele distribution similar to that in other Caucasoid population groups, with alleles of 29 and 30 CGG repeats accounting for 46% of the chromosomes. The combination of Southern analysis and PCR of the (CGG)n repeat is highly efficient for diagnosis, compared with cytogenetic techniques, especially in the detection of female carriers, NTMs, and prenatal diagnosis, enabling accurate genetic counseling to be provided in all cases.