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Background: 15q11.2 deletions and duplications have been linked to autism spectrum disorder, schizophrenia, and intellectual disability. Recent evidence suggests that dysfunctional CYFIP1 (cytoplasmic FMR1 interacting protein 1) contributes to the clinical phenotypes observed in individuals with 15q11.2 deletion/duplication syndrome. CYFIP1 plays crucial roles in neuronal development and brain connectivity, promoting actin polymerization and regulating local protein synthesis. However, information about the impact of single nucleotide variants in CYFIP1 on neurodevelopmental disorders is limited., Methods: Here, we report a family with 2 probands exhibiting intellectual disability, autism spectrum disorder, spastic tetraparesis, and brain morphology defects and who carry biallelic missense point mutations in the CYFIP1 gene. We used skin fibroblasts from one of the probands, the parents, and typically developing individuals to investigate the effect of the variants on the functionality of CYFIP1. In addition, we generated Drosophila knockin mutants to address the effect of the variants in vivo and gain insight into the molecular mechanism that underlies the clinical phenotype., Results: Our study revealed that the 2 missense variants are in protein domains responsible for maintaining the interaction within the wave regulatory complex. Molecular and cellular analyses in skin fibroblasts from one proband showed deficits in actin polymerization. The fly model for these mutations exhibited abnormal brain morphology and F-actin loss and recapitulated the core behavioral symptoms, such as deficits in social interaction and motor coordination., Conclusions: Our findings suggest that the 2 CYFIP1 variants contribute to the clinical phenotype in the probands that reflects deficits in actin-mediated brain development processes., (Copyright © 2023. Published by Elsevier Inc.)

Background: The majority of variants of unknown clinical significance (VUCS) in the CFTR gene are missense variants. While change on the CFTR protein structure or function is often suspected, impact on splicing may be neglected. Such undetected splicing default of variants may complicate the interpretation of genetic analyses and the use of an appropriate pharmacotherapy., Methods: We selected 15 variants suspected to impact CFTR splicing after in silico predictions on 319 missense variants (214 VUCS), reported in the CFTR-France database. Six specialized laboratories assessed the impact of nucleotide substitutions on splicing (minigenes), mRNA expression levels (quantitative PCR), synthesis and maturation (western blot), cellular localization (immunofluorescence) and channel function (patch clamp) of the CFTR protein. We also studied maturation and function of the truncated protein, consecutive to in-frame aberrant splicing, on additional plasmid constructs., Results: Six of the 15 variants had a major impact on CFTR splicing by in-frame (n = 3) or out-of-frame (n = 3) exon skipping. We reclassified variants into: splicing variants; variants causing a splicing defect and the impairment of CFTR folding and/or function related to the amino acid substitution; deleterious missense variants that impair CFTR folding and/or function; and variants with no consequence on the different processes tested., Conclusion: The 15 variants have been reclassified by our comprehensive approach of in vitro experiments that should be used to properly interpret very rare exonic variants of the CFTR gene. Targeted therapies may thus be adapted to the molecular defects regarding the results of laboratory experiments., Competing Interests: Conflict of Interest Statement The authors declare no conflicts of interest., (Copyright © 2022 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Purpose: To report the effect of the ketogenic diet (KD) on non-convulsive status epilepticus (NCSE) due to Angelman syndrome (AS) in two members of a large Georgian family affected by a novel frameshift variant in the UBE3A gene (NM_000462.3)., Methods: We evaluated two members of this family who were affected with clinical and EEG features of AS. Clinical history with special emphasis on development, seizure type, frequency, and treatment was reviewed. Routine and long-term video EEG monitoring were conducted, particularly during NCSE. A non-fasting inpatient KD protocol was implemented using blended food orally with full administration of 4:1 (fat to non-fat) ratio. Urine ketone bodies (KBs), measured with urine ketone acetone strips readings, reached 150 mg/dL in both patients., Results: Patients had characteristic signs of AS and presented with epilepsy between the age of 2-4 years. As methylation tests were negative, next generation sequencing disclosed a c.2365del variant. For both, NCSE was revealed by cognitive deterioration and did not respond to anti-seizure medication. As recommended, IV pyridoxine, benzodiazepines, and valproic acid were administered, but without success. For both patients, NCSE resolved on the second-third day of KD initiation, before the appearance of ketonuria and resulting in improved communication, mood and sleep., Conclusion: KD is safe and effective for the treatment of NCSE due to AS. Resolution before the appearance of ketone bodies points to a possible mechanism of action of KD., (Copyright © 2021 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.)

Purpose: DYRK1A syndrome is among the most frequent monogenic forms of intellectual disability (ID). We refined the molecular and clinical description of this disorder and developed tools to improve interpretation of missense variants, which remains a major challenge in human genetics., Methods: We reported clinical and molecular data for 50 individuals with ID harboring DYRK1A variants and developed (1) a specific DYRK1A clinical score; (2) amino acid conservation data generated from 100 DYRK1A sequences across different taxa; (3) in vitro overexpression assays to study level, cellular localization, and kinase activity of DYRK1A mutant proteins; and (4) a specific blood DNA methylation signature., Results: This integrative approach was successful to reclassify several variants as pathogenic. However, we questioned the involvement of some others, such as p.Thr588Asn, still reported as likely pathogenic, and showed it does not cause an obvious phenotype in mice., Conclusion: Our study demonstrated the need for caution when interpreting variants in DYRK1A, even those occurring de novo. The tools developed will be useful to interpret accurately the variants identified in the future in this gene., (© 2021. The Author(s), under exclusive licence to the American College of Medical Genetics and Genomics.)

Background: Major issues of newborn screening (NBS) for CF are the assessment of disease liability of variants and of the penetrance of clinical CF, notably in inconclusive diagnosis. The penetrance of CF is defined as the risk of a particular genotype to lead to a CF phenotype., Methods: We aimed to get insight into the penetrance of CF for fifteen CFTR variants: 5 frequent CF-causing and 10 classified as of varying clinical consequence (VCC) or associated with a CFTR-related disorder (CFTR-RD) in CFTR2 or CFTR-France databases. The penetrance was approached by: (1) comparison of variant allelic frequencies in CF patients (CFTR2) and in the general population; (2) estimation of the likelihood of a positive NBS test for the 14 compound heterozygous with F508del and the F508del homozygous genotypes, defined as the ratio of detected/expected number of neonates with a given genotype in the 2002-2017 period., Results: A full penetrance was observed for severe CF-causing variants. Five variants were more frequently found in the general population than in CF patients: TG11T5, TG12T5, TG13T5, L997F and R117H;T7. The likelihood of a positive NBS test was 0.03% for TG11T5, 0.3% for TG12T5, 1.9% for TG13T5, 0.6% for L997F, 11.7% for D1152H, and 17.8% for R117H;T7. Penetrance varied greatly for variants with discrepant classification between CFTR2 and CFTR-France: 5.1% for R117C, 12.3% for T338I, 43.5% for D110H and 52.6% for L206W., Conclusion: These results illustrate the contribution of genetics population data to assess the disease liability of variants for diagnosis and genetic counselling purposes., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Widespread random monoallelic gene expression (RMAE) effects influence about 10% of human genes. However, the mechanisms by which RME of autosomal genes is established and those by which it is maintained both remain open questions. Because the choice of allelic expression is randomly performed cell-by-cell, the RMAE mechanism is not observable in non-clonal cell populations or in whole tissues. Several target genes of MeCP2, the gene involved in Rett syndrome (RTT), have been previously described as subject to RMAE, suggesting that MeCP2 may be involved in the establishment and/or maintenance of RME of autosomal genes. To improve our knowledge on this largely unknown phenomenon, and to study the role of MeCP2 in RMAE, we compared RMA gene expression profiles in clonal cell cultures expressing wild-type MeCP2 versus mutant MeCP2 from a RTT patient carrying a pathogenic non-sense variant. Our data clearly demonstrated that MeCP2 deficiency does not affect significantly allelic gene expression of X-linked genes, imprinted genes as well as the RMAE profile in the majority of genes. However, the functional deficiency in MeCP2 appeared to disrupt the mono-allelic or the bi-allelic expression of at least 49 genes allowing us to define a specific signature of MECP2 mutated clones., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Cystic fibrosis (CF) is the most frequent genetic disorder in the Caucasian population benefiting from systematic newborn screening tests. It is also the most frequent indication of prenatal and preimplantation genetic diagnosis for a single gene disorder. During the past thirty years, thanks in part to the evolution of diagnostic techniques, our knowledge on CFTR genetics and pathophysiological mechanisms involved in CF have significantly improved. With the implementation of newborn screening in France and in several countries, the diagnosis now often occurs in clinically asymptomatic infants and this has modified the criteria for CF diagnosis. Recently, guidelines for CF diagnosis have been reformulated in Europe and the US, in regard to sweat chloride usual values and disease terminology. This review describes the methods and molecular approaches that are used in routine practice or are being developed to detect CFTR protein dysfunction and to identify disease-causing CFTR variants. Ultimately, an optimal use of all these functional and genetic resources may improve patient care and therapeutic decision-making. © 2020 French Society of Pediatrics. Published by Elsevier Masson SAS. All rights reserved., (© 2020 Elsevier Masson SAS. Tous droits réservés.)

Background: The CFTR genotype remains incomplete in 1% of Cystic Fibrosis (CF) cases, because only one or no disease-causing variants is detected after extended analysis. This fraction is probably higher in CFTR-Related Disorders (CFTR-RD). Deep-intronic CFTR variants are putative candidates to fill this gap. However, the recurrence, phenotypic spectrum and full molecular characterization of newly reported variants are unknown., Methods: Minigenes and analysis of CFTR transcripts in nasal epithelial cells were used to determine the impact on CFTR splicing of intronic variants that we previously identified by next generation sequencing of the whole CFTR locus. Phenotypic data were collected in 19 patients with CF and CFTR-RD, in whom one of the deep intronic variants has been detected., Results: Three deep-intronic variants promoted the inclusion of pseudo-exons (PE) in the CFTR transcript, hindering the synthesis of a functional protein. The c.2989-313A > T variant, detected in four patients with CF or CFTR-RD from three different families, led to the inclusion of a 118 bp PE. The c.3469-1304C > G variant promoted the inclusion of a 214 bp-PE and was identified in five patients with CF from four families. Haplotype analysis confirmed that this variant was associated with one CF chromosome of African origin. The most represented variant in our cohort was the c.3874-4522A > G, detected in 10 patients with various phenotypes, from male infertility to CF with pancreatic insufficiency., Conclusion: These three deep intronic CFTR variants are associated with a large phenotypic spectrum, including typical CF. They should be included in CF diagnostic testing and carrier screening strategies., (Copyright © 2018. Published by Elsevier B.V.)

Purpose: Variants in IQSEC2, escaping X inactivation, cause X-linked intellectual disability with frequent epilepsy in males and females. We aimed to investigate sex-specific differences., Methods: We collected the data of 37 unpublished patients (18 males and 19 females) with IQSEC2 pathogenic variants and 5 individuals with variants of unknown significance and reviewed published variants. We compared variant types and phenotypes in males and females and performed an analysis of IQSEC2 isoforms., Results: IQSEC2 pathogenic variants mainly led to premature truncation and were scattered throughout the longest brain-specific isoform, encoding the synaptic IQSEC2/BRAG1 protein. Variants occurred de novo in females but were either de novo (2/3) or inherited (1/3) in males, with missense variants being predominantly inherited. Developmental delay and intellectual disability were overall more severe in males than in females. Likewise, seizures were more frequently observed and intractable, and started earlier in males than in females. No correlation was observed between the age at seizure onset and severity of intellectual disability or resistance to antiepileptic treatments., Conclusion: This study provides a comprehensive overview of IQSEC2-related encephalopathy in males and females, and suggests that an accurate dosage of IQSEC2 at the synapse is crucial during normal brain development.

Purpose: Although 97-99% of CFTR mutations have been identified, great efforts must be made to detect yet-unidentified mutations., Methods: We developed a small-scale next-generation sequencing approach for reliably and quickly scanning the entire gene, including noncoding regions, to identify new mutations. We applied this approach to 18 samples from patients suffering from cystic fibrosis (CF) in whom only one mutation had hitherto been identified., Results: Using an in-house bioinformatics pipeline, we could rapidly identify a second disease-causing CFTR mutation for 16 of 18 samples. Of them, c.1680-883A>G was found in three unrelated CF patients. Analysis of minigenes and patients' transcripts showed that this mutation results in aberrantly spliced transcripts because of the inclusion of a pseudoexon. It is located only three base pairs from the c.1680-886A>G mutation (1811+1.6kbA>G), the fourth most frequent mutation in southwestern Europe. We next tested the effect of antisense oligonucleotides targeting splice sites on these two mutations on pseudoexon skipping. Oligonucleotide transfection resulted in the restoration of the full-length, in-frame CFTR transcript, demonstrating the effect of antisense oligonucleotide-induced pseudoexon skipping in CF., Conclusion: Our data confirm the importance of analyzing noncoding regions to find unidentified mutations, which is essential to designing targeted therapeutic approaches.

Background: Cystic Fibrosis (CF) is an autosomal recessive disorder implicating the Cystic Fibrosis Transmembrane Regulator (CFTR). Even though CF is mainly considered an inherited monogenic disease, numerous findings over the last few years argue for a more complicated multifactorial disease involving modifier genes. The 19q13.2-19q13.4 region is suspected to contain genetic modifiers that correlate to the severity of CF., Method: Here we studied a cohort of p.F508del patients for potential SNPs in the hsa-miR-99b/hsa-let-7e/hsa-miR-125a cluster, which is found within the 19q13.2-19q13.4 region., Results: Three polymorphisms were identified in the hsa-miR-99b/hsa-let-7e/hsa-miR-125a cluster. Using a cell based model, we analysed whether expression of DeltaF508-CFTR influences the expression of mature hsa-miR-99b, hsa-let-7e, and hsa-miR-125a. We found that hsa-miR-99b and hsa-miR-125a were significantly increased in DeltaF508-CFTR expressing cells. The three miRNAs appear to be derived from the same precursor but differ in their expression levels suggesting differential maturation of these miRNAs in CF. In silico analysis revealed that two out of the three polymorphisms we identified in a CF p.F508del patients cohort could modulate miRNA maturation and therefore impact on hsa-miR-99b/hsa-let-7e/hsa-miR-125a expression levels., Conclusion: Ingenuity Pathway Analysis indicated that hsa-miR-99b and hsa-miR-125a could be associated with the phenotypes manifested by p.F508del patients. Here we provide novel elements in the mechanism of hsa-miR-99b and hsa-miR-125a biogenesis, and for the role of CFTR and DeltaF508-CFTR on the expression of this miRNA cluster. These findings augment existing data implicating miRNAs as putative CF modifiers., (Copyright © 2015 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

CFTR exon 10 and its flanking regions are duplicated in the human genome. These duplications present mutations compared to the normal exon 10 sequence. Due to the polymorphic sequence of the 3' intron 9 sequence, it may appear difficult to sequence exon 10 and some mutations described in this exon could, in fact, be variations observed in an ectopic duplicated sequence. In our previous work we described a methodology to carry out PCR only of exon 10 and not of ectopic regions. In this work, we analyzed mutations described in the CF data base as being CFTR mutations but also found in ectopic regions: c.1392G>T, c.1338_1339delAT, c.1235delC, and c.1247A>G. We have shown that these mutations appear to be authentic mutations in CFTR exon 10 and not ectopic variations in analyzed patients. These mutations validate the usefulness of our new strategy in the mutation analysis of this region of CFTR., (Copyright © 2012 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Background: The identification by CFTR mRNA studies of a new deep-intronic splicing mutation, c.870-1113_1110delGAAT, in one patient of our series with mild CF symptoms and in three CF patients of an Italian study, led us to evaluate the mutation frequency and phenotype/genotype correlations., Methods: 266 patients with CF and related disorders and having at least one undetected mutation, were tested at the gDNA level in three French reference laboratories., Results: In total, the mutation was found in 13 unrelated patients (5% of those already carrying a mutation) plus 4 siblings, including one homozygote and 12 heterozygotes having a severe CF mutation. The sweat test was positive in 10/14 documented cases, the diagnosis was delayed after 20 years in 9/15 and pancreatic insufficiency was present in 5/16., Conclusion: c.870-1113_1110delGAAT should be considered as CF-causing with phenotype variability and overall delayed diagnosis. Its frequency highlights the potential of mRNA studies., (Copyright © 2011 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Background: Clinical phenotype varies among cystic fibrosis (CF) patients with identical CF transmembrane conductance regulator (CFTR)genotype, suggesting that genetic modifiers exist. Transgenic mice that overexpress SCNN1beta present CF-like lung disease symptoms. Mutations or variants in SCNN1beta may therefore potentially modulate the clinical phenotype in CF patients., Methods: We analysed by DHPLC SCNN1beta and SCNN1gamma genes in 56 patients with classical CF. Patients were classified into two groups according to their CFTR genotype and their severity: 38 patients with severe genotype and an unexpectedly mild lung phenotype, and 18 patients with mild genotype and a severe lung phenotype., Results: We found 3 patients out of 56 carrying at least one missense mutation. Two were novel (p.Thr313Met in SCNN1beta, p.Leu481Gln in SCNN1gamma) and two were previously described (p.Gly589Ser in SCNN1beta and p.Val546Ileu in SCNNgamma). p.Thr313Met has been identified in a CF patient with mild genotype and severe lung phenotype suggesting that it could act in increasing ENaC activity. The three other variants have been identified in CF patients with severe genotype and mild lung phenotype suggesting that they might decrease ENaC activity. However, the function of ENaC in the nasal epithelia of these patients, evaluated by nasal potential difference measurements, did not support the fact that these variants were functional, at least in nasal epithelium., Conclusion: Our results suggest that genetic variants in ENaCbeta and gamma genes do not modulate disease severity in the majority of CF patients.

The MECP2 gene is responsible for 80-85% of typical cases of Rett syndrome with deleterious mutations affecting exons 3 and 4. Recently, an alternate transcript including exon 1 was discovered with a new protein isoform (MeCP2_e1) much more abundant in brain. We screened exon 1 of MECP2 for mutations and for large rearrangements in a panel of 212 typical cases of Rett syndrome and one family case with atypical Rett syndrome. We identified two deleterious mutations (c.48_55dup and c.62+2_62+3del) and four large rearrangements encompassing exon 1 of MECP2. We also identified the c.16_21dup alteration formerly reported as c.3_4insGCCGCC and give additional support to classify this sequence variation as polymorphic. In our large panel of typical Rett, mutations affecting exon 1 of MECP2 represent 1% of the deleterious alleles. This study confirms that mutations in exon 1 of MECP2 are a rare cause of Rett syndrome.

Diabetes mellitus (DM) and abnormal glucose tolerance (IGT) are common in cystic fibrosis (CF). The loss of pancreatic beta-cells due to pancreatic fibrosis is thought to be one of the principal causes of diabetes in CF, but the aetiology of DM remains somewhat puzzling. Genetic factors may contribute to the development of CF related diabetes (CFRD). The purpose of this study was to investigate the role of polymorphisms in six genes on IGT or DM incidence. PCR and dHPLC were used to screen DNA samples for polymorphisms. Using 2-h oral glucose tolerance tests, 163 adult pancreatic insufficient CF patients have been subdivided in 3 groups: 54 NGT (normal glucose tolerance), 33 IGT and 76 CFRD. We found the first evidence for the association between CFRD and UCSNP-19 polymorphism in the CAPN10 gene. The UCSNP-19 genotype distribution differed significantly between NGT, IGT and CFRD groups. The difference reflected an increase in the 22 genotype (3 copies of 32-bp sequence) in IGT and CFRD patients (p=0.05). Odds ratio for the homozygote 22 versus homozygote 11 was 3.4 (p=0.02). All allele and genotype distributions for the other polymorphisms were similar in the three groups. In conclusion, our observations suggest that UCSNP-19 of CAPN10 may be involved in the pathogenesis of diabetes in CF.

Mutations in the MECP2 (Methyl-CpG-binding protein) gene have been reported to cause Rett syndrome (RTT), an X-linked progressive encephalopathy. Recent studies have identified large gene rearrangements that escape the common PCR-based mutation screening strategy and mutations in a novel MeCP2 isoform (named MECP2B). We have collected the results of MECP2 mutational analysis concerning 424 RTT patients conducted in eight laboratories in France. In total, 121 different MECP2 mutations were identified. R168X (11.5%) is the most common of MECP2 mutations, followed by R270X (9%), R255X (8.7%), T158 M (8.3%) and R306C (6.8%). Only eight mutations had relative frequency>3%. Large and complex rearrangements not previously detected using only a PCR-based strategy represent 5.8% of MECP2 mutations. On the contrary, mutation in exon 1 appears to be rare (less than 0.5%). These data demonstrate the high allelic heterogeneity of RTT in France and suggest that routine mutation screening in MECP2 should include quantitative analysis of the MECP2 gene. This study represents an important instrument for molecular diagnosis strategy and genetic counseling in RTT families.

We assessed the contribution of the sweat test, genotyping and nasal potential difference (NPD) in the diagnosis of cystic fibrosis (CF) in adults with diffuse bronchiectasis (DB). Among 601 adults referred for DB from 1992 to 2001, 46 were diagnosed with CF. The sweat test was positive in 37 patients and normal or intermediate in nine patients. Two CF mutations were identified in 18 patients (39%) by screening for 31 mutations and in 36 patients (78%) after complete genetic analysis. NPD was suggestive of CF in 71% of the patients. The combination of the sweat test and genetic analysis led to the diagnosis of CF in 45 patients. In the nine patients with normal or intermediate sweat test, the diagnosis was confirmed by screening for 31 mutations in five, by complete genetic screening in three, and by NPD in the remaining patient. Searching for CF should start with sweat test. If the sweat test is normal or intermediate, screening for 31 mutations may help to diagnose CF. A complete genetic analysis is indicated when only one mutation is detected and/or when other clinical features, such as obstructive azoospermia or pancreatic insufficiency, are suggestive of CF. NPD measurement is indicated in controversial cases.

Objective: To treat cystic fibrosis women who failed to conceive., Patients and Methods: Multidisciplinary coordinated approach in 20 infertile women affected with cystic fibrosis. Advisability of pregnancy was based on the assessment of pulmonary and nutritional status, risks of maternal health deterioration, infertility factors, risk of cystic fibrosis in the offspring. Assisted reproductive techniques in moderately affected women are described., Results: Three patients requested only information, three were discouraged, two are still evaluated, 12 were treated and three delivered healthy children. The pulmonary status of each patient remained unchanged 36 months after delivery, and all three children remain healthy., Discussion and Conclusion: Assisted reproductive techniques are an option in moderately affected infertile cystic fibrosis women. Advisability and management of pregnancy should be provided by a coordinated team of healthcare professionals with knowledge and experience in cystic fibrosis.

Cystic fibrosis (CF) is one of the most common lethal autosomal recessive disease among the Caucasian population. It is caused by defects in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. More than 1000 different CF mutations have been described. This large heterogeneity of mutations could explain in part the great variability of clinical expression of the disease. However, the severity of the lung disease is very different among patients with similar CFTR genotype, even from the same family. These discrepancies in phenotypes within patients of the same genotype suggest the influence of nongenetic environmental factors and genetic modifiers outside the CF locus.

Ten percent of couples trying for a child fail to conceive. In approximately 50% of cases, infertility is due to the inability of the male partner to produce spermatozoa in sufficient numbers to effect conception. Over the past 5 years, molecular studies have suggested that interstitial microdeletions in Yq11 represent an etiological factor for male infertility. Y-microdeletions have been detected in 12% of non-obstructive azoospermia and 6% of severe oligozoospermia. In general, microdeletions were detected by separate multiplex-polymerase chain reaction (PCR) reactions using primer pairs for single tagged sites (STSs) of all three azoospermia factor (AZF) regions (AZFa, AZFb and AZFc). This review describes the molecular methods and laboratory guidelines for molecular diagnosis of Y-chromosomal microdeletions. The diagnostics of Y-chromosomal microdeletions should be performed in two steps: in any case, the routine diagnostic should include six STS loci, two STS loci localised in each AZF region, and once a deletion is detected, the analysis can be extended to STS loci known to cross the proximal and the distal borderlines of each AZF region. Other molecular techniques such as DGGE, Southern blot should be performed to detect partial deletions of gene copies or mosaicism. These different molecular approaches should allow explaining 10% of male infertility, to evaluate the risk to pass the defect onto their male offspring (by intracytoplasmic sperm injection) and improve the genetic counselling of couples undergoing micromanipulative assisted reproduction.

Microdeletions of Yq are associated with azoospermia and severe oligozoospermia. Recently, we described a new molecular genetic strategy based on the denaturing gradient gel electrophoresis (DGGE) to rapidly identify deletions of the Y chromosome that include the DAZ locus. Using this approach, we have shown not a complete deletion but only a reduction in the number of copies of the DAZ gene by PCR-DGGE in two oligozoospermic patients. These results have been confirmed by Southern blot analysis. This finding suggests that partial deletion of the DAZ cluster could be cause of impaired spermatogenesis.

We have recently shown that mutations in oligophrenin-1 (OPHN1) are responsible for non-specific X-linked mental retardation (MRX). The structure of the gene encoding the OPHN1 protein was determined by isolation of genomic DNA clones from the human cosmid library. Genomic fragments containing exons were sequenced, and the sequences of the exons and flanking introns were defined. Knowledge of the genomic structure of the OPHN1 gene, which spans at least 500 kb and consists of 25 exons, will facilitate the search for additional mutations in OPHN1. OPHN1 was screened for mutations in 164 subjects with non-specific mental retardation. Three nucleotide substitutions were identified, one of which was a silent mutation in the codon threonine 301 at position 903 (G-->C). The other substitutions were located in exon 2, a G-->A substitution at position 133 (A45T), and in exon 10, a C-->T substitution at position 902 (T301M), but these are common polymorphisms rather than disease-causing mutations.

Congenital bilateral absence of the vas deferens is a congenital reproductive disorder that affects about one in 1000 male individuals. Screening of the entire coding and flanking sequences of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in 64 males with CBAVD revealed that in only 23% CBAVD was caused by two CFTR mutations. The 5T allele in one copy, that causes reduced levels of the normal CFTR protein, in combination with a CFTR mutation in the other copy, was one of the most common causes of CBAVD. Twenty six per cent of men with CBAVD had the 5T allele. The presence of only one CFTR mutation or the 5T allele in 34% of patients suggests that undetected changes in CFTR may be involved in CBAVD. These molecular defects are probably mutations with partial penetrance. Moreover, the high proportion (20%) of patients with CBAVD who did not have CFTR mutations or the 5T allele allows to propose that another gene or genes could be responsible for CBAVD. In these cases, in vitro fertilization may be required and the genetic counselling appears to be very complex and additional studies, including CFTR mRNA and linkage analyses, are required to resolve these questions.

15-20% of the CF mutation are expected to be rare and escape detection by systems designed to screen for common mutations. The highly polymorphic simple repeats would be particularly useful for genetic diagnosis in CF families where the mutations have not been identified. In this study, we used denaturing gradient gel electrophoresis with psoralen-modified oligonucleotide primers to study the GTnTm polymorphism previously identified at the intron 8 - exon 9 junction of the CFTR gene. Twelve characteristic patterns were identified. The most frequent genotype in CF alleles was GT10T9 and in non-CF alleles GT11T7. In this study, the heterozygous incidence is 70% in unrelated CF carriers. This polymorphism is full informative in 45% and half-informative in 50%. We conclude, that this polymorphism, easy to study by a relatively simple, rapid and cheap procedure, would be particularly useful in genetic counselling for CF and prenatal diagnosis in CF families in which mutations have not been yet identified.