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Abstract Background Aproximately 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. Results Known 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%). Conclusion This 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.

Background: Truncating pathogenic or likely pathogenic variants of CDH1 cause hereditary diffuse gastric cancer (HDGC), a tumour risk syndrome that predisposes carrier individuals to diffuse gastric and lobular breast cancer. Rare CDH1 missense variants are often classified as variants of unknown significance. We conducted a genotype-phenotype analysis in families carrying rare CDH1 variants, comparing cancer spectrum in carriers of pathogenic or likely pathogenic variants (PV/LPV; analysed jointly) or missense variants of unknown significance, assessing the frequency of families with lobular breast cancer among PV/LPV carrier families, and testing the performance of lobular breast cancer-expanded criteria for CDH1 testing., Methods: This genotype-first study used retrospective diagnostic and clinical data from 854 carriers of 398 rare CDH1 variants and 1021 relatives, irrespective of HDGC clinical criteria, from 29 institutions in ten member-countries of the European Reference Network on Tumour Risk Syndromes (ERN GENTURIS). Data were collected from Oct 1, 2018, to Sept 20, 2022. Variants were classified by molecular type and clinical actionability with the American College of Medical Genetics and Association for Molecular Pathology CDH1 guidelines (version 2). Families were categorised by whether they fulfilled the 2015 and 2020 HDGC clinical criteria. Genotype-phenotype associations were analysed by Student's t test, Kruskal-Wallis, χ 2 , and multivariable logistic regression models. Performance of HDGC clinical criteria sets were assessed with an equivalence test and Youden index, and the areas under the receiver operating characteristic curves were compared by Z test., Findings: From 1971 phenotypes (contributed by 854 probands and 1021 relatives aged 1-93 years), 460 had gastric and breast cancer histology available. CDH1 truncating PV/LPVs occurred in 176 (21%) of 854 families and missense variants of unknown significance in 169 (20%) families. Multivariable logistic regression comparing phenotypes occurring in families carrying PV/LPVs or missense variants of unknown significance showed that lobular breast cancer had the greatest positive association with the presence of PV/LPVs (odds ratio 12·39 [95% CI 2·66-57·74], p=0·0014), followed by diffuse gastric cancer (8·00 [2·18-29·39], p=0·0017) and gastric cancer (7·81 [2·03-29·96], p=0·0027). 136 (77%) of 176 families carrying PV/LPVs fulfilled the 2015 HDGC criteria. Of the remaining 40 (23%) families, who did not fulfil the 2015 criteria, 11 fulfilled the 2020 HDGC criteria, and 18 had lobular breast cancer only or lobular breast cancer and gastric cancer, but did not meet the 2020 criteria. No specific CDH1 variant was found to predispose individuals specifically to lobular breast cancer, although 12 (7%) of 176 PV/LPV carrier families had lobular breast cancer only. Addition of three new lobular breast cancer-centred criteria improved testing sensitivity while retaining high specificity. The probability of finding CDH1 PV/LPVs in patients fulfilling the lobular breast cancer-expanded criteria, compared with the 2020 criteria, increased significantly (AUC 0·92 vs 0·88; Z score 3·54; p=0·0004)., Interpretation: CDH1 PV/LPVs were positively associated with HDGC-related phenotypes (lobular breast cancer, diffuse gastric cancer, and gastric cancer), and no evidence for a positive association with these phenotypes was found for CDH1 missense variants of unknown significance. CDH1 PV/LPVs occurred often in families with lobular breast cancer who did not fulfil the 2020 HDGC criteria, supporting the expansion of lobular breast cancer-centred criteria., Funding: European Reference Network on Genetic Tumour Risk Syndromes, European Regional Development Fund, Fundação para a Ciência e a Tecnologia (Portugal), Cancer Research UK, and European Union's Horizon 2020 research and innovation programme., Competing Interests: Declaration of interests DGE declares fees from Astrazeneca and Recursion. ERW declares grants from International Alliance for Cancer Early Detection, for which they are codirector of the research domain. GNR declares receipt of funding for study materials, medical writing, and article processing charges from Italian Ministry of Education (GNR). MJLL declares consulting fees (via the Radboud University Medical Center) from Merck Sharp & Dohme (MSD), AstraZeneca, Lilly, Janssen-Cilag, Illumina, GlaxoSmithKline. PRB declares fees from AstraZeneca, MSD, and Bristol Myers Squibb; and is a scientific committee member for the Geneticancer patients association. JBa declares fees from AstraZeneca, Lilly, and Pfizer. SA is a member of APC subVCEP of the InSiGHT/ClinGen Hereditary Colorectal Cancer/Polyposis Variant Curation Expert Panel; is an unpaid member of the German Gene Diagnostics Commission and speaker of the Centre for Hereditary Tumour Syndromes of the University of Bonn. RH declares grants from SLA Pharma and Janssen Pharmaceuticals; consulting fees from Janssen and One Two Therapeutics; equipment from Fujifilm; is the head of German Consortium for Familial Gastrointestinal Cancer; and is an unpaid advisory board member of the Lynch Syndrome advocacy Group and the Familial Polyposis Group. ES declares grants from NCT/DKTK Master. ES declares honoraria for presentations from AstraZeneca, Georg Thieme Verlag KG, and payment for expert testimony from Illumina; is a member of the board of directors of Deutsche Gesellschaft für HumanGenetik; an advisor for Dresden-concept Genome Center; and is board of directors president (paid) for LNS laboratoire National de Santé. RdP declares support for presentations (via his institution) from MSD and AstraZeneca. GC declares to receive funding for study materials, medical writing, article processing charges from the Spanish Ministry of Science and Innovation, the Instituto de Salud Carlos III CIBERONC, and the Government of Catalonia.; consulting fees from VCN Biosciences Synthetic Biologics; is the chair of the Council of the International Society of Hereditary Gastrointestinal Tumours and the FUREGA Fundació Recerca en Gastroenterologia; and stock in Synthetic Biologics. CLa declares consulting fees and honoraria from AstraZeneca and MSD, and is a paid advisory board member for Illumina., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Sotos syndrome is an autosomal dominant disorder characterized by overgrowth, macrocephaly, distinctive facial features and learning disabilities. Haploinsufficiency of the nuclear receptor SET domain-containing protein 1 ( NSD1 ) gene located on chromosome 5q35 is the major cause of the syndrome. This syndrome shares characteristics with other overgrowth syndromes, which can complicate the differential diagnosis., Methods: Genomic DNA was extracted from peripheral blood samples of members of the same family and targeted exome analysis was performed. In silico study of the variant found by next-generation sequencing was used to predict disruption/creation of splice sites and the identification of potential cryptic splice sites. RNA was extracted from peripheral blood samples of patients and functional analyses were performed to confirm the pathogenicity., Results: We found a novel c.6463 + 5G>A heterozygous NSD1 gene pathogenic variant in a son and his father. Molecular analyses revealed that part of the intron 22 of NSD1 is retained due to the destruction of the splicing donor site, causing the appearance of a premature stop codon in the NSD1 protein., Conclusions: Our findings underline the importance of performing RNA functional assays in order to determine the clinical significance of intronic variants, and contribute to the genetic counseling and clinical management of patients and their relatives. Our work also highlights the relevance of using in silico prediction tools to detect a potential alteration in the splicing process., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Villate, Maortua, Tejada and Llano-Rivas.)

The PTEN tumor suppressor gene is mutated with high incidence in tumors and in the germline of patients with cancer predisposition or with macrocephaly associated with autism. PTEN nonsense mutations generating premature termination codons (PTC) and producing nonfunctional truncated PTEN proteins are frequent in association with human disease. However, there are no studies addressing the restoration of full-length PTEN proteins from the PTC-mutated PTEN gene by translational readthrough. Here, we have performed a global translational and functional readthrough analysis of the complete collection of PTEN PTC somatic or hereditary mutations found in tumors or in the germline of patients (disease-associated PTEN PTCome), and we set standards for the analysis of the potential of readthrough functional reconstitution in disease-relevant genes. Our analysis indicates that prevalent pathogenic PTEN PTC mutations are susceptible to PTEN functional restoration in response to readthrough-inducing compounds. Comprehensive readthrough analyses of disease-associated PTComes will be valuable tools for the implementation of readthrough-based precision interventions in specific groups of patients., (© 2021 Wiley Periodicals LLC.)

Hypomagnesemia, seizures, and intellectual disability (HSMR) syndrome is a rare disorder caused by mutations in the cyclin M2 (CNNM2) gene. Due to the limited number of cases, extensive phenotype analyses of these patients have not been performed, hindering early recognition of patients. In this study, we established the largest cohort of HSMR to date, aiming to improve recognition and diagnosis of this complex disorder. Eleven novel variants in CNNM2 were identified in nine single sporadic cases and in two families with suspected HSMR syndrome. 25 Mg 2+ uptake assays demonstrated loss-of-function in seven out of nine variants in CNNM2. Interestingly, the pathogenic mutations resulted in decreased plasma membrane expression. The phenotype of those affected by pathogenic CNNM2 mutations was compared with five previously reported cases of HSMR. All patients suffered from hypomagnesemia (0.44-0.72 mmol/L), which could not be fully corrected by Mg 2+ supplementation. The majority of patients (77%) experienced generalized seizures and exhibited mild to moderate intellectual disability and speech delay. Moreover, severe obesity was present in most patients (89%). Our data establish hypomagnesemia, seizures, intellectual disability, and obesity as hallmarks of HSMR syndrome. The assessment of these major features offers a straightforward tool for the clinical diagnosis of HSMR., (© 2021 The Authors. Human Mutation published by Wiley Periodicals LLC.)

There are four classes of CGG repeat alleles in the FMR1 gene: normal alleles have up to 44 repeats; patients with Fragile X Syndrome have more than 200 repeats; those between 55 and 200 CGGs are considered FMR1 premutation alleles, because they are associated with maternal expansions of the number of CGGs in the next generation and finally, alleles between 45 and 54 CGGs are called intermediate or gray zone alleles. In these last categories, the stability depends on the presence of AGG interruptions, which usually occurs between 9 and 10 CGGs. In this context, we have studied retrospectively 66 women with CGG repeats between 45 and 65, and their offspring. In total 87 transmissions were analyzed with triplet repeat primed PCR using AmplideX® FMR1 PCR (Asuragen, Austin, TX, USA) and we found that alleles with CGG repeats between 45 and 58 do not expand in the next generation except two cases with 56 repeats and 0 AGG interruptions. Furthermore, we have found four females with alleles with more than 59 CGG repeats and 2 AGG interruptions that do not expand either. Alleles from 56 CGG repeats without AGGs expand in all cases. In light of these results and those of the literature, we consider that the risk of unstable transmissions should be based on the presence or absence of AGG interruptions and not on the classical cutoffs which define each category of FMR1 alleles. The application of these results in the genetic and reproductive counseling is essential and AGG interruptions should always be studied., (Copyright © 2020 Villate, Ibarluzea, Maortua, de la Hoz, Rodriguez-Revenga, Izquierdo-Álvarez and Tejada.)

Since the discovery of the FMR1 gene and the clinical and molecular characterization of Fragile X Syndrome in 1991, more than 141 genes have been identified in the X-chromosome in these 28 years thanks to applying continuously evolving molecular techniques to X-linked intellectual disability (XLID) families. In the past decade, array comparative genomic hybridization and next generation sequencing technologies have accelerated gene discovery exponentially. Classically, XLID has been subdivided in syndromic intellectual disability (S-XLID)-where intellectual disability (ID) is always associated with other recognizable physical and/or neurological features-and non-specific or non-syndromic intellectual disability (NS-XLID) where the only common feature is ID. Nevertheless, new advances on the study of these entities have showed that this classification is not always clear-cut because distinct variants in several of these XLID genes can result in S-XLID as well as in NS-XLID. This review focuses on the current knowledge on the XLID genes involved in non-syndromic forms, with the emphasis on their pathogenic mechanism, thus allowing the possibility to elucidate why some of them can give both syndromic and non-syndromic phenotypes., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder that appears in at least one-third of adult carriers of FMR1 premutation. Several studies have shown that mitochondrial dysfunction may play a role in neurodegenerative disorders. In order to assess whether mitochondrial DNA variants are involved in the risk of developing FXTAS we evaluated the frequency of mitochondrial haplogroups in 132 unrelated Spanish FMR1 premutation carriers. In addition, the entire mitogenome of 26 FMR1 premutation carriers was sequenced using massively parallel sequencing technologies to analyze mitochondrial DNA variants. Statistical analyses reveal a significant difference in the frequency of T haplogroup. Data analysis of mitochondrial DNA sequences evidence an association between FXTAS and the burden of heteroplasmic variants as well as their distribution. Our results suggest that haplogroup T might be a potential protective factor for FXTAS and that FXTAS individuals accumulate higher rates of heteroplasmic variants in compromised regions of the mitochondrial genome. These results may explain, in part, the role of mitochondrial DNA in the development of FXTAS., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Hereditary spastic paraplegias (HSPs) are a heterogeneous group of genetic disorders with spastic paraparesis as the main clinical feature. Complex forms may co-occur with other motor, sensory, and cognitive impairment. A growing number of loci and genes are being identified, but still more than 50% of the patients remain without molecular diagnosis. We present a Spanish family with autosomal dominant HSP and intellectual disability (ID) in which we found a possible dual genetic diagnosis with incomplete penetrance and variable expressivity in the parents and three siblings: a heterozygous duplication of 15q11.2-q13.1 found by array CGH and a novel missense heterozygous change in REEP1 [c.73A>G; p.(Lys25Glu)] found by whole exome sequencing (WES). Following the standard genetic diagnosis approach in ID, array CGH analysis was first performed in both brothers affected by spastic paraparesis and ID from school age, and a heterozygous duplication of 15q11.2-q13.1 was found. Subsequently, the duplication was also found in the healthy mother and in the sister, who presented attention deficit/hyperactivity disorder (ADHD) symptoms from school age and pes cavus with mild pyramidal signs at 22 years of age. Methylation analysis revealed that the three siblings carried the duplication unmethylated in the maternal allele, whereas their mother harbored it methylated in her paternal allele. Functional studies revealed an overexpression of UBE3A and ATP10A in the three siblings, and the slightest cognitive phenotype of the sister seems to be related to a lower expression of ATP10A . Later, searching for the cause of HSP, WES was performed revealing the missense heterozygous variant in REEP1 in all three siblings and the father, who presented subtle pyramidal signs in the lower limbs as well as the sister. Our findings reinforce the association of maternally derived UBE3A overexpression with neurodevelopmental disorders and support that a spectrum of clinical severity is present within families. They also reveal that a dual genetic diagnosis is possible in patients with presumed complex forms of HSP and cognitive impairment., (Copyright © 2020 Aguilera-Albesa, de la Hoz, Ibarluzea, Ordóñez-Castillo, Busto-Crespo, Villate, Ibiricu-Yanguas, Yoldi-Petri, García de Gurtubay, Perez de Nanclares, Pereda and Tejada.)

X-linked intellectual disability (XLID) is known to contribute up to 10% of intellectual disability (ID) in males and could explain the increased ratio of affected males observed in patients with ID. Over the past decade, next-generation sequencing has clearly stimulated the gene discovery process and has become part of the diagnostic procedure. We have performed targeted next-generation sequencing of 82 XLID genes on 61 non-related male patients with suggestive non-syndromic XLID. These patients were initially referred to the molecular genetics laboratory to exclude Fragile X Syndrome. The cohort includes 47 male patients with suggestive X-linked family history of ID meaning that they had half-brothers or maternal cousins or uncles affected; and 14 male patients with ID and affected brothers whose mothers show skewed X-inactivation. Sequencing data analysis identified 17 candidate variants in 16 patients. Seven families could be re-contacted and variant segregation analysis of the respective eight candidate variants was performed: HUWE1 , IQSEC2 , MAOA , MED12 , PHF8 , SLC6A8 , SLC9A6 , and SYN1 . Our results show the utility of targeted next-generation sequencing in unravelling the genetic origin of XLID, especially in retrospective cases. Variant segregation and additional studies like RNA sequencing and biochemical assays also helped in re-evaluating and further classifying the genetic variants found., Competing Interests: The authors declare no conflict of interest.

X-linked intellectual disability (XLID) is known to explain up to 10% of the intellectual disability in males. A large number of families in which intellectual disability is the only clinically consistent manifestation have been described. While linkage analysis and candidate gene testing were the initial approaches to find genes and variants, next generation sequencing (NGS) has accelerated the discovery of more and more XLID genes. Using NGS, we resolved the genetic cause of MRX82 (OMIM number 300518), a large Spanish Basque family with five affected males with intellectual disability and a wide phenotypic variability among them despite having the same pathogenic variant. Although the previous linkage study had mapped the locus to an interval of 7.6Mb in Xq24-Xq25 of the X chromosome, this region contained too many candidate genes to be analysed using conventional approaches. NGS revealed a novel nonsense variant: c.118C > T; p.Gln40* in UPF3B , a gene previously implicated in XLID that encodes a protein involved in nonsense-mediated mRNA decay (NMD). Further molecular studies showed that the mRNA transcript was not completely degraded by NMD. However, UPF3B protein was not detected by conventional Western Blot analysis at least downstream of the 40 residue demonstrating that the phenotype could be due to the loss of functional protein. This is the first report of a premature termination codon before the three functional domains of the UPF3B protein and these results directly implicate the absence of these domains with XLID, autism and some dysmorphic features., (Copyright © 2019 Tejada, Villate, Ibarluzea, de la Hoz, Martínez-Bouzas, Beristain, Martínez, Friez, Sobrino and Barros.)

FMR1 premutation carriers (55-200 CGGs) are at risk of developing Fragile X-associated primary ovarian insufficiency as well as Fragile X-associated tremor/ataxia syndrome. FMR1 premutation alleles are also associated with a variety of disorders, including psychiatric, developmental, and neurological problems. However, there is a major concern regarding clinical implications of smaller CGG expansions known as intermediate alleles (IA) or gray zone alleles (45-54 CGG). Although several studies have hypothesized that IA may be involved in the etiology of FMR1 premutation associated phenotypes, this association still remains unclear. The aim of this study was to provide new data on the clinical implications of IA. We reviewed a total of 17 011 individuals: 1142 with primary ovarian insufficiency, 478 with movement disorders, 14 006 with neurodevelopmental disorders and 1385 controls. Similar IA frequencies were detected in all the cases and controls (cases 1.20% vs controls 1.39%, P = .427). When comparing the allelic frequencies of IA ≥ 50CGGs, a greater, albeit not statistically significant, number of alleles were detected in all the cohorts of patients. Therefore, IA below 50 CGGs should not be considered as risk factors for FMR1 premutation-associated phenotypes, at least in our population. However, the clinical implication of IA ≥ 50CGGs remains to be further elucidated., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Mutations in CHD7 have been shown to be a major cause of CHARGE syndrome, which presents many symptoms and features common to other syndromes making its diagnosis difficult. Next generation sequencing (NGS) of a panel of intellectual disability related genes was performed in an adult patient without molecular diagnosis. A splice donor variant in CHD7 (c.5665 + 1G > T) was identified. To study its potential pathogenicity, exons and flanking intronic sequences were amplified from patient DNA and cloned into the pSAD ® splicing vector. HeLa cells were transfected with this construct and a wild-type minigene and functional analysis were performed. The construct with the c.5665 + 1G > T variant produced an aberrant transcript with an insert of 63 nucleotides of intron 28 creating a premature termination codon (TAG) 25 nucleotides downstream. This would lead to the insertion of 8 new amino acids and therefore a truncated 1896 amino acid protein. As a result of this, the patient was diagnosed with CHARGE syndrome. Functional analyses underline their usefulness for studying the pathogenicity of variants found by NGS and therefore its application to accurately diagnose patients.

Most common breast cancer susceptibility variants have been identified through genome-wide association studies (GWAS) of predominantly estrogen receptor (ER)-positive disease. We conducted a GWAS using 21,468 ER-negative cases and 100,594 controls combined with 18,908 BRCA1 mutation carriers (9,414 with breast cancer), all of European origin. We identified independent associations at P < 5 × 10 -8 with ten variants at nine new loci. At P < 0.05, we replicated associations with 10 of 11 variants previously reported in ER-negative disease or BRCA1 mutation carrier GWAS and observed consistent associations with ER-negative disease for 105 susceptibility variants identified by other studies. These 125 variants explain approximately 16% of the familial risk of this breast cancer subtype. There was high genetic correlation (0.72) between risk of ER-negative breast cancer and breast cancer risk for BRCA1 mutation carriers. These findings may lead to improved risk prediction and inform further fine-mapping and functional work to better understand the biological basis of ER-negative breast cancer.

Introduction: Congenital haemolytic anaemia (CHA) refers to a group of genetically heterogeneous disorders, mainly caused by changes in genes encoding globin chains, cytoskeletal proteins and red cell enzymes, in which accurate diagnosis can be challenging with conventional techniques., Methods: To set-up a comprehensive assay for detecting mutations that could improve aetiological diagnosis, we designed a custom panel for sequencing coding regions from 40 genes known to be involved in the pathogenesis of CHA, using the Ion Torrent ™ (Thermo Fisher Scientific, S.L. Waltham, MA, USA) Personal Genome Machine (PGM) Sequencer. A control group of 16 samples with previously known mutations and a test group of 10 patients with unknown mutations were included for assay validation and application, respectively., Results: In the test group, we identified pathogenic mutations in all cases: four patients had novel mutations in genes related to membrane defects (SPTB, ANK1, SLC4A1 and EPB41), four were homozygous or compound heterozygous for mutations in genes related to enzyme deficiencies (GPI, TPI1 and GSS), one had a mutation in the HBB gene and another presented a homozygous mutation in the ADAMTS13 gene., Conclusions: Ion PGM sequencing with our custom panel is a highly efficient way to detect mutations causing haemolytic anaemia, including new variations. It is a high-throughput detection method that is ready for application in clinical laboratories., (© 2016 John Wiley & Sons Ltd.)

Fragile X-associated tremor/ataxia syndrome (FXTAS) and fragile X-associated primary ovarian insufficiency (FXPOI) are definitely related to the fragile X mental retardation 1 ( FMR1 ) premutation (PM). Additional medical problems have also been associated with the PM, such as fibromyalgia, endocrine, and psychiatric disorders. To improve our understanding in the field, we reviewed all PM carriers and their reasons for any medical referrals from 104 fragile X families molecularly diagnosed in our laboratory and living in the Spanish Basque Country. After signing the written informed consent, we studied their electronic medical records in order to identify the disorders associated with the PM and their frequencies. We obtained clinical data in 188 PM carriers (147 women and 41 men). In women, the frequency of FXPOI (22.61%) was similar to that previously reported in PM carriers. In men, the frequency of definite FXTAS (28.57%) was lower than reported elsewhere. Furthermore, thyroid pathology was associated with the PM, the frequency of hypothyroidism being much higher in the studied region than in the general population (8.84% vs. 0.93%). Finally, we found no association with fibromyalgia or psychiatric problems. These findings represent another population contribution in this field and may be useful for the clinical management of PM carriers., Competing Interests: The authors declare no conflict of interest. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Missense MECP2 variants can have various phenotypic effects ranging from a normal phenotype to typical Rett syndrome (RTT). In females, the phenotype can also be influenced by the X-inactivation pattern. In this study, we present detailed clinical descriptions of six patients with a rare base-pair substitution affecting Arg309 at the C-terminal end of the transcriptional repression domain (TRD). All patients have intellectual disability and present with some RTT features, but they do not fulfill the clinical criteria for typical or atypical RTT. Most of the patients also have mild facial dysmorphism. Intriguingly, the mother of an affected male patient is an asymptomatic carrier of this variant. It is therefore likely that the p.(Arg309Trp) variation does not necessarily lead to male lethality, and it results in a wide range of clinical features in females, probably influenced by different X-inactivation patterns in target tissues., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objective: Endometrial cancer is the second most frequent neoplasm in women with Lynch syndrome (LS). We sought to assess whether analyzing women with endometrial cancer would identify families with LS not identified with current clinical criteria., Methods: We included women diagnosed with endometrial cancer younger than 50 years and also older if they had a family cancer history associated with LS. In blood samples obtained, we analyzed mutations in mismatch repair (MMR) genes, as well as protein expression by immunohistochemistry and microsatellite instability (MSI) in tumour tissue., Results: A total of 103 patients were enrolled. We detected 14 pathogenic mutations and 4 genetic variants of unknown clinical significance in MMR genes. We found MSI in 41.66% of the women with a pathogenic mutation. In this group, 76.92% showed loss of at least one MMR protein. Women with mutations were younger at diagnosis, but all of them had a family history compatible with LS., Conclusions: Analysis of the MMR genes, in particular MSH6, seems to be appropriate in women with endometrial cancer and a family history of tumours associated with LS., (© 2016 S. Karger AG, Basel.)

To identify genetic causes of intellectual disability (ID), we screened a cohort of 986 individuals with moderate to severe ID for variants in 565 known or candidate ID-associated genes using targeted next-generation sequencing. Likely pathogenic rare variants were found in ∼11% of the cases (113 variants in 107/986 individuals: ∼8% of the individuals had a likely pathogenic loss-of-function [LoF] variant, whereas ∼3% had a known pathogenic missense variant). Variants in SETD5, ATRX, CUL4B, MECP2, and ARID1B were the most common causes of ID. This study assessed the value of sequencing a cohort of probands to provide a molecular diagnosis of ID, without the availability of DNA from both parents for de novo sequence analysis. This modeling is clinically relevant as 28% of all UK families with dependent children are single parent households. In conclusion, to diagnose patients with ID in the absence of parental DNA, we recommend investigation of all LoF variants in known genes that cause ID and assessment of a limited list of proven pathogenic missense variants in these genes. This will provide 11% additional diagnostic yield beyond the 10%-15% yield from array CGH alone., (© 2015 The Authors. **Human Mutation published by Wiley Periodicals, Inc.)

Background: People with fragile X syndrome (FXS) have an intellectual dysfunction that can range from very mild to severe. Symptoms can include speech and language delays and behavioural difficulties such as aggression or self injurious behaviours, emotional lability, and anxiety-related problems (for example obsessive-compulsive symptoms and perseverative behaviours). In some cases, affected people may have an additional diagnosis of attention deficit hyperactivity disorder or an autism spectrum disorder., Objectives: To review the efficacy and safety of L-acetylcarnitine in improving the psychological, intellectual, and social performance of people with FXS., Search Methods: In May 2015 we searched the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, Embase, PsycINFO, Web of Science, and two other databases. We also searched three trials registers, four theses databases, and the reference lists of relevant studies and reviews., Selection Criteria: Randomised controlled trials (RCTs) that assessed the efficacy of L-acetylcarnitine, at any dose, in people of any age diagnosed with FXS compared with placebo., Data Collection and Analysis: For each trial, two review authors independently extracted data on the children included and interventions compared, and assessed the risk of bias of the studies across the following domains: randomisation sequence generation, allocation concealment, blinding (of participants, personnel, and outcome assessors), incomplete outcome data, selective outcome reporting, and other potential sources of bias., Main Results: We found only two RCTs that compared oral L-acetylcarnitine (LAC) with oral placebo in children with FXS. The studies included a total of 83 participants, all of them male, who were treated and followed for one year. The age of participants at the start of treatment ranged from 6 to 13 years, with a mean age of 9 years. Neither study provided information on randomisation, allocation concealment procedures, or blinding of outcome assessment, and we received no responses from the authors we emailed for clarification. We therefore rated studies as being at unclear risk of bias on these domains. We judged both studies to be at low risk of bias for blinding of participants and personnel, incomplete outcome data, and selective reporting, but to be at high risk of other bias, as at least one study was funded by a drug company, and in both studies people working for the company were part of the research team.We used the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach to rate the quality of the available evidence. Overall, the quality of the evidence was low due to the imprecision of results and high risk of other bias.Regarding the primary outcome of psychological and learning capabilities, both studies assessed the effect of interventions on children's verbal and non-verbal intellectual functioning using the Wechsler Intelligence Scale for Children - Revised. The authors did not provide detailed data on those results but said that they found no important differences between treatment and placebo.Both studies evaluated the impact of the treatment on hyperactive behaviour using the Conners' Abbreviated Parent-Teacher Questionnaire. In one study, teachers' assessments of the children found no clear evidence of a difference (mean difference (MD) 0.50, 95% confidence interval (CI) -5.08 to 6.08, n = 51; low-quality evidence). The other study stated that there were no differences between treated and untreated participants, but did not provide detailed data for inclusion in the meta-analysis.Parents' assessments favoured LAC in one study (MD -0.57, 95% CI -0.94 to -0.19, n = 17; low-quality evidence), but not in the other (MD -2.80, 95% CI -7.61 to 2.01, n = 51; low-quality evidence), though changes were not large enough to be considered clinically relevant.Regarding social skills, one study reported no clear evidence of a difference in Vineland Adaptive Behavior composite scores (MD 8.20, 95% CI -0.02 to 16.42, n = 51; low-quality evidence), yet results in the socialisation domain favoured LAC (MD 11.30, 95% CI 2.52 to 20.08, n = 51; low-quality evidence).Both studies assessed the safety of the active treatment and recorded no side effects. Neither of the included studies assessed the secondary outcome of caregiver burden., Authors' Conclusions: Low-quality evidence from two small trials showed that when compared to placebo, LAC may not improve intellectual functioning or hyperactive behaviour in children with FXS.

Importance: Limited information about the relationship between specific mutations in BRCA1 or BRCA2 (BRCA1/2) and cancer risk exists., Objective: To identify mutation-specific cancer risks for carriers of BRCA1/2., Design, Setting, and Participants: Observational study of women who were ascertained between 1937 and 2011 (median, 1999) and found to carry disease-associated BRCA1 or BRCA2 mutations. The international sample comprised 19,581 carriers of BRCA1 mutations and 11,900 carriers of BRCA2 mutations from 55 centers in 33 countries on 6 continents. We estimated hazard ratios for breast and ovarian cancer based on mutation type, function, and nucleotide position. We also estimated RHR, the ratio of breast vs ovarian cancer hazard ratios. A value of RHR greater than 1 indicated elevated breast cancer risk; a value of RHR less than 1 indicated elevated ovarian cancer risk., Exposures: Mutations of BRCA1 or BRCA2., Main Outcomes and Measures: Breast and ovarian cancer risks., Results: Among BRCA1 mutation carriers, 9052 women (46%) were diagnosed with breast cancer, 2317 (12%) with ovarian cancer, 1041 (5%) with breast and ovarian cancer, and 7171 (37%) without cancer. Among BRCA2 mutation carriers, 6180 women (52%) were diagnosed with breast cancer, 682 (6%) with ovarian cancer, 272 (2%) with breast and ovarian cancer, and 4766 (40%) without cancer. In BRCA1, we identified 3 breast cancer cluster regions (BCCRs) located at c.179 to c.505 (BCCR1; RHR = 1.46; 95% CI, 1.22-1.74; P = 2 × 10(-6)), c.4328 to c.4945 (BCCR2; RHR = 1.34; 95% CI, 1.01-1.78; P = .04), and c. 5261 to c.5563 (BCCR2', RHR = 1.38; 95% CI, 1.22-1.55; P = 6 × 10(-9)). We also identified an ovarian cancer cluster region (OCCR) from c.1380 to c.4062 (approximately exon 11) with RHR = 0.62 (95% CI, 0.56-0.70; P = 9 × 10(-17)). In BRCA2, we observed multiple BCCRs spanning c.1 to c.596 (BCCR1; RHR = 1.71; 95% CI, 1.06-2.78; P = .03), c.772 to c.1806 (BCCR1'; RHR = 1.63; 95% CI, 1.10-2.40; P = .01), and c.7394 to c.8904 (BCCR2; RHR = 2.31; 95% CI, 1.69-3.16; P = .00002). We also identified 3 OCCRs: the first (OCCR1) spanned c.3249 to c.5681 that was adjacent to c.5946delT (6174delT; RHR = 0.51; 95% CI, 0.44-0.60; P = 6 × 10(-17)). The second OCCR spanned c.6645 to c.7471 (OCCR2; RHR = 0.57; 95% CI, 0.41-0.80; P = .001). Mutations conferring nonsense-mediated decay were associated with differential breast or ovarian cancer risks and an earlier age of breast cancer diagnosis for both BRCA1 and BRCA2 mutation carriers., Conclusions and Relevance: Breast and ovarian cancer risks varied by type and location of BRCA1/2 mutations. With appropriate validation, these data may have implications for risk assessment and cancer prevention decision making for carriers of BRCA1 and BRCA2 mutations.

MECP2 (methyl CpG binding protein 2) duplication causes syndromic intellectual disability. Patients often suffer from life-threatening infections, suggesting an additional immunodeficiency. We describe for the first time the detailed infectious and immunological phenotype of MECP2 duplication syndrome. 17/27 analyzed patients suffered from pneumonia, 5/27 from at least one episode of sepsis. Encapsulated bacteria (S.pneumoniae, H.influenzae) were frequently isolated. T-cell immunity showed no gross abnormalities in 14/14 patients and IFNy-secretion upon ConA-stimulation was not decreased in 6/7 patients. In 6/21 patients IgG2-deficiency was detected - in 4/21 patients accompanied by IgA-deficiency, 10/21 patients showed low antibody titers against pneumococci. Supra-normal IgG1-levels were detected in 11/21 patients and supra-normal IgG3-levels were seen in 8/21 patients - in 6 of the patients as combined elevation of IgG1 and IgG3. Three of the four patients with IgA/IgG2-deficiency developed multiple severe infections. Upon infections pronounced acute-phase responses were common: 7/10 patients showed CRP values above 200 mg/l. Our data for the first time show systematically that increased susceptibility to infections in MECP2 duplication syndrome is associated with IgA/IgG2-deficiency, low antibody titers against pneumococci and elevated acute-phase responses. So patients with MECP2 duplication syndrome and low IgA/IgG2 may benefit from prophylactic substitution of sIgA and IgG.

To date, few cases of 3p proximal interstitial deletions have been reported and the phenotype and genotype correlation is not well understood. Here, we report a new case of a 3p proximal interstitial deletion. The patient is an 11-year-old female with speech and social interaction difficulties, learning disability, and slight facial dysmorphism, but no other major malformations. An 8 Mb de novo interstitial deletion at 3p14.2-p14.1, from position 60.461.316 to 68.515.453, was revealed by means of array comparative genomic hybridization and confirmed using quantitative reverse-transcription polymerase chain reaction assays. This region includes six genes: FEZF2, CADPS, SYNPR, ATXN7, PRICKLE, and MAGI1, that are known to have a role in neurodevelopment. These genes are located on the proximal side of the deletion. We compare our case with previously well-defined patients reported in the literature and databases.

Fragile X-associated primary ovarian insufficiency (FXPOI) is among the family of disorders caused by the expansion of a CGG repeat sequence in the 5' untranslated region of the X-linked gene FMR1. About 20% of women who carry the premutation allele (55 to 200 unmethylated CGG repeats) develop hypergonadotropic hypogonadism and cease menstruating before age 40. Some proportion of those who are still cycling show hormonal profiles indicative of ovarian dysfunction. FXPOI leads to subfertility and an increased risk of medical conditions associated with early estrogen deficiency. Little progress has been made in understanding the etiology of this clinically significant disorder. Understanding the molecular mechanisms of FXPOI requires a detailed knowledge of ovarian FMR1 mRNA and FMRP's function. In humans, non-invasive methods to discriminate the mechanisms of the premutation on ovarian function are not available, thus necessitating the development of model systems. Vertebrate (mouse and rat) and invertebrate (Drosophila melanogaster) animal studies for the FMR1 premutation and ovarian function exist and have been instrumental in advancing our understanding of the disease phenotype. For example, rodent models have shown that FMRP is highly expressed in oocytes where it is important for folliculogenesis. The two premutation mouse models studied to date show evidence of ovarian dysfunction and, together, suggest that the long repeat in the transcript itself may have some pathological effect quite apart from any effect of the toxic protein. Further, ovarian morphology in young animals appears normal and the primordial follicle pool size does not differ from that of wild-type animals. However, there is a progressive premature decline in the levels of most follicle classes. Observations also include granulosa cell abnormalities and altered gene expression patterns. Further comparisons of these models are now needed to gain insight into the etiology of the ovarian dysfunction. Premutation model systems in non-human primates and those based on induced pluripotent stem cells show particular promise and will complement current models. Here, we review the characterization of the current models and describe the development and potential of the new models. Finally, we will discuss some of the molecular mechanisms that might be responsible for FXPOI.

The MECP2 gene located on Xq28 is one of the most important genes contributing to the spectrum of neurodevelopmental disorders. Therefore, we present our experience in the molecular study of this gene. MECP2 was thoroughly tested for the presence of mutations (sequencing of four exons and rearrangements) in 120 female patients: 28 with classic Rett syndrome, five with atypical Rett syndrome, and 87 with heterogeneous phenotypes with some Rett-like features. Another 120 female patients with intellectual disability of unknown origin were also studied, but in these cases we only tested exons 3 and 4. Finally, 861 healthy controls (519 females and 342 males) were also studied for exon 3 and 4. Eighteen different pathological mutations were found, five of them previously undescribed, and four large deletions detected by multiplex ligation-dependent probe amplification. All were de novo mutations not present in the parents. In conclusion, i) MECP2 is one of the most important genes in the diagnosis of genetic intellectual disability in females; ii) MECP2 must be studied not only in patients with classical/atypical Rett syndrome but also in patients with other phenotypes related to Rett syndrome; and iii) for the new variants, it is important to perform complementary studies, including the analysis of large populations of healthy individuals and the use of in silico programs., (Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

We present a clinical and molecular cytogenetic characterization of two new patients with a complex supernumerary marker consisting of the entire short arm of chromosome 18 with a chromosome 13/21 centromere. One patient is a girl with a nonsyndromic intellectual disability and the second is a prenatally diagnosed fetus. To our knowledge, these are the fourth and fifth such cases to be described in the literature, suggesting the existence of a possible recurring constitutional structural chromosome abnormality., (Copyright © 2013 Wiley Periodicals, Inc.)

Background: The PALB2 gene, also known as FANCN, forms a bond and co-localizes with BRCA2 in DNA repair. Germline mutations in PALB2 have been identified in approximately 1% of familial breast cancer and 3-4% of familial pancreatic cancer. The goal of this study was to determine the prevalence of PALB2 mutations in a population of BRCA1/BRCA2 negative breast cancer patients selected from either a personal or family history of pancreatic cancer., Methods: 132 non-BRCA1/BRCA2 breast/ovarian cancer families with at least one pancreatic cancer case were included in the study. PALB2 mutational analysis was performed by direct sequencing of all coding exons and intron/exon boundaries, as well as multiplex ligation-dependent probe amplification., Results: Two PALB2 truncating mutations, the c.1653T>A (p.Tyr551Stop) previously reported, and c.3362del (p.Gly1121ValfsX3) which is a novel frameshift mutation, were identified. Moreover, several PALB2 variants were detected; some of them were predicted as pathological by bioinformatic analysis. Considering truncating mutations, the prevalence rate of our population of BRCA1/2-negative breast cancer patients with pancreatic cancer is 1.5%., Conclusions: The prevalence rate of PALB2 mutations in non-BRCA1/BRCA2 breast/ovarian cancer families, selected from either a personal or family pancreatic cancer history, is similar to that previously described for unselected breast/ovarian cancer families. Future research directed towards identifying other gene(s) involved in the development of breast/pancreatic cancer families is required.

Background: Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) located in the Xp22 region have been shown to cause a subset of atypical Rett syndrome with infantile spasms or early seizures starting in the first postnatal months., Methods: We performed mutation screening of CDKL5 in 60 female patients who had been identified as negative for the methyl CpG-binding protein 2 gene (MECP2) mutations, but who had current or past epilepsy, regardless of the age of onset, type, and severity. All the exons in the CDKL5 gene and their neighbouring sequences were examined, and CDKL5 rearrangements were studied by multiplex ligation-dependent probe amplification (MLPA)., Results: Six previously unidentified DNA changes were detected, two of which were disease-causing mutations in the catalytic domain: a frameshift mutation (c.509&#95;510insGT; p.Glu170GlyfsX36) and a complete deletion of exon 10. Both were found in patients with seizures that started in the first month of life., Conclusions: This study demonstrated the importance of CDKL5 mutations as etiological factors in neurodevelopmental disorders, and indicated that a thorough analysis of the CDKL5 gene sequence and its rearrangements should be considered in females with Rett syndrome-like phenotypes, severe encephalopathy and epilepsy with onset before 5 months of age. This study also confirmed the usefulness of MLPA as a diagnostic screening method for use in clinical practice.

Duplications leading to functional disomy of chromosome Xq28, including MECP2 as the critical dosage-sensitive gene, are associated with a distinct clinical phenotype in males, characterized by severe mental retardation, infantile hypotonia, progressive neurologic impairment, recurrent infections, bladder dysfunction, and absent speech. Female patients with Xq duplications including MECP2 are rare. Only recently submicroscopic duplications of this region on Xq28 have been recognized in four females, and a triplication in a fifth, all in combination with random X-chromosome inactivation (XCI). Based on this small series, it was concluded that in females with MECP2 duplication and random XCI, the typical symptoms of affected boys are not present. We present clinical and molecular data on a series of five females with an Xq28 duplication including the MECP2 gene, both isolated and as the result of a translocation, and compare them with the previously reported cases of small duplications in females. The collected data indicate that the associated phenotype in females is distinct from males with similar duplications, but the clinical effects may be as severe as seen in males., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Background: Genome-wide association studies (GWAS) identified variants at 19p13.1 and ZNF365 (10q21.2) as risk factors for breast cancer among BRCA1 and BRCA2 mutation carriers, respectively. We explored associations with ovarian cancer and with breast cancer by tumor histopathology for these variants in mutation carriers from the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA)., Methods: Genotyping data for 12,599 BRCA1 and 7,132 BRCA2 mutation carriers from 40 studies were combined., Results: We confirmed associations between rs8170 at 19p13.1 and breast cancer risk for BRCA1 mutation carriers [HR, 1.17; 95% confidence interval (CI), 1.07-1.27; P = 7.42 × 10(-4)] and between rs16917302 at ZNF365 (HR, 0.84; 95% CI, 0.73-0.97; P = 0.017) but not rs311499 at 20q13.3 (HR, 1.11; 95% CI, 0.94-1.31; P = 0.22) and breast cancer risk for BRCA2 mutation carriers. Analyses based on tumor histopathology showed that 19p13 variants were predominantly associated with estrogen receptor (ER)-negative breast cancer for both BRCA1 and BRCA2 mutation carriers, whereas rs16917302 at ZNF365 was mainly associated with ER-positive breast cancer for both BRCA1 and BRCA2 mutation carriers. We also found for the first time that rs67397200 at 19p13.1 was associated with an increased risk of ovarian cancer for BRCA1 (HR, 1.16; 95% CI, 1.05-1.29; P = 3.8 × 10(-4)) and BRCA2 mutation carriers (HR, 1.30; 95% CI, 1.10-1.52; P = 1.8 × 10(-3))., Conclusions: 19p13.1 and ZNF365 are susceptibility loci for ovarian cancer and ER subtypes of breast cancer among BRCA1 and BRCA2 mutation carriers., Impact: These findings can lead to an improved understanding of tumor development and may prove useful for breast and ovarian cancer risk prediction for BRCA1 and BRCA2 mutation carriers., (©2012 AACR.)

Germline mutations in BRCA1 and BRCA2 are associated with increased risks of breast and ovarian cancer. A genome-wide association study (GWAS) identified six alleles associated with risk of ovarian cancer for women in the general population. We evaluated four of these loci as potential modifiers of ovarian cancer risk for BRCA1 and BRCA2 mutation carriers. Four single-nucleotide polymorphisms (SNPs), rs10088218 (at 8q24), rs2665390 (at 3q25), rs717852 (at 2q31), and rs9303542 (at 17q21), were genotyped in 12,599 BRCA1 and 7,132 BRCA2 carriers, including 2,678 ovarian cancer cases. Associations were evaluated within a retrospective cohort approach. All four loci were associated with ovarian cancer risk in BRCA2 carriers; rs10088218 per-allele hazard ratio (HR) = 0.81 (95% CI: 0.67-0.98) P-trend = 0.033, rs2665390 HR = 1.48 (95% CI: 1.21-1.83) P-trend = 1.8 × 10(-4), rs717852 HR = 1.25 (95% CI: 1.10-1.42) P-trend = 6.6 × 10(-4), rs9303542 HR = 1.16 (95% CI: 1.02-1.33) P-trend = 0.026. Two loci were associated with ovarian cancer risk in BRCA1 carriers; rs10088218 per-allele HR = 0.89 (95% CI: 0.81-0.99) P-trend = 0.029, rs2665390 HR = 1.25 (95% CI: 1.10-1.42) P-trend = 6.1 × 10(-4). The HR estimates for the remaining loci were consistent with odds ratio estimates for the general population. The identification of multiple loci modifying ovarian cancer risk may be useful for counseling women with BRCA1 and BRCA2 mutations regarding their risk of ovarian cancer., (© 2012 Wiley Periodicals, Inc.)

It has been demonstrated that monoallelic PALB2 (Partner and Localizer of BRCA2) gene mutations predispose to familial breast cancer. Some of the families reported with germline PALB2 mutations presented male breast cancer as a characteristic clinical feature. Therefore, we wanted to investigate the contribution of germline PALB2 mutations in a set of 131 Spanish BRCA1/BRCA2-negative breast/ovarian cancer families with at least one male breast cancer case. The analysis included direct sequencing of all coding exons and intron/exon boundaries as well as a Multiplex Ligation-dependent Probe Amplification-based analysis of genomic rearrangements. For the first time we have identified a genomic rearrangement of PALB2 gene involving a large deletion from exon 7 to 11 in a breast cancer family. We have also identified several PALB2 variants, but no other obvious deleterious PALB2 mutation has been found. Thus, our study does not support an enrichment of PALB2 germline mutations in the subset of breast cancer families with male breast cancer cases. The identification of intronic and exonic variants indicates the necessity of assessing the implications of variants that do not lead to PALB2 truncation in the pathoghenicity of the PALB2 gene.

Multiplex ligation-dependent probe amplification (MLPA) and array- comparative genomic hybridization analysis have been proven to be useful in the identification of submicroscopic copy-number imbalances in families with nonsyndromic X-linked intellectual disability (NS-XLID). Here we report the first description of a child with mild intellectual disability and a submicroscopic duplication at Xp22.12 identified by MLPA with a P106 MRX kit (MRC-Holland, Amsterdam, Netherlands) and further confirmed and characterized with a custom 244-k oligo-array, fluorescence in situ hybridization, quantitative polymerase chain reaction (qPCR), and immunoblotting. This 1.05-megabase duplication encompasses 7 genes, RPS6KA3 being the only of these genes known to be related to ID. The proband was an 8-year-old boy referred to the genetics unit for psychomotor retardation and learning disabilities. Both maternal brothers also showed learning difficulties and delayed language during childhood in a similar way to the proband. These boys also carried the duplication, as did the healthy mother and grandmother of the proband. The same duplication was also observed in the 5-year-old younger brother who presented with features of developmental delay and learning disabilities during the previous year. Increased RPS6KA3/RSK2 levels were demonstrated in the proband by qPCR and immunoblotting. To our knowledge, this is the first family identified with a submicroscopic duplication including the entire RPS6KA3/RSK2 gene, and our findings suggest that an increased dose of this gene is responsible for a mild form of NS-XLID.

Background: It has been argued that individuals with fragile X syndrome could have low folate levels in their bodies and that supplementing their dietary intake might remediate the adverse developmental and behavioural effects of the condition., Objectives: To review the efficacy and safety of folic acid in the treatment of people with fragile X syndrome., Search Strategy: We searched four databases in November 2010: CENTRAL, PubMed, EMBASE and PsycINFO., Selection Criteria: Randomised controlled trials., Data Collection and Analysis: Two review authors independently extracted data and assessed risk of bias using the Cochrane 'Risk of bias' tool., Main Results: We included five trials, which were published between 1986 and 1992. Overall, they included 67 patients, all male, with ages ranging from one to 54 years. Intellectual disability in participants varied from borderline to severe and some studies included patients with an additional diagnosis of autism or autistic behaviour. Four of the studies were placebo-controlled cross-over trials and one study was a parallel design. The duration of follow-up ranged from two months to 12 months and the period on folic acid or placebo ranged from two to eight months. Doses of folic acid ranged from 10 mg to 250 mg per day, 10 mg per day being the most common. Most of the younger patients involved were also taking part in special education programmes (usually involving language and occupational therapy).We were not able to perform meta-analysis to combine results but none of the individual studies found evidence of clinical benefit with the use of folic acid medication in fragile X syndrome patients on any of the areas of interest, either psychological and learning capabilities or behaviour and social performance, as measured with standardised tools. Separate analysis of evidence for patients of different age groups, i.e. prepubertal children and postpubertal young people, found some statistically significant results, but did not show clear evidence of benefit for either group. Adverse effects of folic acid treatment were rare, not serious and transient.Studies were generally poorly reported and we classified only one study as being at low risk of bias., Authors' Conclusions: The quality of available evidence is low and not suitable for drawing conclusions about the effect of folic acid on fragile X syndrome patients. It consists of few studies with small samples of patients, all of them male, with little statistical power to detect anything other than huge effects.

Background: Germline mutations in the BRCA1 and BRCA2 genes are associated with increased risks of breast and ovarian cancers. Although several common variants have been associated with breast cancer susceptibility in mutation carriers, none have been associated with ovarian cancer susceptibility. A genome-wide association study recently identified an association between the rare allele of the single-nucleotide polymorphism (SNP) rs3814113 (ie, the C allele) at 9p22.2 and decreased risk of ovarian cancer for women in the general population. We evaluated the association of this SNP with ovarian cancer risk among BRCA1 or BRCA2 mutation carriers by use of data from the Consortium of Investigators of Modifiers of BRCA1/2., Methods: We genotyped rs3814113 in 10,029 BRCA1 mutation carriers and 5837 BRCA2 mutation carriers. Associations with ovarian and breast cancer were assessed with a retrospective likelihood approach. All statistical tests were two-sided., Results: The minor allele of rs3814113 was associated with a reduced risk of ovarian cancer among BRCA1 mutation carriers (per-allele hazard ratio of ovarian cancer = 0.78, 95% confidence interval = 0.72 to 0.85; P = 4.8 × 10(-9)) and BRCA2 mutation carriers (hazard ratio of ovarian cancer = 0.78, 95% confidence interval = 0.67 to 0.90; P = 5.5 × 10(-4)). This SNP was not associated with breast cancer risk among either BRCA1 or BRCA2 mutation carriers. BRCA1 mutation carriers with the TT genotype at SNP rs3814113 were predicted to have an ovarian cancer risk to age 80 years of 48%, and those with the CC genotype were predicted to have a risk of 33%., Conclusion: Common genetic variation at the 9p22.2 locus was associated with decreased risk of ovarian cancer for carriers of a BRCA1 or BRCA2 mutation.

The known breast cancer susceptibility polymorphisms in FGFR2, TNRC9/TOX3, MAP3K1, LSP1, and 2q35 confer increased risks of breast cancer for BRCA1 or BRCA2 mutation carriers. We evaluated the associations of 3 additional single nucleotide polymorphisms (SNPs), rs4973768 in SLC4A7/NEK10, rs6504950 in STXBP4/COX11, and rs10941679 at 5p12, and reanalyzed the previous associations using additional carriers in a sample of 12,525 BRCA1 and 7,409 BRCA2 carriers. Additionally, we investigated potential interactions between SNPs and assessed the implications for risk prediction. The minor alleles of rs4973768 and rs10941679 were associated with increased breast cancer risk for BRCA2 carriers (per-allele HR = 1.10, 95% CI: 1.03-1.18, P = 0.006 and HR = 1.09, 95% CI: 1.01-1.19, P = 0.03, respectively). Neither SNP was associated with breast cancer risk for BRCA1 carriers, and rs6504950 was not associated with breast cancer for either BRCA1 or BRCA2 carriers. Of the 9 polymorphisms investigated, 7 were associated with breast cancer for BRCA2 carriers (FGFR2, TOX3, MAP3K1, LSP1, 2q35, SLC4A7, 5p12, P = 7 × 10(-11) - 0.03), but only TOX3 and 2q35 were associated with the risk for BRCA1 carriers (P = 0.0049, 0.03, respectively). All risk-associated polymorphisms appear to interact multiplicatively on breast cancer risk for mutation carriers. Based on the joint genotype distribution of the 7 risk-associated SNPs in BRCA2 mutation carriers, the 5% of BRCA2 carriers at highest risk (i.e., between 95th and 100th percentiles) were predicted to have a probability between 80% and 96% of developing breast cancer by age 80, compared with 42% to 50% for the 5% of carriers at lowest risk. Our findings indicated that these risk differences might be sufficient to influence the clinical management of mutation carriers.

Germline mutations in BRCA1 and BRCA2 confer high risks of breast and ovarian cancer, and their identification allows genetic testing of at-risk relatives. However, estimates of these risks illustrate controversies, depending on the published series. The penetrance, the earlier onset of the disease and the effect of mutations on the risk of developing breast and ovarian cancer were evaluated in 344 females belonging to 34 families from the Basque Country in Spain, in which BRCA1 or BRCA2 mutations were transmitted. Kaplan-Meier survival curves were used to derive cumulative probability curves for breast and ovarian cancer by mutation status, birth cohort and mutation position, and significance of the differences was assessed using the log-rank test. The estimated probability for breast cancer by age 70 is about 64% in BRCA1 and 69% in BRCA2, whereas the probability of developing ovarian cancer is about 37% and 25% for BRCA1 and BRCA2, respectively. There is a marginally significant higher risk of developing ovarian cancer in BRCA1 families than in BRCA2 families. The effect of birth cohort on breast cancer cumulative incidence presents an increased risk for females born after 1966 and a decreased risk for those born before 1940. There is no association between mutation position and breast cancer; however, ovarian cancer is associated to BRCA1, presenting exon 11 as an ovarian cluster. These results are important for the breast and ovarian cancer diagnosis and prevention in at-risk families.

The distribution of BRCA1 and BRCA2 germ line mutations in breast/ovarian cancer families varies among different populations, which typically present a wide spectrum of unique mutations. Splicing mutation 5272-1G>A of BRCA1 and frameshift mutation 5374delTATG of BRCA2 are highly prevalent mutations in Castilla-León (Spain), accounting for 18.4% and 13.6% of BRCA1 and BRCA2 positive families, respectively. To test the presence of founder effects, 9 Spanish 5272-1G>A and 13 5374delTATG families were genotyped with polymorphic markers linked to BRCA1 or BRCA2. All the 5272-1G>A families shared a common haplotype in eight markers (1.1 Mb region) and the mutation age was estimated in 15 generations (approximately 380 years). A conserved haplotype associated to 5374delTATG was observed in four markers (0.82 Mb). The mutation occurred approximately 48 generations ago (approximately 1200 years). Each mutation likely arose from a common ancestor that could be traced to a small area of Castilla-León and expanded to other Spanish regions. They can have a significant impact on the clinical management of asymptomatic carriers as well as on the genetic screening strategy to be followed in populations with Spanish ancestries.