RNA Analysis and Clinical Characterization of a Novel Splice Variant in the NSD1 Gene Causing Familial Sotos Syndrome.

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.

The phenotypic and genetic spectrum of patients with heterozygous mutations in cyclin M2 (CNNM2).

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 Mg2+ 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 Mg2+ 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.

Effect of AGG Interruptions on FMR1 Maternal Transmissions.

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.

Role of mitochondrial DNA variants in the development of fragile X-associated tremor/ataxia syndrome.

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.

Hereditary Spastic Paraplegia and Intellectual Disability: Clinicogenetic Lessons From a Family Suggesting a Dual Genetics Diagnosis.

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.

Non-syndromic X linked intellectual disability: Current knowledge in light of the recent advances in molecular and functional studies.

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.

Targeted Next-Generation Sequencing in Patients with Suggestive X-Linked Intellectual Disability.

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.

Molecular and Clinical Characterization of a Novel Nonsense Variant in Exon 1 of the UPF3B Gene Found in a Large Spanish Basque Family (MRX82).

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.

Functional Analyses of a Novel Splice Variant in the CHD7 Gene, Found by Next Generation Sequencing, Confirm Its Pathogenicity in a Spanish Patient and Diagnose Him with CHARGE Syndrome.

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.

Associated Clinical Disorders Diagnosed by Medical Specialists in 188 FMR1 Premutation Carriers Found in the Last 25 Years in the Spanish Basque Country: A Retrospective Study.

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.

Analysis of Lynch Syndrome Mismatch Repair Genes in Women with Endometrial Cancer.

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.

Targeted Next-Generation Sequencing Analysis of 1,000 Individuals with Intellectual Disability.

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.

3p14 De Novo Interstitial Microdeletion in a Patient with Intellectual Disability and Autistic Features with Language Impairment: A Comparison with Similar Cases.

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.

L-acetylcarnitine for treating fragile X syndrome.

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.

Infectious and immunologic phenotype of MECP2 duplication syndrome.

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.

Use of model systems to understand the etiology of fragile X-associated primary ovarian insufficiency (FXPOI).

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.

Molecular testing for fragile X: analysis of 5062 tests from 1105 fragile X families--performed in 12 clinical laboratories in Spain.

Fragile X syndrome is the most common inherited form of intellectual disability. Here we report on a study based on a collaborative registry, involving 12 Spanish centres, of molecular diagnostic tests in 1105 fragile X families comprising 5062 individuals, of whom, 1655 carried a full mutation or were mosaic, three cases had deletions, 1840 had a premutation, and 102 had intermediate alleles. Two patients with the full mutation also had Klinefelter syndrome. We have used this registry to assess the risk of expansion from parents to children. From mothers with premutation, the overall rate of allele expansion to full mutation is 52.5%, and we found that this rate is higher for male than female offspring (63.6% versus 45.6%; P < 0.001). Furthermore, in mothers with intermediate alleles (45-54 repeats), there were 10 cases of expansion to a premutation allele, and for the smallest premutation alleles (55-59 repeats), there was a 6.4% risk of expansion to a full mutation, with 56 repeats being the smallest allele that expanded to a full mutation allele in a single meiosis. Hence, in our series the risk for alleles of <59 repeats is somewhat higher than in other published series. These findings are important for genetic counselling.

Analysis of PALB2 gene in BRCA1/BRCA2 negative Spanish hereditary breast/ovarian cancer families with pancreatic cancer cases.

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.

MECP2 gene study in a large cohort: testing of 240 female patients and 861 healthy controls (519 females and 342 males).

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.