Hybrid Minigene Assay: An Efficient Tool to Characterize mRNA Splicing Profiles of NF1 Variants.

Neurofibromatosis type 1 (NF1) is caused by heterozygous loss of function mutations in the NF1 gene. Although patients are diagnosed according to clinical criteria and few genotype-phenotype correlations are known, molecular analysis remains important. NF1 displays allelic heterogeneity, with a high proportion of variants affecting splicing, including deep intronic alleles and changes outside the canonical splice sites, making validation problematic. Next Generation Sequencing (NGS) technologies integrated with multiplex ligation-dependent probe amplification (MLPA) have largely overcome RNA-based techniques but do not detect splicing defects. A rapid minigene-based system was set up to test the effects of NF1 variants on splicing. We investigated 29 intronic and exonic NF1 variants identified in patients during the diagnostic process. The minigene assay showed the coexistence of multiple mechanisms of splicing alterations for seven variants. A leaky effect on splicing was documented in one de novo substitution detected in a sporadic patient with a specific phenotype without neurofibromas. Our splicing assay proved to be a reliable and fast method to validate novel NF1 variants potentially affecting splicing and to detect hypomorphic effects that might have phenotypic consequences, avoiding the requirement of patient's RNA.

Setup and Validation of a Targeted Next-Generation Sequencing Approach for the Diagnosis of Lysosomal Storage Disorders.

Lysosomal storage disorders (LSDs) are monogenic diseases, due to accumulation of specific undegraded substrates into lysosomes. LSD diagnosis could take several years because of both poor knowledge of these diseases and shared clinical features. The diagnostic approach includes clinical evaluations, biochemical tests, and genetic analysis of the suspected gene. In this study, we evaluated an LSD targeted sequencing panel as a tool capable to potentially reverse this classic diagnostic route. The panel includes 50 LSD genes and 230 intronic sequences conserved among 33 placental mammals. For the validation phase, 56 positive controls, 13 biochemically diagnosed patients, and nine undiagnosed patients were analyzed. Disease-causing variants were identified in 66% of the positive control alleles and in 62% of the biochemically diagnosed patients. Three undiagnosed patients were diagnosed. Eight patients undiagnosed by the panel were analyzed by whole exome sequencing: for two of them, the disease-causing variants were identified. Five patients, undiagnosed by both panel and exome analyses, were investigated through array comparative genomic hybridization: one of them was diagnosed. Conserved intronic fragment analysis, performed in cases unresolved by the first-level analysis, evidenced no candidate intronic variants. Targeted sequencing has low sequencing costs and short sequencing time. However, a coverage >60× to 80× must be ensured and/or Sanger validation should be performed. Moreover, it must be supported by a thorough clinical phenotyping.

The Arg1038Gly missense variant in the NF1 gene causes a mild phenotype without neurofibromas.

BACKGROUND: Neurofibromatosis type 1 (NF1) is an autosomal dominant condition caused by inactivating mutations of the NF1 gene. The wide allelic heterogeneity of this condition, with more than 3,000 pathogenic variants reported so far, is paralleled by its high clinical variability, which is observed even within the same family. The definition of genotype-phenotype correlations has been hampered by the complexity of the NF1 gene and, although a few exceptions have been recognized, the clinical course remains unpredictable in most patients. METHODS: Sequencing of NF1 in patients with cafè-au-lait spots identified the c.3112A>G variant. RNA analysis and a minigene assay were employed to investigate splicing. RESULTS: Here we report a novel genotype-phenotype correlation in NF1: the identification of the missense variant NM_000267.3:c.3112A>G p.(Arg1038Gly) in seven individuals from two unrelated families with a mild phenotype. All the patients manifest cafè-au-lait spots without neurofibromas or other NF1-associated complications, and Noonan syndrome features in most cases. The missense variant was not previously reported in available databases, segregates with the phenotype and involves a highly conserved residue. Both a minigene assay and patient's RNA analysis excluded an effect on splicing. CONCLUSION: Our data support the correlation of the p.Arg1038Gly missense substitution with the cutaneous phenotype without neurofibromas or other complications. This finding may have relevant implications for patients and genetic counseling, but also to get insights into the function of neurofibromin.

Catechol-O-Methyltransferase (COMT) Val158Met Polymorphism and Eating Disorders: Data From a New Biobank and Meta-Analysis of Previously Published Studies.

OBJECTIVES: We investigated whether catechol-O-methyltransferase (COMT) Val158Met polymorphism is associated with eating disorders (EDs). METHODS: We conducted a systematic literature search of studies published until 15 January 2017 and added data from the Italian 'Biobanca Veneta per i Disturbi Alimentari' biobank, performing a meta-analysis comparing COMT Val158Met genotype and allele frequencies in EDs and anorexia nervosa (AN) or bulimia nervosa (BN) patients versus controls. RESULTS: Ten studies plus Biobanca Veneta per i Disturbi Alimentari (ED: n = 920, controls: n = 261 controls) with 3541 ED patients (AN = 2388; BN = 233) and 3684 controls were included. There were no significant group differences in COMT Val158Met alleles and genotype frequencies between patients and controls, for all EDs pooled together [range of odds ratios (ORs): 0.96-1.04, p-values: 0.46-0.97, I2 = 0%] and when analysing separately patients with AN (ORs: 0.94-1.04, p-values: 0.31-0.61, I2 = 0%) or BN (ORs: 0.80-1.09, p-values: 0.28-0.64, I2 = 0-44%). CONCLUSIONS: Meta-analysing data results from 11 studies and 7225 subjects show that COMT Val158Met polymorphism is not associated with EDs. Copyright © 2017 John Wiley & Sons, Ltd and Eating Disorders Association.

ATXN2 trinucleotide repeat length correlates with risk of ALS.

We investigated a CAG trinucleotide repeat expansion in the ATXN2 gene in amyotrophic lateral sclerosis (ALS). Two new case-control studies, a British dataset of 1474 ALS cases and 567 controls, and a Dutch dataset of 1328 ALS cases and 691 controls were analyzed. In addition, to increase power, we systematically searched PubMed for case-control studies published after 1 August 2010 that investigated the association between ATXN2 intermediate repeats and ALS. We conducted a meta-analysis of the new and existing studies for the relative risks of ATXN2 intermediate repeat alleles of between 24 and 34 CAG trinucleotide repeats and ALS. There was an overall increased risk of ALS for those carrying intermediate sized trinucleotide repeat alleles (odds ratio 3.06 [95% confidence interval 2.37-3.94]; p = 6 × 10-18), with an exponential relationship between repeat length and ALS risk for alleles of 29-32 repeats (R2 = 0.91, p = 0.0002). No relationship was seen for repeat length and age of onset or survival. In contrast to trinucleotide repeat diseases, intermediate ATXN2 trinucleotide repeat expansion in ALS does not predict age of onset but does predict disease risk.

Functional connectivity correlates of response inhibition impairment in anorexia nervosa.

Anorexia nervosa (AN) is a disorder characterized by high levels of cognitive control and behavioral perseveration. The present study aims at exploring inhibitory control abilities and their functional connectivity correlates in patients with AN. Inhibitory control - an executive function that allows the realization of adaptive behavior according to environmental contingencies - has been assessed by means of the Stop-Signal paradigm. The study involved 155 patients with lifetime AN and 102 healthy women. A subsample underwent resting-state functional magnetic resonance imaging and was genotyped for COMT and 5-HTTLPR polymorphisms. AN patients showed an impaired response inhibition and a disruption of the functional connectivity of the ventral attention circuit, a neural network implicated in behavioral response when a stimulus occurs unexpected. The 5-HTTLPR genotype appears to significantly interact with the functional connectivity of ventral attention network in explaining task performance in both patients and controls, suggesting a role of the serotoninergic system in mechanisms of response selection. The disruption of the ventral attention network in patients with AN suggests lower efficiency of bottom-up signal filtering, which might be involved in difficulties to adapt behavioral responses to environmental needs. Our findings deserve further research to confirm their scientific and therapeutic implications.

Clinical and genetic correlates of decision making in anorexia nervosa.

INTRODUCTION: Decision-making (DM) abilities have been found to be impaired in anorexia nervosa (AN), but few data are available about the characteristics and correlates of this cognitive function. The aim of the present study was to provide data on DM functioning in AN using both veridical and adaptive paradigms. While in veridical DM tasks, the individual's ability to predict a true/false response is measured, adaptive DM is the ability to consider both internal and external demands in order to make a good choice, in the absence of a single true "correct" answer. METHOD: The participants were 189 women, of whom 91 were eating-disordered patients with a lifetime diagnosis of anorexia nervosa, and 98 were healthy women. All the participants underwent clinical, neuropsychological, and genetic assessment. The cognitive evaluation included a set of neuropsychological tasks and two decision-making tests: The Iowa Gambling Task and the Cognitive Bias Task. RESULTS: Anorexia nervosa patients showed significantly poorer performances on both decision-making tasks than healthy women. The Cognitive Bias Task revealed that anorexia nervosa patients employed significantly more context-independent decision-making strategies, which were independent from diagnostic subtype, handedness, education, and psychopathology. In the whole sample (patients and controls), Cognitive Bias Task performance was independently predicted by lifetime anorexia nervosa diagnosis, body mass index at assessment, and 5-HTTLPR genotype. CONCLUSIONS: Patients displayed poor decision-making functioning in both veridical and adaptive situations. The difficulties detected in anorexia nervosa individuals may affect not only the ability to consider the future outcomes of their actions (leading to "myopia for the future"), but also the capacity to update and review one's own mindset according to new environmental stimuli.

p.Arg1809Cys substitution in neurofibromin is associated with a distinctive NF1 phenotype without neurofibromas.

Analysis of 786 NF1 mutation-positive subjects with clinical diagnosis of neurofibromatosis type 1 (NF1) allowed to identify the heterozygous c.5425C>T missense variant (p.Arg1809Cys) in six (0.7%) unrelated probands (three familial and three sporadic cases), all exhibiting a mild form of disease. Detailed clinical characterization of these subjects and other eight affected relatives showed that all individuals had multiple cafè-au-lait spots, frequently associated with skinfold freckling, but absence of discrete cutaneous or plexiform neurofibromas, Lisch nodules, typical NF1 osseous lesions or symptomatic optic gliomas. Facial features in half of the individuals were suggestive of Noonan syndrome. Our finding and revision of the literature consistently indicate that the c.5425C>T change is associated with a distinctive, mild form of NF1, providing new data with direct impact on genetic counseling and patient management.

Neural signatures of the interaction between the 5-HTTLPR genotype and stressful life events in healthy women.

A change in neural connectivity of brain structures implicated in the memory of negative life events has been hypothesized to explain the enhancement of memory encoding during the processing of negative stimuli in depressed patients. Here, we investigated the effects of the interaction between negative life events and the 5-HTTLPR genotype - a polymorphism of the serotonin transporter gene - on the functional and structural connectivity of the hippocampal area in 34 healthy women. All participants were genotyped for the presence of the 5-HTTLPR short variant and for the A/G single-nucleotide polymorphism; they underwent clinical assessment including structured diagnostic interviews to exclude the presence of psychiatric disorders and to assess the presence of stressful life events. Resting state functional magnetic resonance imaging and diffusion tensor imaging scans were performed. We found significant interactions between stressful events and the 5-HTTLPR genotype in both the functional connectivity of the parahippocampus with the posterior cingulate cortex and the structural connectivity between the hippocampus and both the amygdala and the putamen. In addition, we found several genotype-related differences in the relationship between functional/structural connectivity of the hippocampal area and the ability to update expectations or stress-related phenotypes, such as anxiety symptoms. If confirmed by future studies, these mechanisms may clarify the role of the 5HTTLPR genotype as a risk factor for depression, in interaction with negative events.

Catechol-O-methyltransferase genotype modifies executive functioning and prefrontal functional connectivity in women with anorexia nervosa.

BACKGROUND: Anorexia nervosa is characterized by high levels of perseveration and inflexibility, which interfere with successful treatments. Dopamine (DA) signalling seems to play a key role in modulating the prefrontal cortex, since both DA deficiency and excess nega tively influence the efficiency of cognitive functions. The present study explores the effect of a functional polymorphism (Val158Met) in the catechol-O-methyltransferase (COMT) gene on the set-shifting abilities and prefrontal functional connectivity of patients with anorexia nervosa. METHODS: All participants performed the Wisconsin Card Sorting Task, and a subsample underwent resting-state functional magnetic resonance imaging. RESULTS: We included 166 patients with DSM-IV lifetime anorexia nervosa and 140 healthy women in our study. Both underweight and weight-recovered patients with anorexia nervosa showed high levels of perseveration, but only in the underweight group did the Val158Met polymorphism affect cognitive performance, showing the U-shaped curve characteristic of increased DA signalling in the prefrontal cortex. Underweight patients with anorexia nervosa who are Met homozygotes had significantly higher levels of perseveration and increased prefrontal functional connectivity than underweight patients in the other genotype groups, indicating abnormal regional cortical processing. LIMITATIONS: Although our data show that grey matter reduction in starving patients with anorexia nervosa did not explain our findings, the cross-sectional design of the present study did not allow us to distinguish between the effects of starvation and those of low estrogen levels. CONCLUSION: Starvation affects DA release in the prefrontal cortex of patients with anorexia nervosa with different effects on executive functioning and prefrontal functional connectivity according to the COMT genotype. This observation has several therapeutic implications that need to be addressed by future studies.

No difference in 5-HTTLPR and Stin2 polymorphisms frequency between premature ejaculation patients and controls.

INTRODUCTION: Premature ejaculation (PE) is defined as the inability of men to control ejaculation and it is the most prevalent male sexual dysfunction. The neurobiogenesis of ejaculation is very complex and involves the serotoninergic (5-hydroxytryptamine [5-HT]) system. A genetic etiology of PE in humans was stated accounting for around 30%. Recently, genetic polymorphisms located on SLC6A4 gene codifying for 5-HT transporter (5-HTT or serotonin transporter [SERT]), the major regulator of serotonergic neurotransmission, have been linked with the pathogenesis of PE and associated with the clinical response to therapy with contrasting results. AIM: In order to establish a possible pathogenetic link between PE and SLC6A4 polymorphisms, we analyzed the 5-HTT-linked polymorphic region (5-HTTLPR), rs25531, and STin2 polymorphisms in 121 patients affected by lifelong and acquired PE. METHODS: Polymerase chain reaction (PCR)-based technology followed by restriction fragment length polymorphism (RFLP) analysis. MAIN OUTCOME MEASURES: Intravaginal ejaculatory latency time was measured by stopwatch in order to diagnose PE, and the results of the SLC6A4 polymorphisms analysis in PE patients was compared with the control group. RESULTS: Genotype frequencies for 5-HTTLPR, rs25531, and STin2 for both patients and controls showed no significant deviation from Hardy-Weinberg equilibrium. No statistically significant differences were found in the frequency of SLC6A4 gene polymorphisms in PE patients vs. controls, or in lifelong PE patients vs. controls, or acquired PE patients vs. controls, or lifelong PE vs. acquired PE patients. The obtained data were contrasting with three out of four previously published reports. CONCLUSIONS: The present results indicate that no difference exists in SLC6A4 polymorphisms frequency between PE patients and controls. A comparison with the previously published reports on this field is reported.

Association study of AMH and AMHRII polymorphisms with unexplained infertility.

OBJECTIVE: To investigate the association of AMH and AMHRII polymorphisms with reproductive abilities in a sample of women with idiopathic infertility. DESIGN: Case-control study. SETTING: University Department of Obstetrics and Gynecology, and University Unit of Clinical Genetics. PATIENT(S): 76 women with idiopathic sterility and 100 fertile women as controls. INTERVENTION(S): Genotyping was performed by high-resolution melt analysis. MAIN OUTCOME MEASURE(S): Genotype distribution and allele frequency of AMH and AMHRII polymorphisms. Reconstruction of haplotype alleles to evaluate the linkage disequilibrium between single nucleotide polymorphisms. RESULT(S): Allele frequencies of -482 A>G, IVS 5-6 C>T, IVS 10+77 A>G, 146T>G polymorphisms are statistically significantly different in infertile patients compared with controls. CONCLUSION(S): Genetic variants of AMH and AMHRII genes seem to be associated with infertility, suggesting a role in the pathophysiology of normo-estrogenic and normo-ovulatory infertility. A clearer understanding of their function in ovarian physiology may help clinicians to find a role for antimüllerian hormone measurement in the field of reproductive medicine.

Is CFTR 621+3 A>G a cystic fibrosis causing mutation?

The 621+3 A>G variant of the CFTR gene was initially detected in four Greek patients with a severe form of cystic fibrosis, and it is reported to impair CFTR mRNA splicing. We present three lines of evidence that argue against the pathogenicity of this variant. First, its allelic frequency in the Italian population was 0.4%. Even considering the lowest value in the confidence interval we would expect 10% of Italian CF patients to be heterozygotes for this variant, whereas it has been reported only in one patient (0.04% of Italian CF patients). Second, expression of the 621+3 A>G variant in HeLa cells using a hybrid minigene showed that 39.5+/-1.1% of transcripts were correctly spliced, indicating that its effects on mRNA splicing are similar to those of the CFTR intron 8 5T variant, associated with congenital bilateral absence of vas deferens (CBAVD), but not with CF. Third, we have identified an asymptomatic individual who harbored the 621+3 A>G variant in trans with the Q552X mutation. Because 621+3 A>G is often included in population-screening programs, this information is critical to provide adequate counseling to patients. Further work should be aimed at investigating whether this variant may have a role in CBAVD or atypical CF.