Systematic Collaborative Reanalysis of Genomic Data Improves Diagnostic Yield in Neurologic Rare Diseases.

Many patients experiencing a rare disease remain undiagnosed even after genomic testing. Reanalysis of existing genomic data has shown to increase diagnostic yield, although there are few systematic and comprehensive reanalysis efforts that enable collaborative interpretation and future reinterpretation. The Undiagnosed Rare Disease Program of Catalonia project collated previously inconclusive good quality genomic data (panels, exomes, and genomes) and standardized phenotypic profiles from 323 families (543 individuals) with a neurologic rare disease. The data were reanalyzed systematically to identify relatedness, runs of homozygosity, consanguinity, single-nucleotide variants, insertions and deletions, and copy number variants. Data were shared and collaboratively interpreted within the consortium through a customized Genome-Phenome Analysis Platform, which also enables future data reinterpretation. Reanalysis of existing genomic data provided a diagnosis for 20.7% of the patients, including 1.8% diagnosed after the generation of additional genomic data to identify a second pathogenic heterozygous variant. Diagnostic rate was significantly higher for family-based exome/genome reanalysis compared with singleton panels. Most new diagnoses were attributable to recent gene-disease associations (50.8%), additional or improved bioinformatic analysis (19.7%), and standardized phenotyping data integrated within the Undiagnosed Rare Disease Program of Catalonia Genome-Phenome Analysis Platform functionalities (18%).

Variability in Phelan-McDermid Syndrome in a Cohort of 210 Individuals.

Phelan-McDermid syndrome (PMS, OMIM# 606232) results from either different rearrangements at the distal region of the long arm of chromosome 22 (22q13.3) or pathogenic sequence variants in the SHANK3 gene. SHANK3 codes for a structural protein that plays a central role in the formation of the postsynaptic terminals and the maintenance of synaptic structures. Clinically, patients with PMS often present with global developmental delay, absent or severely delayed speech, neonatal hypotonia, minor dysmorphic features, and autism spectrum disorders (ASD), among other findings. Here, we describe a cohort of 210 patients with genetically confirmed PMS. We observed multiple variant types, including a significant number of small deletions (<0.5 Mb, 64/189) and SHANK3 sequence variants (21 cases). We also detected multiple types of rearrangements among microdeletion cases, including a significant number with post-zygotic mosaicism (9.0%, 17/189), ring chromosome 22 (10.6%, 20/189), unbalanced translocations (de novo or inherited, 6.4%), and additional rearrangements at 22q13 (6.3%, 12/189) as well as other copy number variations in other chromosomes, unrelated to 22q deletions (14.8%, 28/189). We compared the clinical and genetic characteristics among patients with different sizes of deletions and with SHANK3 variants. Our findings suggest that SHANK3 plays an important role in this syndrome but is probably not uniquely responsible for all the spectrum features in PMS. We emphasize that only an adequate combination of different molecular and cytogenetic approaches allows an accurate genetic diagnosis in PMS patients. Thus, a diagnostic algorithm is proposed.

Identifying susceptibility genes for primary ovarian insufficiency on the high-risk genetic background of a fragile X premutation.

OBJECTIVE: To identify modifying genes that explains the risk of fragile X-associated primary ovarian insufficiency (FXPOI). DESIGN: Gene-based, case/control association study, followed by a functional screen of highly ranked genes using a Drosophila model. SETTING: Participants were recruited from academic and clinical settings. PATIENT(S): Women with a premutation (PM) who experienced FXPOI at the age of 35 years or younger (n = 63) and women with a PM who experienced menopause at the age of 50 years or older (n = 51) provided clinical information and a deoxyribonucleic acid sample for whole genome sequencing. The functional screen was on the basis of Drosophila TRiP lines. INTERVENTION(S): Clinical information and a DNA sample were collected for whole genome sequencing. MAIN OUTCOME MEASURES: A polygenic risk score derived from common variants associated with natural age at menopause was calculated and associated with the risk of FXPOI. Genes associated with the risk of FXPOI were identified on the basis of the P-value from gene-based association test and an altered level of fecundity when knocked down in the Drosophila PM model. RESULTS: The polygenic risk score on the basis of common variants associated with natural age at menopause explained approximately 8% of the variance in the risk of FXPOI. Further, SUMO1 and KRR1 were identified as possible modifying genes associated with the risk of FXPOI on the basis of an untargeted gene analysis of rare variants. CONCLUSIONS: In addition to the large genetic effect of a PM on ovarian function, the additive effects of common variants associated with natural age at menopause and the effect of rare modifying variants appear to play a role in FXPOI risk.

Chromosome microarray analysis should be offered to all invasive prenatal diagnostic testing following a normal rapid aneuploidy test result.

Chromosomal microarray analysis (CMA) has now replaced karyotyping in the analysis of prenatal cases with a fetal structural anomaly, whereas in those pregnancies undergoing invasive prenatal diagnosis with a normal fetal ultrasound, conventional karyotyping is still performed. The aims of this study were to establish the diagnostic yield of CMA in prenatal diagnosis, and to provide new data that might contribute to reconsider current practices. We reviewed 2905 prenatal samples with a normal rapid aneuploidy detection test referred for evaluation by CMA testing. Our study revealed pathogenic and reported susceptibility copy number variants associated with syndromic disorders in 4.8% (n = 138/2905) of cases, being 2.8% (n = 81/2905) the estimated added diagnostic value of CMA over karyotyping. Clinically significant CMA abnormality was detected in 5.4% (107/1975) of the fetuses with ultrasound anomalies and in 1.4% (5/345) of those considered as low-risk pregnancies. Our series shows that in prenatal samples, CMA increases 2-fold the diagnostic yield achieved by conventional karyotyping.

An exome-wide exploration of cases of primary ovarian insufficiency uncovers novel sequence variants and candidate genes.

Primary ovarian insufficiency (POI) implies the cessation of menstruation for several months in women before the age of 40 years and is a major cause of infertility. The study of the contribution of genetic factors to POI has been fueled by the use of whole exome sequencing (WES). Here, to uncover novel causative pathogenic variants and risk alleles, WES has been performed in 12 patients with familial POI (eight unrelated index cases and two pairs of sisters) and six women with early menopause and family history of POI (four index cases and one pair of sisters). Likely causative variants in NR5A1 and MCM9 genes were identified as well as a variant in INHA that requires further investigation. Moreover, we have identified more than one candidate variant in 3 out of 15 familial cases. Taken together, our results highlight the genetic heterogeneity of POI and early menopause and support the hypothesis of an oligogenic inheritance of such conditions, in addition to monogenic inheritance.

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.

A phase 1, randomized double-blind, placebo controlled trial to evaluate safety and efficacy of epigallocatechin-3-gallate and cognitive training in adults with Fragile X syndrome.

BACKGROUND & AIMS: Despite the wide spectrum of experimental compounds tested in clinical trials, there is still no proven pharmacological treatment available for Fragile-X syndrome (FXS), since several targeted clinical trials with high expectations of success have failed to demonstrate significant improvements. Here we tested epigallocatechin-3-gallate (EGCG) as a treatment option for ameliorating core cognitive and behavioral features in FXS. METHODS: We conducted preclinical studies in Fmr1 knockout mice (Fmr1-/y) using novel object-recognition memory paradigm upon acute EGCG (10 mg/kg) administration. Furthermore we conducted a double-blind placebo-controlled phase I clinical trial (TESXF; NCT01855971). Twenty-seven subjects with FXS (18-55 years) were administered of EGCG (5-7 mg/kg/day) combined with cognitive training (CT) during 3 months with 3 months of follow-up after treatment discontinuation. RESULTS: Preclinical studies showed an improvement in memory using the Novel Object Recognition paradigm. We found that FXS patients receiving EGCG + CT significantly improved cognition (visual episodic memory) and functional competence (ABAS II-Home Living skills) in everyday life compared to subjects receiving Placebo + CT. CONCLUSIONS: Phase 2 clinical trials in larger groups of subjects are necessary to establish the therapeutic potential of EGCG for the improvement of cognition and daily life competences in FXS.

Expansions and contractions of the FMR1 CGG repeat in 5,508 transmissions of normal, intermediate, and premutation alleles.

Instability of the FMR1 repeat, commonly observed in transmissions of premutation alleles (55-200 repeats), is influenced by the size of the repeat, its internal structure and the sex of the transmitting parent. We assessed these three factors in unstable transmissions of 14/3,335 normal (~5 to 44 repeats), 54/293 intermediate (45-54 repeats), and 1561/1,880 premutation alleles. While most unstable transmissions led to expansions, contractions to smaller repeats were observed in all size classes. For normal alleles, instability was more frequent in paternal transmissions and in alleles with long 3' uninterrupted repeat lengths. For premutation alleles, contractions also occurred more often in paternal than maternal transmissions and the frequency of paternal contractions increased linearly with repeat size. All paternal premutation allele contractions were transmitted as premutation alleles, but maternal premutation allele contractions were transmitted as premutation, intermediate, or normal alleles. The eight losses of AGG interruptions in the FMR1 repeat occurred exclusively in contractions of maternal premutation alleles. We propose a refined model of FMR1 repeat progression from normal to premutation size and suggest that most normal alleles without AGG interruptions are derived from contractions of maternal premutation alleles.

Fragile X-associated tremor/ataxia syndrome: Regional decrease of mitochondrial DNA copy number relates to clinical manifestations.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder that appears in at least one-third of adult carriers of a premutation (55-200 CGG repeats) in the fragile X mental retardation 1 (FMR1) gene. Several studies have shown that mitochondrial dysfunction may play a central role in aging and also in neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease as well as in FXTAS. It has been recently proposed that mtDNA copy number, measured by the number of mitochondrial genomes per nuclear genome (diploid), could be a useful biomarker of mitochondrial dysfunction. In order to elucidate the role of mtDNA variation in the pathogenesis of FXTAS, mtDNA copy number was quantified by digital droplet Polymerase chain reaction. In human brain samples, mtDNA levels were measured in the cerebellar vermis, dentate nucleus, parietal and temporal cortex, thalamus, caudate nucleus and hippocampus from a female FXTAS patient, a FMR1 premutation male carrier without FXTAS and from three male controls. The mtDNA copy number was further analyzed using this technology in dermal fibroblasts primary cultures derived from three FXTAS patients and three controls as well as in cortex and cerebellum of a CGG knock in FXTAS mice model. Finally, qPCR was carried out in human blood samples. Results indicate reduced mtDNA copy number in the specific brain region associated with disease progression in FXTAS patients, providing new insights into the role of mitochondrial dysfunction in the pathogenesis of FXTAS.

Genetic linkage analysis of a large family identifies FIGN as a candidate modulator of reduced penetrance in heritable pulmonary arterial hypertension.

BACKGROUND: Mapping the genetic component of molecular mechanisms responsible for the reduced penetrance (RP) of rare disorders constitutes one of the most challenging problems in human genetics. Heritable pulmonary arterial hypertension (PAH) is one such disorder characterised by rare mutations mostly occurring in the bone morphogenetic protein receptor type 2 (BMPR2) gene and a wide heterogeneity of penetrance modifier mechanisms. Here, we analyse 32 genotyped individuals from a large Iberian family of 65 members, including 22 carriers of the pathogenic BMPR2 mutation c.1472G>A (p.Arg491Gln), 8 of them diagnosed with PAH by right-heart catheterisation, leading to an RP rate of 36.4%. METHODS: We performed a linkage analysis on the genotyping data to search for genetic modifiers of penetrance. Using functional genomics data, we characterised the candidate region identified by linkage analysis. We also predicted the haplotype segregation within the family. RESULTS: We identified a candidate chromosome region in 2q24.3, 38 Mb upstream from BMPR2, with significant linkage (LOD=4.09) under a PAH susceptibility model. This region contains common variants associated with vascular aetiology and shows functional evidence that the putative genetic modifier is located in the upstream distal promoter of the fidgetin (FIGN) gene. CONCLUSION: Our results suggest that the genetic modifier acts through FIGN transcriptional regulation, whose expression variability would contribute to modulating heritable PAH. This finding may help to advance our understanding of RP in PAH across families sharing the p.Arg491Gln pathogenic mutation in BMPR2.

Quantitative analysis of somatically acquired and constitutive uniparental disomy in gastrointestinal cancers.

Somatically acquired uniparental disomies (aUPDs) are frequent events in solid tumors and have been associated with cancer-related genes. Studies assessing their functional consequences across several cancer types are therefore necessary. Here, we aimed at integrating aUPD profiles with the mutational status of cancer-related genes in a tumor-type specific manner. Using TCGA datasets for 1,032 gastrointestinal cancers, including colon (COAD), rectum (READ), stomach (STAD), esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC), we show a non-random distribution of aUPD, suggesting the existence of a cancer-specific landscape of aUPD events. Our analysis indicates that aUPD acts as a "second hit" in Knudson's model in order to achieve biallelic inactivation of tumor suppressor genes. In particular, APC, ARID1A and NOTCH1 were recurrently inactivated by the presence of homozygous mutation as a consequence of aUPD in COAD and READ, STAD and ESCC, respectively. Furthermore, while TP53 showed inactivation caused by aUPD at chromosome arm 17p across all tumor types, copy number losses at this genomic position were also frequent. By experimental and computationally inferring genome ploidy, we demonstrate that an increased number of aUPD events, both affecting the whole chromosome or segments of it, were present in highly aneuploid genomes compared to near-diploid tumors. Finally, the presence of mosaic UPD was detected at a higher frequency in DNA extracted from peripheral blood lymphocytes of patients with colorectal cancer compared to healthy individuals. In summary, our study defines specific profiles of aUPD in gastrointestinal cancers and provides unequivocal evidence of their relevance in cancer.

Paternal transmission of a FMR1 full mutation allele.

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability (ID) and autism. In most of cases, the molecular basis of this syndrome is a CGG repeat expansion in the 5' untranslated region of the FMR1 gene. It is inherited as an X linked dominant trait, with a reduced penetrance (80% for males and 30% for females). Full mutation (FM) expansion from premutated alleles (PM) is only acquired via maternal meiosis, while paternal transmission always remains in the PM range. We present a 16-year-old girl with a mild fragile X syndrome phenotype. FMR1 gene study showed that the patient inherited a mosaic premutation-full mutation with an unmethylated uninterrupted allele (175, >200 CGG) from her father. The father showed an 88 CGG uninterrupted unmethylated allele in blood and sperm cells. To our knowledge, this is the first case of a FMR1 mosaic premutation-full mutation allele inherited from a PM father. In our opinion, the most likely explanation could be a postzygotic somatic expansion. We can conclude that in rare cases of a child with a full mutation whose mother does not carry a premutation, the possibility of paternal transmission should be considered.

New insights into the regulatory function of CYFIP1 in the context of WAVE- and FMRP-containing complexes.

Cytoplasmic FMRP interacting protein 1 (CYFIP1) is a candidate gene for intellectual disability (ID), autism, schizophrenia and epilepsy. It is a member of a family of proteins that is highly conserved during evolution, sharing high homology with its Drosophila homolog, dCYFIP. CYFIP1 interacts with the Fragile X mental retardation protein (FMRP, encoded by the FMR1 gene), whose absence causes Fragile X syndrome, and with the translation initiation factor eIF4E. It is a member of the WAVE regulatory complex (WRC), thus representing a link between translational regulation and the actin cytoskeleton. Here, we present data showing a correlation between mRNA levels of CYFIP1 and other members of the WRC. This suggests a tight regulation of the levels of the WRC members, not only by post-translational mechanisms, as previously hypothesized. Moreover, we studied the impact of loss of function of both CYFIP1 and FMRP on neuronal growth and differentiation in two animal models - fly and mouse. We show that these two proteins antagonize each other's function not only during neuromuscular junction growth in the fly but also during new neuronal differentiation in the olfactory bulb of adult mice. Mechanistically, FMRP and CYFIP1 modulate mTor signaling in an antagonistic manner, likely via independent pathways, supporting the results obtained in mouse as well as in fly at the morphological level. Collectively, our results illustrate a new model to explain the cellular roles of FMRP and CYFIP1 and the molecular significance of their interaction.

The p. R151C Polymorphism in MC1R Gene Modifies the Age of Onset in Spanish Huntington's Disease Patients.

The expansion of CAG repeats (≥36 CAG) in the HTT gene is the only known genetic cause of Huntington's disease (HD) and the main determinant of the course of the disease. The length of the expanded CAG repeats correlates inversely with the age of onset (AOO) but does not completely determine it. We investigated the role of the melanocortin 1 receptor (MC1R) gene as a modifier factor of AOO in 600 HD patients from Spain. We sequenced the entire region of the MC1R gene and analyzed all the nonsynonymous MC1R genetic variants with a minor allele frequency of at least 0.01 in HD patients. The variability in AOO attributable to the CAG repeats and MC1R polymorphisms was evaluated using a multiple linear regression model. We found that the loss-of-function p. R151C MC1R polymorphism has a significant influence on the AOO (P = 0.004; Bonferroni-corrected P = 0.032) which explains 1.42% of the variance in AOO that cannot be accounted for by the expanded CAG repeat. Our results suggest that the MC1R gene could modify the AOO in Spanish HD patients and encourage the evaluation of loss-of-function MC1R polymorphisms in other HD populations with a higher frequency of these MC1R polymorphisms.

Update on the Clinical, Radiographic, and Neurobehavioral Manifestations in FXTAS and FMR1 Premutation Carriers.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a progressive neurodegenerative disorder caused by a repeat expansion in the fragile X mental retardation 1 (FMR1) gene. The disorder is characterized by kinetic tremor and cerebellar ataxia, shows age-dependent penetrance, and occurs more frequently in men. This paper summarizes the key emerging issues in FXTAS as presented at the Second International Conference on the FMR1 Premutation: Basic Mechanisms & Clinical Involvement in 2015. The topics discussed include phenotype-genotype relationships, neurobehavioral function, and updates on FXTAS genetics and imaging.

Carriage of One or Two FMR1 Premutation Alleles Seems to Have No Effect on Illness Severity in a FXTAS Female with an Autozygous FMR1 Premutation Allele.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder that occurs in FMR1 premutation carriers. The prevalence of FMR1 premutation carriers in the general population is relatively high, and although rare, a premutation in both X chromosomes may occur in females inheriting a premutation allele from each of both parent carriers. Here, we report the first female with an autozygous (homozygous by descendent) FMR1 premutation allele, who fulfills neurological and radiological FXTAS findings/criteria. Molecular characterization included CGG repeat length, AGG interruption pattern, FMR1 messenger RNA (mRNA), fragile X mental retardation protein (FMRP) level quantification, and single-nucleotide polymorphism (SNP) microarray. Neuroradiological assessment of 3-T magnetic resonance imaging and neurological and cognitive/neuropsychological evaluations were performed. Neurological and neuroradiological examination of the female with the same FMR1 allele in the premutation range (77 CGGs) demonstrated FXTAS features. Further familial evaluation showed a similar neuropsychiatric profile, with impairments in cognitive flexibility and visuospatial function, mainly. A unique family with an autozygous FMR1 premutation female is presented. Neurological/cognitive and neuroradiological examinations revealed FXTAS-specific findings in the female with the autozygous FMR1 premutation allele. The consistent molecular and cognitive/psychiatric phenotype in family members suggests that carrying one or two FMR1 premutation alleles has no effect on illness severity.

FMR1 Premutation: Basic Mechanisms and Clinical Involvement.

The wide spectrum of clinical phenotypes associated with the FMR1 premutation affect more than two million people worldwide. The clinical implications have only been recognized recently despite this disorder constitutes a relevant health problem. The present issue of The Cerebellum is focused on the "2(nd) International Conference on the FMR1 Premutation: Basic Mechanisms and Clinical Involvement" held in Sitges, Barcelona (Spain), from September 30th to October 2nd, 2015. The conference was attended by professionals from different countries in Europe, the USA, Chile, Israel, Australia, and Indonesia and covered the latest clinical and molecular findings resulting from FMR1 premutation studies. Although the pathologies associated with the FMR1 premutation are considered as rare diseases, seventy abstracts were presented. This reflects the relevance of this topic in the medical community and the growing interest among professionals from other disciplines. The major topics discussed included why and how the mRNA toxicity due to a gain of function and non-canonical RAN are responsible for disorders associated with the premutation. Several presentations addressed the impact of these mechanisms in FXTAS and FXPOI, two clinical presentations caused by the FMR1 premutation. Interestingly, a deterioration of the DNA repair machinery was first proposed as the pathogenicity cause of premutation alleles. Communications related to FXTAS and FXPOI animal models were also presented. These models facilitate studies aimed to understand disease progression and early treatment interventions. Finally, there were presentations related to psychiatric, psychological, neurological, and radiological aspects. Interesting discussion on intermediate alleles and their involvement in clinical and reproductive aspects was generated. In this regards, genetic counselling is improved by taking into account the AGG interruptions and including information about the FMR1 premutation associated pathologies although there are still some uncertainties linked to the spectrum of these pathologies. Overall, the meeting covered all aspects of the different pathologies associated with the premutation of the FMR1 gene.

Heterogeneity of a Constitutional Complex Chromosomal Rearrangement in 2q.

Complex chromosome rearrangements (CCRs) are unusual structural chromosome alterations found in humans, and to date only a few have been characterized molecularly. New mechanisms, such as chromothripsis, have been proposed to explain the presence of the CCRs in cancer cells and in patients with congenital disorders and/or mental retardation. The aim of the present study was the molecular characterization of a constitutional CCR in a girl with multiple congenital disorders and intellectual disability in order to determine the genotype-phenotype relation and to clarify whether the CCR could have been caused by chromosomal catastrophic events. The present CCR was characterized by G-banding, high-resolution CGH, multiplex ligation-dependent probe amplification and subtelomeric 2q-FISH analyses. Preliminary results indicate that the de novo CCR is unbalanced showing a 2q37.3 deletion and 2q34q37.2 partial trisomy. Our patient shows some of the typical traits and intellectual disability described in patients with 2q37 deletion and also in carriers of 2q34q37.2 partial trisomy; thus, the clinical disorders could be explained by additional effects of both chromosome alterations (deletions and duplications). A posterior, sequential FISH study using BAC probes revealed the unexpected presence of at least 17 different reorganizations affecting 2q34q37.2, suggesting the existence of chromosome instability in this region. The present CCR is the first case described in the literature of heterogeneity of unbalanced CCRs affecting a small region of 2q, indicating that the mechanisms involved in constitutional chromosome rearrangement may be more complex than previously thought.