New Insights into Endogenous Retrovirus-K Transcripts in Amyotrophic Lateral Sclerosis.

Retroviral reverse transcriptase activity and the increased expression of human endogenous retroviruses (HERVs) are associated with amyotrophic lateral sclerosis (ALS). We were interested in confirming HERVK overexpression in the ALS brain, its use as an accessory diagnostic marker for ALS, and its potential interplay with neuroinflammation. Using qPCR to analyze HERVK expression in peripheral blood mononuclear cells (PBMCs) and in postmortem brain samples from ALS patients, no significant differences were observed between patients and control subjects. By contrast, we report alterations in the expression patterns of specific HERVK copies, especially in the brainstem. Out of 27 HERVK copies sampled, the relative expression of 17 loci was >1.2-fold changed in samples from ALS patients. In particular, the relative expression of two HERVK copies (Chr3-3 and Chr3-5) was significantly different in brainstem samples from ALS patients compared with controls. Further qPCR analysis of inflammation markers in brain samples revealed a significant increase in NLRP3 levels, while TNFA, IL6, and GZMB showed slight decreases. We cannot confirm global HERVK overexpression in ALS, but we can report the ALS-specific overexpression of selected HERVK copies in the ALS brain. Our data are compatible with the requirement for better patient stratification and support the potential importance of particular HERVK copies in ALS.

Burden of Rare Variants in ALS and Axonal Hereditary Neuropathy Genes Influence Survival in ALS: Insights from a Next Generation Sequencing Study of an Italian ALS Cohort.

Although the genetic architecture of amyotrophic lateral sclerosis (ALS) is incompletely understood, recent findings suggest a complex model of inheritance in ALS, which is consistent with a multistep pathogenetic process. Therefore, the aim of our work is to further explore the architecture of ALS using targeted next generation sequencing (NGS) analysis, enriched in motor neuron diseases (MND)-associated genes which are also implicated in axonal hereditary motor neuropathy (HMN), in order to investigate if disease expression, including the progression rate, could be influenced by the combination of multiple rare gene variants. We analyzed 29 genes in an Italian cohort of 83 patients with both familial and sporadic ALS. Overall, we detected 43 rare variants in 17 different genes and found that 43.4% of the ALS patients harbored a variant in at least one of the investigated genes. Of note, 27.9% of the variants were identified in other MND- and HMN-associated genes. Moreover, multiple gene variants were identified in 17% of the patients. The burden of rare variants is associated with reduced survival and with the time to reach King stage 4, i.e., the time to reach the need for percutaneous endoscopic gastrostomy (PEG) positioning or non-invasive mechanical ventilation (NIMV) initiation, independently of known negative prognostic factors. Our data contribute to a better understanding of the molecular basis of ALS supporting the hypothesis that rare variant burden could play a role in the multistep model of disease and could exert a negative prognostic effect. Moreover, we further extend the genetic landscape of ALS to other MND-associated genes traditionally implicated in degenerative diseases of peripheral axons, such as HMN and CMT2.

Collagen XIX Alpha 1 Improves Prognosis in Amyotrophic Lateral Sclerosis.

The identification of more reliable diagnostic or prognostic biomarkers in age-related neurodegenerative diseases, such as Amyotrophic Lateral Sclerosis (ALS), is urgently needed. The objective in this study was to identify more reliable prognostic biomarkers of ALS mirroring neurodegeneration that could be of help in clinical trials. A total of 268 participants from three cohorts were included in this study. The muscle and blood cohorts were analyzed in two cross-sectional studies, while the serial blood cohort was analyzed in a longitudinal study at 6-monthly intervals. Fifteen target genes and fourteen proteins involved in muscle physiology and differentiation, metabolic processes and neuromuscular junction dismantlement were studied in the three cohorts. In the muscle biopsy cohort, the risk for a higher mortality in an ALS patient that showed high Collagen type XIX, alpha 1 (COL19A1) protein levels and a fast progression of the disease was 70.5% (P < 0.05), while in the blood cohort, this risk was 20% (P < 0.01). In the serial blood cohort, the linear mixed model analysis showed a significant association between increasing COL19A1 gene levels along disease progression and a faster progression during the follow-up period of 24 months (P < 0.05). Additionally, higher COL19A1 levels and a faster progression increased 17.9% the mortality risk (P < 0.01). We provide new evidence that COL19A1 can be considered a prognostic biomarker that could help the selection of homogeneous groups of patients for upcoming clinical trial and may be pointed out as a promising therapeutic target in ALS.

Combined evaluation of genotype and phenotype of thiopurine S-methyltransferase (TPMT) in the clinical management of patients in chronic therapy with azathioprine.

Background The thiopurine S-methyltransferase (TPMT)/azathioprine (AZA) gene-drug pair is one of the most well-known pharmacogenetic markers. Despite this, few studies investigated the implementation of TPMT testing and the combined evaluation of genotype and phenotype in multidisciplinary clinical settings where patients are undergoing chronic therapy with AZA. Methods A total of 356 AZA-treated patients for chronic autoimmune diseases were enrolled. DNA was isolated from whole blood and the samples were analyzed for the c.460G>A and c.719A>G variants by the restriction fragment length polymorphism (RFLP) technique and sequenced for the c.238G>C variant. The TPMT enzyme activity was determined in erythrocytes by a high-performance liquid chromatography (HPLC) assay. Results All the patients enrolled were genotyped while the TPMT enzyme activity was assessed in 41 patients. Clinical information was available on 181 patients. We found no significant difference in the odds of having adverse drug reactions (ADRs) in wild-type patients and variant allele carriers, but the latter had an extra risk of experiencing hematologically adverse events. The enzyme activity was significantly associated to genotype. Conclusions TPMT variant allele carriers have an extra risk of experiencing hematologically adverse events compared to wild-type patients. Interestingly, only two out of 30 (6.6%) patients had discordant results between genotype, phenotype and onset of ADRs.

Increase in DNA methylation in patients with amyotrophic lateral sclerosis carriers of not fully penetrant SOD1 mutations.

OBJECTIVE: More than 180 different superoxide dismutase 1 (SOD1) mutations have been described to date in amyotrophic lateral sclerosis (ALS) patients, including not completely penetrant ones leading to phenotypic heterogeneity among carriers. We collected DNA samples from five ALS families with not fully penetrant SOD1 mutations (p.Asn65Ser, p.Gly72Ser, p.Gly93Asp, and p.Gly130_Glu133del) searching for epigenetic differences among ALS patients, asymptomatic/paucisymptomatic carriers and non-carrier family members. METHODS: Global DNA methylation levels (5-methylcytosine levels) were determined in blood DNA samples with an enzyme-linked immunosorbent assay (ELISA), and the methylation analysis of SOD1, FUS, TARDBP and C9orf72 genes was performed using Methylation-Sensitive High-Resolution Melting (MS-HRM) technique. RESULTS: Global DNA methylation levels were significantly higher in blood DNA of ALS patients than in asymptomatic/paucisymptomatic carriers or family members non-carriers of SOD1 mutations, and a positive correlation between global DNA methylation levels and disease duration (months) was observed. SOD1, FUS, TARDBP and C9orf72 gene promoters were demethylated in all subjects. CONCLUSIONS: The present study suggests that global changes in DNA methylation might contribute to the ALS phenotype in carriers of not fully penetrant SOD1 mutations, thus reinforcing the role of epigenetic factors in modulating the phenotypic expression of the disease.

Serum Proteome in a Sporadic Amyotrophic Lateral Sclerosis Geographical Cluster.

This study is meant to characterize the serum proteome in a small geographical cluster of sporadic ALS subjects originating from a restricted geographical area and sharing the same environmental exposure, in a broader context of evaluating the relevance of environmental factors to disease onset, status, and progression. An Artificial Neural Network based software is used to compare the relative abundance of proteins identified as different (by means of bi-dimensional electrophoresis and mass spectrometry) in the serum proteome of patients and age-matched healthy controls. The patient's group is characterized by altered levels of acute phase reactants and of proteins involved in lipid homeostasis, along with over-representation of the APOE*4 allele. Characterization of the serum proteome in a small cluster of sporadic ALS patients, originating from a geographically restricted area with a high prevalence of the disease and evaluation of the results with software based on artificial neural networks, highlights the association of the relative abundance of some proteins (most notably, acute phase reactants and lipid homeostasis proteins) with the disease presence and status.

TBK1 mutations in Italian patients with amyotrophic lateral sclerosis: genetic and functional characterisation.

BACKGROUND: TANK-binding kinase 1 (TBK1) gene has been recently identified as a causative gene of amyotrophic lateral sclerosis (ALS). METHODS: We sequenced the TBK1 gene in a cohort of 154 Italian patients with ALS with unclear genetic aetiology. We subsequently assessed the pathogenic potential of novel identified TBK1 variants using functional in vitro studies: expression, targeting and activity were evaluated in patient-derived fibroblasts and in cells transfected with mutated-TBK1 plasmids. RESULTS: We identified novel genomic TBK1 variants including two loss-of-function (LoF) (p.Leu59Phefs*16 and c.358+5G>A), two missense (p.Asp118Asn and p.Ile397Thr) and one intronic variant (c.1644-5_1644-2delAATA), in addition to two previously reported pathogenetic missense variants (p.Lys291Glu and p.Arg357Gln). Functional studies in patient-derived fibroblasts revealed that the c.358+5G>A causes aberrant pre-mRNA processing leading TBK1 haploinsufficiency. Biochemical studies in cellular models showed that the truncating variant p.Leu59Phefs*16 abolishes TBK1 protein expression, whereas the p.Asp118Asn variant severely impairs TBK1 phosphorylation activity. Conversely, the p.Ile397Thr variant displayed enhanced phosphorylation activity, whose biological relevance is not clear. CONCLUSION: The observed frequency of TBK1 LoF variants was 1.3% (2/154), increasing up to 3.2% (5/154) by taking into account also the functional missense variants that we were able to classify as potentially pathogenic, supporting the relevance of TBK1 in the Italian population with ALS.

Genetic Counseling Dilemmas for a Patient with Sporadic Amyotrophic Lateral Sclerosis, Frontotemporal Degeneration & Parkinson's Disease.

Amyotrophic lateral sclerosis (ALS), frontotemporal degeneration and Parkinson's disease may be different expressions of the same neurodegenerative disease. However, association between ALS and parkinsonism-dementia complex (ALS-PDC) has only rarely been reported apart from the cluster detected in Guam. We report a patient presenting with ALS-PDC in whom pathological mutations/expansions were investigated. No other family members were reported to have any symptoms of a neurological condition. Our case demonstrates that ALS-PDC can occur as a sporadic disorder, even though the coexistence of the three clinical features in one patient suggests a single underlying genetic cause. It is known that genetic testing should be preferentially offered to patients with ALS who have affected first or second-degree relatives. However, this case illustrates the importance of genetic counseling for family members of patients with sporadic ALC-PDC in order to provide education on the low recurrence risk. Here, we dicuss the ethical, psychological and practical consequences for patients and their relatives.

Blood trace metals in a sporadic amyotrophic lateral sclerosis geographical cluster.

Amyotrophic lateral sclerosis (ALS) is a fatal disorder with unknown etiology, in which genetic and environmental factors interplay to determine the onset and the course of the disease. Exposure to toxic metals has been proposed to be involved in the etiology of the disease either through a direct damage or by promoting oxidative stress. In this study we evaluated the concentration of a panel of metals in serum and whole blood of a small group of sporadic patients, all living in a defined geographical area, for which acid mine drainage has been reported. ALS prevalence in this area is higher than in the rest of Italy. Results were analyzed with software based on artificial neural networks. High concentrations of metals (in particular Se, Mn and Al) were associated with the disease group. Arsenic serum concentration resulted lower in ALS patients, but it positively correlated with disease duration. Comet assay was performed to evaluate endogenous DNA damage that resulted not different between patients and controls. Up to now only few studies considered geographically well-defined clusters of ALS patients. Common geographical origin among patients and controls gave us the chance to perform metallomic investigations under comparable conditions of environmental exposure. Elaboration of these data with software based on machine learning processes has the potential to be extremely useful to gain a comprehensive view of the complex interactions eventually leading to disease, even in a small number of subjects.

The genetic and clinical spectrum of a large cohort of patients with distal renal tubular acidosis.

Primary distal renal tubular acidosis is a rare genetic disease. Mutations in SLC4A1, ATP6V0A4, and ATP6V1B1 genes have been described as the cause of the disease, transmitted as either an autosomal dominant or recessive trait. Particular clinical features, such as sensorineural hearing loss, have been mainly described in association with mutations in one gene instead of the others. Nevertheless, the diagnosis of distal renal tubular acidosis is essentially based on clinical and laboratory findings, and the series of patients described so far are usually represented by small cohorts. Therefore, a strict genotype-phenotype correlation is still lacking, and questions about whether clinical and laboratory data should direct the genetic analysis remain open. Here, we applied next-generation sequencing in 89 patients with a clinical diagnosis of distal renal tubular acidosis, analyzing the prevalence of genetic defects in SLC4A1, ATP6V0A4, and ATP6V1B1 genes and the clinical phenotype. A genetic cause was determined in 71.9% of cases. In our group of sporadic cases, clinical features, including sensorineural hearing loss, are not specific indicators of the causal underlying gene. Mutations in the ATP6V0A4 gene are quite as frequent as mutations in ATP6V1B1 in patients with recessive disease. Chronic kidney disease was frequent in patients with a long history of the disease. Thus, our results suggest that when distal renal tubular acidosis is suspected, complete genetic testing could be considered, irrespective of the clinical phenotype of the patient.

Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) as a model of small vessel disease: update on clinical, diagnostic, and management aspects.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common and best known monogenic small vessel disease. Here, we review the clinical, neuroimaging, neuropathological, genetic, and therapeutic aspects based on the most relevant articles published between 1994 and 2016 and on the personal experience of the authors, all directly involved in CADASIL research and care. We conclude with some suggestions that may help in the clinical practice and management of these patients.

A CGH array procedure to detect PAX6 gene structural defects.

Aniridia is a rare congenital disease characterized by eye development defects, in which the more evident clinical manifestation is iris absence or malformation. In most of the patients, aniridia is associated to PAX6 gene point mutations or deletions. When these deletions are large and involve other genes, a more complex disease, named WAGR syndrome, arises. In order to develop a new tool to analyze aniridia and WAGR subjects, a CGH array (CGHa) of the PAX6 genomic region was set up. We generated a custom microarray kit using an oligonucleotide-based platform that allows high resolution molecular profiling of genomic aberrations in 20 Mb of the 11p13 chromosomal region, centered on the PAX6 gene. The average probe spacing was 100 bp. Thirty-five subjects have been analyzed. The major advantage of CGHa compared to MLPA was the knowledge of the deletions borders. Our approach identifies patients harboring deletions including the WT1 gene and, therefore, at risk for kidney tumors. The CGHa assay confirmed that several aniridia patients show a deletion at the level of ELP4 gene, without involvement of the PAX6 exonic regions. In all these patients, deletions include the PAX6 transcriptional enhancer SIMO. This finding further highlights the role of mutation/deletion of long-range enhancers in monogenic human pathology.

Clinical evaluation and molecular screening of a large consecutive series of albino patients.

Oculocutaneous albinism (OCA) is characterized by hypopigmentation of the skin, hair and eye, and by ophthalmologic abnormalities caused by a deficiency in melanin biosynthesis. In this study we recruited 321 albino patients and screened them for the genes known to cause oculocutaneous albinism (OCA1-4 and OCA6) and ocular albinism (OA1). Our purpose was to detect mutations and genetic frequencies of the main causative genes, offering to albino patients an exhaustive diagnostic assessment within a multidisciplinary approach including ophthalmological, dermatological, audiological and genetic evaluations. We report 70 novel mutations and the frequencies of the major causative OCA genes that are as follows: TYR (44%), OCA2 (17%), TYRP1 (1%), SLC45A2 (7%) and SLC24A5 (<0.5%). An additional 5% of patients had GPR143 mutations. In 19% of cases, a second reliable mutation was not detected, whereas 7% of our patients remain still molecularly undiagnosed. This comprehensive study of a consecutive series of OCA/OA1 patients allowed us to perform a clinical evaluation of the different OCA forms.

Expanding the clinical spectrum of COL1A1 mutations in different forms of glaucoma.

BACKGROUND: Primary congenital glaucoma (PCG) and early onset glaucomas are one of the major causes of children and young adult blindness worldwide. Both autosomal recessive and dominant inheritance have been described with involvement of several genes including CYP1B1, FOXC1, PITX2, MYOC and PAX6. However, mutations in these genes explain only a small fraction of cases suggesting the presence of further candidate genes. METHODS: To elucidate further genetic causes of these conditions whole exome sequencing (WES) was performed in an Italian patient, diagnosed with PCG and retinal detachment, and his unaffected parents. Sanger sequencing of the complete coding region of COL1A1 was performed in a total of 26 further patients diagnosed with PCG or early onset glaucoma. Exclusion of pathogenic variations in known glaucoma genes as CYP1B1, MYOC, FOXC1, PITX2 and PAX6 was additionally done per Sanger sequencing and Multiple Ligation-dependent Probe Amplification (MLPA) analysis. RESULTS: In the patient diagnosed with PCG and retinal detachment, analysis of WES data identified compound heterozygous variants in COL1A1 (p.Met264Leu; p.Ala1083Thr). Targeted COL1A1 screening of 26 additional patients detected three further heterozygous variants (p.Arg253*, p.Gly767Ser and p.Gly154Val) in three distinct subjects: two of them diagnosed with early onset glaucoma and mild form of osteogenesis imperfecta (OI), one patient with a diagnosis of PCG at age 4 years. All five variants affected evolutionary, highly conserved amino acids indicating important functional restrictions. Molecular modeling predicted that the heterozygous variants are dominant in effect and affect protein stability and thus the amount of available protein, while the compound heterozygous variants act as recessive alleles and impair binding affinity to two main COL1A1 binding proteins: Hsp47 and fibronectin. CONCLUSIONS: Dominant inherited mutations in COL1A1 are known causes of connective tissues disorders such as OI. These disorders are also associated with different ocular abnormalities, although recognition of the common pathology for both features is seldom being recognized. Our results expand the role of COL1A1 mutations in different forms of early-onset glaucoma with and without signs of OI. Thus, we suggest including COL1A1 mutation screening in the genetic work-up of glaucoma cases and detailed ophthalmic examinations with fundus analysis in patients with OI.

Clinical Pregenetic Screening for Stroke Monogenic Diseases: Results From Lombardia GENS Registry.

BACKGROUND AND PURPOSE: Lombardia GENS is a multicentre prospective study aimed at diagnosing 5 single-gene disorders associated with stroke (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, Fabry disease, MELAS [mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes], hereditary cerebral amyloid angiopathy, and Marfan syndrome) by applying diagnostic algorithms specific for each clinically suspected disease METHODS: We enrolled a consecutive series of patients with ischemic or hemorrhagic stroke or transient ischemic attack admitted in stroke units in the Lombardia region participating in the project. Patients were defined as probable when presenting with stroke or transient ischemic attack of unknown etiopathogenic causes, or in the presence of <3 conventional vascular risk factors or young age at onset, or positive familial history or of specific clinical features. Patients fulfilling diagnostic algorithms specific for each monogenic disease (suspected) were referred for genetic analysis. RESULTS: In 209 patients (57.4±14.7 years), the application of the disease-specific algorithm identified 227 patients with possible monogenic disease. Genetic testing identified pathogenic mutations in 7% of these cases. Familial history of stroke was the only significant specific feature that distinguished mutated patients from nonmutated ones. The presence of cerebrovascular risk factors did not exclude a genetic disease. CONCLUSIONS: In patients prescreened using a clinical algorithm for monogenic disorders, we identified monogenic causes of events in 7% of patients in comparison to the 1% to 5% prevalence reported in previous series.

A novel OTX2 gene frameshift mutation in a child with microphthalmia, ectopic pituitary and growth hormone deficiency.

OTX2 mutations are reported in patients with eye maldevelopment and in some cases with brain or pituitary abnormalities. We describe a child carrying a novel OTX2 heterozygous mutation. She presented microphthalmia, absence of retinal vascularization, vitreal spots and optic nerve hypoplasia in the right eye and mild macular dystrophy in the left eye. Midline brain structures and cerebral parenchyma were normal, except for the ectopic posterior pituitary gland. OTX2 sequencing showed a heterozygous c.402del mutation. Most of OTX2 mutations are nonsense or frameshift introducing a premature termination codon and resulting in a truncated protein. More rarely missense mutations occur. Our novel OTX2 mutation (c.402del) is a frameshift mutation (p.S135Lfs*43), never reported before, causing a premature codon stop 43 amino-acids downstream, which is predicted to generate a premature truncation. The mutation was associated with microphthalmia and ectopic posterior pituitary.

Screening of PAX6 gene in Italian congenital aniridia patients revealed four novel mutations.

PURPOSE: To uncover underlying mutations in a cohort of Italian patients with aniridia, a rare congenital panocular condition with an incidence ranging from 1:64,000 to 1:100,000. The disease may be found isolated or in association with other syndromes characterized by partial or complete absence of the iris and iris hypoplasia. METHODS: We analyzed the PAX6 gene in 11 patients with aniridia fulfilling the following inclusion criteria: partial or complete absence of the iris and age < 18 years at the time of diagnosis. DNA sequence analysis was integrated with Multiple Ligation Probe Assay (MLPA) analysis. RESULTS: We identified seven PAX6 mutations, including four novel ones. The majority of mutations lie in the DNA-binding domain and all produce a truncated protein. All tested patients did not have WT1 gene deletions thus excluding the WAGR syndrome. We present the clinical findings in the four cases harboring novel mutations. We were unable to identify mutations in four cases with complete aniridia thus indicating that other gene/s could be involved in the disease. CONCLUSIONS: It is important to establish the molecular diagnosis early to avoid repeated and long-term screening for Wilms tumor. Our work further emphasizes that a wide range of ocular phenotypes are associated with loss of function PAX6 mutations. In addition to the possibility of stochastic variations, other genetic variations could play a role as modifier genes, thus giving rise to the observed different ocular phenotypes.