ABSTRACT
OBJECTIVES: To implement non-invasive prenatal testing (NIPT) for fetal aneuploidies with semiconductor sequencing in an academic cytogenomic laboratory and to evaluate the first 15-month experience on clinical samples. METHODS: We validated a NIPT protocol for cell-free fetal DNA sequencing from maternal plasma for the detection of trisomy 13, 18 and 21 on a semiconductor sequencing instrument. Fetal DNA fraction calculation for all samples and several quality parameters were implemented in the workflow. One thousand eighty-one clinical NIPT samples were analysed, following the described protocol. RESULTS: Non-invasive prenatal testing was successfully implemented and validated on 201 normal and 74 aneuploid samples. From 1081 clinical samples, 17 samples showed an abnormal result: 14 trisomy 21 samples, one trisomy 18 and one trisomy 16 were detected. Also a maternal copy number variation on chromosome 13 was observed, which could potentially lead to a false positive trisomy 13 result. One sex discordant result was reported, possibly attributable to a vanishing twin. Moreover, our combined fetal fraction calculation enabled a more reliable risk estimate for trisomy 13, 18 and 21. CONCLUSIONS: Non-invasive prenatal testing for trisomy 21, 18 and 13 has a very high specificity and sensitivity. Because of several biological phenomena, diagnostic invasive confirmation of abnormal results remains required. © 2016 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.
Subject(s)
Chromosome Disorders/diagnosis , DNA/blood , Prenatal Diagnosis/methods , Semiconductors , Sequence Analysis, DNA/methods , Chromosome Disorders/genetics , Chromosomes, Human, Pair 13/genetics , Chromosomes, Human, Pair 18/genetics , DNA/analysis , DNA Copy Number Variations , Down Syndrome/diagnosis , Down Syndrome/genetics , False Positive Reactions , Female , Genetic Testing/methods , Humans , Pregnancy , Reproducibility of Results , Sensitivity and Specificity , Trisomy/diagnosis , Trisomy/genetics , Trisomy 13 Syndrome , Trisomy 18 SyndromeABSTRACT
Glycinergic neurotransmission is a major inhibitory influence in the CNS and its disruption triggers a paediatric and adult startle disorder, hyperekplexia. The postsynaptic α(1)-subunit (GLRA1) of the inhibitory glycine receptor (GlyR) and the cognate presynaptic glycine transporter (SLC6A5/GlyT2) are well-established genes of effect in hyperekplexia. Nevertheless, 52% of cases (117 from 232) remain gene negative and unexplained. Ligand-gated heteropentameric GlyRs form chloride ion channels that contain the α(1) and ß-subunits (GLRB) in a 2α(1):3ß configuration and they form the predominant population of GlyRs in the postnatal and adult human brain, brainstem and spinal cord. We screened GLRB through 117 GLRA1- and SLC6A5-negative hyperekplexia patients using a multiplex-polymerase chain reaction and Sanger sequencing approach. The screening identified recessive and dominant GLRB variants in 12 unrelated hyperekplexia probands. This primarily yielded homozygous null mutations, with nonsense (n = 3), small indel (n = 1), a large 95 kb deletion (n = 1), frameshifts (n = 1) and one recurrent splicing variant found in four cases. A further three cases were found with two homozygous and one dominant GLRB missense mutations. We provide strong evidence for the pathogenicity of GLRB mutations using splicing assays, deletion mapping, cell-surface biotinylation, expression studies and molecular modelling. This study describes the definitive assignment of GLRB as the third major gene for hyperekplexia and impacts on the genetic stratification and biological causation of this neonatal/paediatric disorder. Driven principally by consanguineous homozygosity of GLRB mutations, the study reveals long-term additive phenotypic outcomes for affected cases such as severe apnoea attacks, learning difficulties and developmental delay.
Subject(s)
Epilepsy/genetics , Genetic Diseases, X-Linked/genetics , Muscle Hypertonia/genetics , Receptors, Glycine/genetics , Reflex, Abnormal/genetics , Adolescent , Adult , Amino Acid Sequence , Child , Child, Preschool , Epilepsy/physiopathology , Female , Genetic Diseases, X-Linked/physiopathology , Genetic Predisposition to Disease , Glycine Plasma Membrane Transport Proteins/genetics , Glycine Plasma Membrane Transport Proteins/metabolism , Homozygote , Humans , Male , Molecular Sequence Data , Muscle Hypertonia/physiopathology , Mutation , Pedigree , Protein Conformation , RNA Splice Sites/genetics , Receptors, Glycine/chemistry , Receptors, Glycine/metabolism , Structure-Activity RelationshipABSTRACT
Hyperekplexia is a syndrome of readily provoked startle responses, alongside episodic and generalized hypertonia, that presents within the first month of life. Inhibitory glycine receptors are pentameric ligand-gated ion channels with a definitive and clinically well stratified linkage to hyperekplexia. Most hyperekplexia cases are caused by mutations in the α1 subunit of the human glycine receptor (hGlyR) gene (GLRA1). Here we analyzed 68 new unrelated hyperekplexia probands for GLRA1 mutations and identified 19 mutations, of which 9 were novel. Electrophysiological analysis demonstrated that the dominant mutations p.Q226E, p.V280M, and p.R414H induced spontaneous channel activity, indicating that this is a recurring mechanism in hGlyR pathophysiology. p.Q226E, at the top of TM1, most likely induced tonic activation via an enhanced electrostatic attraction to p.R271 at the top of TM2, suggesting a structural mechanism for channel activation. Receptors incorporating p.P230S (which is heterozygous with p.R65W) desensitized much faster than wild type receptors and represent a new TM1 site capable of modulating desensitization. The recessive mutations p.R72C, p.R218W, p.L291P, p.D388A, and p.E375X precluded cell surface expression unless co-expressed with α1 wild type subunits. The recessive p.E375X mutation resulted in subunit truncation upstream of the TM4 domain. Surprisingly, on the basis of three independent assays, we were able to infer that p.E375X truncated subunits are incorporated into functional hGlyRs together with unmutated α1 or α1 plus ß subunits. These aberrant receptors exhibit significantly reduced glycine sensitivity. To our knowledge, this is the first suggestion that subunits lacking TM4 domains might be incorporated into functional pentameric ligand-gated ion channel receptors.
Subject(s)
Gene Expression Regulation , Muscle Rigidity/metabolism , Mutation, Missense , Receptors, Glycine/metabolism , Amino Acid Substitution , Female , Humans , Male , Muscle Rigidity/genetics , Protein Structure, Secondary , Protein Structure, Tertiary , Receptors, Glycine/geneticsABSTRACT
Congenital hyperekplexia is a rare, potentially treatable neuromotor disorder. Three major genes of effect are known, and all three affect glycinergic neurotransmission. Two genes encode for subunits of the postsynaptic inhibitory glycine receptor, GLRA1 encoding the α1 subunit and GLRB encoding the ß subunit. The third, SLC6A5, encodes the cognate presynaptic glycine transporter 2. Ninety-seven individuals had a clinical diagnosis of hyperekplexia confirmed by genetic testing: 61 cases had mutations in GLRA1, 24 cases in SLC6A5 and 12 in GLRB. Detailed retrospective clinical analysis ascertained that all gene-positive cases present in the neonatal period (occasionally prenatally) and that clonazepam is the treatment of choice (95% found it to be efficacious). We confirm that hyperekplexia is predominantly a recessive condition but dominant cases are seen (16%). We found no genetic evidence for 'major' or 'minor' forms of hyperekplexia on a population basis. Thirty-five gene-negative cases were studied for comparison, their cardinal feature was presentation after the first month of life (P < 0.001). In addition to the characteristic 'stiffness, startles and stumbles' of hyperekplexia, apnoea attacks (50 of 89) and delayed development (47 of 92) were frequently reported. Patients with SLC6A5 mutations were significantly more likely to have had recurrent infantile apnoeas (RR1.9; P < 0.005) than those with GLRA1 mutations. Patients with GLRB and SLC6A5 mutations were more likely to have developmental delay (RR1.5 P < 0.01; RR1.9 P < 0.03) than those with GLRA1 mutations; 92% of GLRB cases reported a mild to severe delay in speech acquisition. Molecular modelling of pathogenic mutations demonstrates specific patterns of protein disruption that can be used to predict phenotype severity. The developmental delay in hyperekplexia, and speech acquisition in particular, may represent failure of developmental neural networks or subtle neurogenic migration defects in the absence of presynaptic glycine release. We recommend early genetic testing for symptomatic neonates and possibly preconception counselling for those at risk for GLRB and SLC6A5 mutations, because of the more challenging phenotype.
Subject(s)
Epilepsy/genetics , Genetic Diseases, X-Linked/genetics , Language Development Disorders/genetics , Mutation/genetics , Reflex, Abnormal/genetics , Adolescent , Adult , Female , Genetic Association Studies , Genetic Predisposition to Disease , Humans , Learning/physiology , Male , Phenotype , Receptors, Glycine/genetics , Retrospective Studies , Young AdultABSTRACT
Multiple endocrine neoplasia type 1 (MEN1) is characterized by tumors of the parathyroid, enteropancreas, and anterior pituitary. The MEN1 gene encodes the tumor suppressor menin of 610 amino acids that has multiple protein partners and activities. The particular pathways that, when lost, lead to tumorigenesis are not known. We demonstrated that members of a three-generation MEN1 kindred are heterozygous for a donor splice site mutation at the beginning of intron 3 (IVS3 + 1GâA). Lymphoblastoid cells of a mutant gene carrier had, in addition to the wild-type menin transcript, an aberrant transcript resulting from use of a cryptic splice site within exon III that splices to the start of exon IV. The predicted menin Δ(184-218) mutant has an in-frame deletion of 35 amino acids but is otherwise of wild-type sequence. The transfected menin Δ(184-218) mutant was well expressed and fully able to mediate the normal inhibition of the activity of the transcriptional regulators JunD and NF-κB. However, it was defective in mediating TGF-ß-stimulated Smad3 action in promoter-reporter assays in insulinoma cells. Importantly, lymphoblastoid cells from an individual heterozygous for the mutation had reduced TGF-ß-induced (Smad3) transcriptional activity but normal JunD and NF-κB function. In addition, the mutant gene carrier lymphoblastoid cells proliferated faster and were less responsive to the cytostatic effects of TGF-ß than cells from an unaffected family member. In conclusion, the menin mutant exhibits selective loss of the TGF-ß signaling pathway and loss of cell proliferation control contributing to the development of MEN1.
Subject(s)
Amino Acid Sequence/genetics , Multiple Endocrine Neoplasia Type 1/metabolism , Proto-Oncogene Proteins/metabolism , RNA Splice Sites/genetics , Sequence Deletion , Signal Transduction , Transforming Growth Factor beta/metabolism , Adolescent , Adult , Aged, 80 and over , Animals , Cell Line, Tumor , Cell Proliferation , Female , Humans , Introns , Male , Middle Aged , Multiple Endocrine Neoplasia Type 1/genetics , Multiple Endocrine Neoplasia Type 1/pathology , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-jun/genetics , Proto-Oncogene Proteins c-jun/metabolism , Rats , Smad3 Protein/genetics , Smad3 Protein/metabolism , Transforming Growth Factor beta/geneticsABSTRACT
Hereditary hyperekplexia or startle disease is characterized by an exaggerated startle response, evoked by tactile or auditory stimuli, leading to hypertonia and apnea episodes. Missense, nonsense, frameshift, splice site mutations, and large deletions in the human glycine receptor α1 subunit gene (GLRA1) are the major known cause of this disorder. However, mutations are also found in the genes encoding the glycine receptor ß subunit (GLRB) and the presynaptic Na(+)/Cl(-)-dependent glycine transporter GlyT2 (SLC6A5). In this study, systematic DNA sequencing of SLC6A5 in 93 new unrelated human hyperekplexia patients revealed 20 sequence variants in 17 index cases presenting with homozygous or compound heterozygous recessive inheritance. Five apparently unrelated cases had the truncating mutation R439X. Genotype-phenotype analysis revealed a high rate of neonatal apneas and learning difficulties associated with SLC6A5 mutations. From the 20 SLC6A5 sequence variants, we investigated glycine uptake for 16 novel mutations, confirming that all were defective in glycine transport. Although the most common mechanism of disrupting GlyT2 function is protein truncation, new pathogenic mechanisms included splice site mutations and missense mutations affecting residues implicated in Cl(-) binding, conformational changes mediated by extracellular loop 4, and cation-π interactions. Detailed electrophysiology of mutation A275T revealed that this substitution results in a voltage-sensitive decrease in glycine transport caused by lower Na(+) affinity. This study firmly establishes the combination of missense, nonsense, frameshift, and splice site mutations in the GlyT2 gene as the second major cause of startle disease.
Subject(s)
Genetic Diseases, Inborn , Glycine Plasma Membrane Transport Proteins , Glycine/metabolism , Mutation , Nerve Tissue Proteins , Neurodegenerative Diseases , Animals , DNA Mutational Analysis , Female , Genetic Diseases, Inborn/genetics , Genetic Diseases, Inborn/metabolism , Glycine/genetics , Glycine Plasma Membrane Transport Proteins/genetics , Glycine Plasma Membrane Transport Proteins/metabolism , Heterozygote , Homozygote , Humans , Ion Transport/genetics , Male , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurodegenerative Diseases/genetics , Neurodegenerative Diseases/metabolism , Protein Structure, Tertiary , Receptors, Glycine/genetics , Receptors, Glycine/metabolism , Xenopus laevisABSTRACT
Episodic falling syndrome (EFS) is a canine paroxysmal hypertonicity disorder found in Cavalier King Charles spaniels. Episodes are triggered by exercise, stress or excitement and characterized by progressive hypertonicity throughout the thoracic and pelvic limbs, resulting in a characteristic 'deer-stalking' position and/or collapse. We used a genome-wide association strategy to map the EFS locus to a 3.48 Mb critical interval on canine chromosome 7. By prioritizing candidate genes on the basis of biological plausibility, we found that a 15.7 kb deletion in BCAN, encoding the brain-specific extracellular matrix proteoglycan brevican, is associated with EFS. This represents a compelling causal mutation for EFS, since brevican has an essential role in the formation of perineuronal nets governing synapse stability and nerve conduction velocity. Mapping of the deletion breakpoint enabled the development of Multiplex PCR and Multiplex Ligation-dependent Probe Amplification (MLPA) genotyping tests that can accurately distinguish normal, carrier and affected animals. Wider testing of a larger population of CKCS dogs without a history of EFS from the USA revealed that carriers are extremely common (12.9%). The development of molecular genetic tests for the EFS microdeletion will allow the implementation of directed breeding programs aimed at minimizing the number of animals with EFS and enable confirmatory diagnosis and pharmacotherapy of affected dogs.
Subject(s)
Brevican/genetics , Dog Diseases/genetics , Genotype , Muscle Hypertonia/veterinary , Animals , Breeding , Dogs , Gene Deletion , Genome-Wide Association Study , Muscle Hypertonia/geneticsABSTRACT
Hyperekplexia is a rare, but potentially fatal, neuromotor disorder characterized by exaggerated startle reflexes and hypertonia in response to sudden, unexpected auditory or tactile stimuli. This disorder is primarily caused by inherited mutations in the genes encoding the glycine receptor (GlyR) alpha1 subunit (GLRA1) and the presynaptic glycine transporter GlyT2 (SLC6A5). In this study, systematic DNA sequencing of GLRA1 in 88 new unrelated human hyperekplexia patients revealed 19 sequence variants in 30 index cases, of which 21 cases were inherited in recessive or compound heterozygote modes. This indicates that recessive hyperekplexia is far more prevalent than previous estimates. From the 19 GLRA1 sequence variants, we have investigated the functional effects of 11 novel and 2 recurrent mutations. The expression levels and functional properties of these hyperekplexia mutants were analyzed using a high-content imaging system and patch-clamp electrophysiology. When expressed in HEK293 cells, either as homomeric alpha1 or heteromeric alpha1beta GlyRs, subcellular localization defects were the major mechanism underlying recessive mutations. However, mutants without trafficking defects typically showed alterations in the glycine sensitivity suggestive of disrupted receptor function. This study also reports the first hyperekplexia mutation associated with a GlyR leak conductance, suggesting tonic channel opening as a new mechanism in neuronal ligand-gated ion channels.
Subject(s)
Muscle Hypertonia/genetics , Receptors, Glycine/genetics , Reflex, Abnormal/genetics , Reflex, Startle/genetics , Cell Line , Female , Genetic Variation , Humans , Male , Mutation/genetics , Phenotype , TransfectionABSTRACT
Defects in glycinergic synaptic transmission in humans, cattle, and rodents result in an exaggerated startle reflex and hypertonia in response to either acoustic or tactile stimuli. Molecular genetic studies have determined that mutations in the genes encoding the postsynaptic glycine receptor (GlyR) α1 and ß subunits (GLRA1 and GLRB) and the presynaptic glycine transporter GlyT2 (SLC6A5) are the major cause of these disorders. Here, we report the first genetically confirmed canine cases of startle disease. A litter of seven Irish wolfhounds was identified in which two puppies developed muscle stiffness and tremor in response to handling. Although sequencing of GLRA1 and GLRB did not reveal any pathogenic mutations, analysis of SLC6A5 revealed a homozygous 4.2kb microdeletion encompassing exons 2 and 3 in both affected animals. This results in the loss of part of the large cytoplasmic N-terminus and all subsequent transmembrane domains due to a frameshift. This genetic lesion was confirmed by defining the deletion breakpoint, Southern blotting, and multiplex ligation-dependent probe amplification (MLPA). This analysis enabled the development of a rapid genotyping test that revealed heterozygosity for the deletion in the dam and sire and three other siblings, confirming recessive inheritance. Wider testing of related animals has identified a total of 13 carriers of the SLC6A5 deletion as well as non-carrier animals. These findings will inform future breeding strategies and enable a rational pharmacotherapy of this new canine disorder.
Subject(s)
Dog Diseases/genetics , Dogs/genetics , Epilepsy, Reflex/genetics , Epilepsy, Reflex/veterinary , Gene Deletion , Glycine Plasma Membrane Transport Proteins/deficiency , Reflex, Startle/genetics , Animals , Base Sequence , Female , Frameshift Mutation/genetics , Glycine Plasma Membrane Transport Proteins/genetics , Male , Molecular Sequence Data , Muscle Tonus/genetics , Pedigree , Tremor/genetics , Tremor/veterinaryABSTRACT
BACKGROUND: Long-term immune recovery in older patients given hematopoietic cell transplantation after non-myeloablative conditioning remains poorly understood. This prompted us to investigate long-term lymphocyte reconstitution and thymic function in 80 patients given allogeneic peripheral blood stem cells after non-myeloablative conditioning. DESIGN AND METHODS: Median age at transplant was 57 years (range 10-71). Conditioning regimen consisted of 2 Gy total body irradiation (TBI) with (n=46) or without (n=20) added fludarabine, 4 Gy TBI with fludarabine (n=6), or cyclophosphamide plus fludarabine (n=8). Stem cell sources were unmanipulated (n=56), CD8-depleted (n=19), or CD34-selected (n=5) peripheral blood stem cells. Immune recovery was assessed by signal-joint T-cell receptor excision circle quantification and flow cytometry. RESULTS: Signal-joint T-cell receptor excision circle levels increased from day 100 to one and two years after transplantation in patients under 50 years of age (n=23; P=0.02 and P=0.04, respectively), and in those aged 51-60 years (n=35; P=0.17 and P=0.06, respectively), but not in patients aged over 60 (n=22; P=0.3 and P=0.3, respectively). Similarly, CD4(+)CD45RA(+) (naïve) T-cell counts increased from day 100 to one and two years after transplantation in patients aged 50 years and under 50 (P=0.002 and P=0.02, respectively), and in those aged 51-60 (P=0.4 and P=0.001, respectively), but less so in patients aged over 60 (P=0.3 and P=0.06, respectively). In multivariate analyses, older patient age (P<0.001), extensive chronic GVHD (P<0.001), and prior (resolved) extensive chronic graft-versus-host disease (P=0.008) were associated with low signal-joint T-cell receptor excision circle levels one year or more after HCT. CONCLUSIONS: In summary, our data suggest that thymic neo-generation of T cells occurred from day 100 onwards in patients under 60 while signal-joint T-cell receptor excision circle levels remained low for patients aged over 60. Further, chronic graft-versus-host disease had a dramatic impact on thymic function, as observed previously in patients given grafts after myeloablative conditioning.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Graft vs Host Disease/prevention & control , Hematopoietic Stem Cell Transplantation , T-Lymphocytes/metabolism , Thymus Gland/metabolism , Transplantation Conditioning , Whole-Body Irradiation , Adolescent , Adult , Aged , Child , Chronic Disease , Cyclophosphamide/administration & dosage , Female , Graft vs Host Disease/immunology , Humans , Male , Middle Aged , Prognosis , Receptors, Antigen, T-Cell/metabolism , Survival Rate , T-Lymphocytes/immunology , Thymus Gland/immunology , Transplantation, Homologous , Vidarabine/administration & dosage , Vidarabine/analogs & derivatives , Young AdultABSTRACT
Recent studies have suggested that coinfusion of mesenchymal stem cells (MSCs) the day of hematopoietic cell transplantation (HCT) might promote engraftment and prevent graft-versus-host disease (GVHD) after myeloablative allogeneic HCT. This prompted us to investigate in a pilot study whether MSC infusion before HCT could allow nonmyeloablative (NMA) HCT (a transplant strategy based nearly exclusively on graft-versus-tumor effects for tumor eradication) from HLA-mismatched donors to be performed safely. Twenty patients with hematologic malignancies were given MSCs from third party unrelated donors 30-120 minutes before peripheral blood stem cells (PBSCs) from HLA-mismatched unrelated donors, after conditioning with 2 Gy total body irradiation (TBI) and fludarabine. The primary endpoint was safety, defined as a 100-day incidence of nonrelapse mortality (NRM) <35%. One patient had primary graft rejection, whereas the remaining 19 patients had sustained engraftment. The 100-day cumulative incidence of grade II-IV acute GVHD (aGVHD) was 35%, whereas 65% of the patients experienced moderate/severe chronic GVHD (cGVHD). One-year NRM (10%), relapse (30%), overall survival (OS) (80%) and progression-free survival (PFS) (60%), and 1-year incidence of death from GVHD or infection with GVHD (10%) were encouraging. These figures compare favorably with those observed in a historic group of 16 patients given HLA-mismatched PBSCs (but no MSCs) after NMA conditioning, which had a 1-year incidence of NRM of 37% (P = .02), a 1-year incidence of relapse of 25% (NS), a 1-year OS and PFS of 44% (P = .02), and 38% (P = .1), respectively, and a 1-year rate of death from GVHD or infection with GVHD of 31% (P = .04). In conclusion, our data suggest that HLA-mismatched NMA HCT with MSC coinfusion appeared to be safe.
Subject(s)
Graft vs Host Disease/prevention & control , Graft vs Tumor Effect , HLA Antigens/immunology , Histocompatibility/immunology , Mesenchymal Stem Cell Transplantation/methods , Peripheral Blood Stem Cell Transplantation/methods , Transplantation Conditioning/methods , Adolescent , Adult , Aged , Child , Disease Progression , Disease-Free Survival , Female , Graft Rejection , Graft vs Host Disease/diagnosis , Hematologic Neoplasms/surgery , Humans , Male , Mesenchymal Stem Cell Transplantation/adverse effects , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/immunology , Mesenchymal Stem Cells/metabolism , Middle Aged , Peripheral Blood Stem Cell Transplantation/adverse effects , Pilot Projects , Survival Analysis , Transplantation Chimera/blood , Transplantation, Homologous , Treatment Outcome , Young AdultSubject(s)
Myoclonus/etiology , Stiff-Person Syndrome/diagnosis , Anticonvulsants/therapeutic use , Clonazepam/therapeutic use , Consanguinity , Diagnosis, Differential , Electroencephalography , Epilepsy/diagnosis , Exons , Gene Deletion , Humans , Infant, Newborn , Male , Receptors, Glycine/genetics , Stiff-Person Syndrome/drug therapy , Stiff-Person Syndrome/geneticsABSTRACT
Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant neurodegenerative disease that results from the expansion of an unstable trinucleotide CAG repeat encoding for a polyglutamine tract. In normal individuals, alleles contain between 14 and 31 CAG repeats, whereas the pathological alleles have more than 35 CAG repeats. The clinical phenotype of SCA2 includes a progressive cerebellar ataxia with additional features such as ophthalmoplegia, extra-pyramidal or pyramidal signs and peripheral neuropathy. We report a SCA2 large African family with several affected individuals. A major pathological allele carrying 43 CAG repeats was identified in the proband. To our knowledge, this is a first report of a SCA disorder described in Central African patients, thus indicating the need to consider this diagnosis in young African ataxic patients.
Subject(s)
Spinocerebellar Ataxias/diagnosis , Spinocerebellar Ataxias/genetics , Trinucleotide Repeats , Adolescent , Alleles , Female , Gene Frequency , Genetic Testing , Humans , Movement Disorders/etiology , Ophthalmoplegia/etiology , Pedigree , Peripheral Nervous System Diseases/etiology , Phenotype , Rwanda , Spinocerebellar Ataxias/complicationsABSTRACT
CONTEXT: Limited screening suggests that three germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene are not involved in sporadic pituitary tumorigenesis. Multiple novel mutations of this gene have since been identified in familial isolated pituitary adenoma cohorts. OBJECTIVE: The objective of the study was to undertake full AIP coding sequence screening to assess for the presence of germline and somatic mutations in European Union subjects with sporadic pituitary tumors. DESIGN: The study design was the analysis of DNA from peripheral blood lymphocytes and analysis of exons 1-6 and paraexonic intron sequences of AIP. Multiplex ligation-dependent probe amplification was used to screen separate sporadic pituitary tumor tissue samples for discrete and extensive deletions or mutations of the AIP gene. SETTING: The study was conducted in university tertiary referral Clinical Genetics, Molecular Biology, and Endocrinology Departments. RESULTS: In 107 patients [prolactinomas (n =49), nonfunctioning tumors (n = 29), somatotropinomas (n = 26), ACTH-secreting tumors (n = 2), TSH-secreting tumors (n = 1)], no germline mutations of AIP were demonstrated. Among a group of 41 tumor samples from other subjects, a novel AIP mutation (R22X) was found in one sample in which the corresponding allele was deleted; follow-up screening of the patient demonstrated a germline R22X AIP mutation. CONCLUSIONS: AIP mutations do not appear to play a prominent role in sporadic pituitary tumorigenesis in this population of European subjects.
Subject(s)
Adenoma/genetics , Mutation/genetics , Pituitary Neoplasms/genetics , Proteins/genetics , Adult , DNA, Neoplasm/genetics , Female , Gene Frequency , Genotype , Humans , Intracellular Signaling Peptides and Proteins , Male , Middle Aged , Multiple Endocrine Neoplasia Type 1/genetics , Mutation/physiology , Polymorphism, Genetic/geneticsABSTRACT
CONTEXT: An association between germline aryl hydrocarbon receptor-interacting protein (AIP) gene mutations and pituitary adenomas was recently shown. OBJECTIVE: The objective of the study was to assess the frequency of AIP gene mutations in a large cohort of patients with familial isolated pituitary adenoma (FIPA). DESIGN: This was a multicenter, international, collaborative study. SETTING: The study was conducted in 34 university endocrinology and genetics departments in nine countries. PATIENTS: Affected members from each FIPA family were studied. Relatives of patients with AIP mutations underwent AIP sequence analysis. MAIN OUTCOME MEASURES: Presence/absence and description of AIP gene mutations were the main outcome measures. INTERVENTION: There was no intervention. RESULTS: Seventy-three FIPA families were identified, with 156 patients with pituitary adenomas; the FIPA cohort was evenly divided between families with homogeneous and heterogeneous tumor expression. Eleven FIPA families had 10 germline AIP mutations. Nine mutations, R16H, G47_R54del, Q142X, E174frameshift, Q217X, Q239X, K241E, R271W, and Q285frameshift, have not been described previously. Tumors were significantly larger (P = 0.0005) and diagnosed at a younger age (P = 0.0006) in AIP mutation-positive vs. mutation-negative subjects. Somatotropinomas predominated among FIPA families with AIP mutations, but mixed GH/prolactin-secreting tumors, prolactinomas, and nonsecreting adenomas were also noted. Approximately 85% of the FIPA cohort and 50% of those with familial somatotropinomas were negative for AIP mutations. CONCLUSIONS: AIP mutations, of which nine new mutations have been described here, occur in approximately 15% of FIPA families. Although pituitary tumors occurring in association with AIP mutations are predominantly somatotropinomas, other tumor types are also seen. Further study of the impact of AIP mutations on protein expression and activity is necessary to elucidate their role in pituitary tumorigenesis in FIPA.
Subject(s)
Adenoma/genetics , Pituitary Neoplasms/genetics , Proteins/genetics , Adenoma/pathology , Adult , Aged , Cohort Studies , Female , Gene Frequency , Germ-Line Mutation/genetics , Growth Hormone/metabolism , Humans , Immunohistochemistry , Intracellular Signaling Peptides and Proteins , Male , Middle Aged , Molecular Sequence Data , Mutation/physiology , Pituitary Neoplasms/pathology , Prolactinoma/genetics , Prolactinoma/metabolism , Prolactinoma/pathologyABSTRACT
SETTING: Early diagnosis of sickle cell disease decreases morbidity. However, cost-effective screening programmes are not yet available. METHODS: We explored the feasibility of systematic screening performed on dried blood harvested from five-day-old newborns. RESULTS: A total of 27,010 samples were collected in Belgian maternity units between June 2003 and February 2005, and the presence of haemoglobin (Hb) C or S in the eluted blood was examined by an enzyme-linked immunosorbent assay (ELISA) test performed with a monoclonal antibody detecting both mutated forms. As this antibody slightly cross-reacts with Hb A, better specificity is achieved if the test is performed not later than day 5. Among the 27,010 samples, 132 (0.49%) were positive. Molecular biology tests performed on dried blood from positive samples showed that 106 of these babies were heterozygotes for the Hb S mutation and three were heterozygotes for the Hb C mutation, while three newborns were SS homozygotes (0.011%). Seventeen samples (0.063%) were false-positives as we could not detect any mutation. CONCLUSIONS: We have developed a new immunological approach in the field of haemoglobinopathy neonatal screening. This ELISA test is cheap (0.2 euro/test or 1800 euro/detected SS homozygote) and could be centralized. Its cost-effectiveness in the whole Belgian population is comparable with that of screening for phenylketonuria or congenital adrenal hyperplasia. Further improvements should obviously be achieved in order to better discriminate heterozygotes and homozygotes, but the accessibility and the low cost of the test are relevant arguments for the screening extension in a wide range of countries, especially in Central Africa.
Subject(s)
Anemia, Sickle Cell/diagnosis , Hematologic Tests/methods , Neonatal Screening/methods , Anemia, Sickle Cell/blood , Anemia, Sickle Cell/epidemiology , Belgium , Chromatography, High Pressure Liquid , Cost-Benefit Analysis , Enzyme-Linked Immunosorbent Assay , False Positive Reactions , Hematologic Tests/economics , Hemoglobin C/analysis , Hemoglobin C/immunology , Hemoglobin, Sickle/analysis , Hemoglobin, Sickle/immunology , Humans , Infant, Newborn , Sensitivity and SpecificitySubject(s)
Complement C1 Inhibitor Protein/genetics , Contraceptives, Oral/adverse effects , Ethinyl Estradiol/adverse effects , Genotype , Hereditary Angioedema Types I and II/diagnosis , Lymphocytes/physiology , Mosaicism , Mutation, Missense/genetics , Spermatozoa/physiology , Adolescent , Asymptomatic Diseases , Complement C4/metabolism , Contraceptives, Oral/therapeutic use , DNA Mutational Analysis , Ethinyl Estradiol/therapeutic use , Female , Genetic Counseling , Humans , Male , Pedigree , Polymorphism, Single Nucleotide , SiblingsABSTRACT
CONTEXT: In multiple endocrine neoplasia type 1 (MEN1) characterized by tumors of parathyroid, enteropancreas, and anterior pituitary, missense mutations in the MEN1 gene product, menin, occur in a subset of cases. The mutant proteins are degraded by the proteasome. However, whether their expression and activity can be restored is not known. OBJECTIVE: Our objective was to functionally characterize a panel of 16 menin missense mutants, including W423R and S443Y identified in new MEN1 families, with respect to protein stability, targeting to the proteasome and restoration of expression by proteasome inhibitors and expression and function by small interfering RNA technology. METHODS: Flag-tagged wild-type (WT) and missense menin mutant expression vectors were transiently transfected in human embryonic kidney (HEK293) and/or rat insulinoma (Rin-5F) cells. RESULTS: The majority of mutants were short-lived, whereas WT menin was stable. Proteasome inhibitors MG132 and PS-341 and inhibition of the chaperone, heat-shock protein 70 (Hsp70), or the ubiquitin ligase, COOH terminus of Hsp70-interacting protein (CHIP), by specific small interfering RNA, restored the levels of the mutants, whereas that of WT menin was largely unaffected. Inhibition of CHIP restored the ability of mutants to mediate normal functions of menin: TGF-ß up-regulation of the promoters of its target genes, the cyclin-dependent kinase inhibitors p15 and p21 as well as TGF-ß inhibition of cell numbers. CONCLUSION: When the levels of missense menin mutants that are targeted to the proteasome are normalized they may function similarly to WT menin. Potentially, targeting specific components of the proteasome chaperone pathway could be beneficial in treating a subset of MEN1 cases.
Subject(s)
Proteasome Endopeptidase Complex/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , RNA, Small Interfering/pharmacology , Ubiquitin-Protein Ligases/antagonists & inhibitors , Animals , Enzyme Activation/drug effects , Enzyme Activation/genetics , Family , Gene Expression Regulation/drug effects , HEK293 Cells , Humans , Mutant Proteins/genetics , Mutant Proteins/metabolism , Mutation, Missense/physiology , Proteasome Endopeptidase Complex/drug effects , Proteasome Endopeptidase Complex/physiology , Protein Transport/drug effects , Protein Transport/genetics , Proteolysis/drug effects , Rats , Tumor Cells, Cultured , Ubiquitin-Protein Ligases/geneticsABSTRACT
OBJECTIVE: To describe a patient who developed a young-onset, dopa-responsive parkinsonism linked to a de novo heterozygous interstitial duplication 4q. DESIGN: Case report. SETTING: Movement Disorder Outpatient Clinic at the University Hospital Centre, Liège, Belgium. PATIENT: A 31-year-old woman. MAIN OUTCOME MEASURES: Clinical, neuroimaging, and genetic data. RESULTS: The duplicated region contains 150 known genes, including the α-synuclein (SNCA) gene locus. Motor and 6-[(18)F]fluoro-L-dopa positron emission tomography features are similar to those previously reported in heterozygote SNCA duplication carriers. Altered expression of other genes contained in the duplicated region may contribute to clinical features that are uncommon in the phenotypic spectrum of SNCA multiplications such as delayed developmental psychomotor milestones during infancy and musculoskeletal abnormalities. CONCLUSION: This case report provides new insights on the genetic basis of parkinsonism.
Subject(s)
Antiparkinson Agents/therapeutic use , Chromosomes, Human, Pair 4 , Levodopa/therapeutic use , Parkinson Disease/drug therapy , Parkinson Disease/genetics , Trisomy/physiopathology , Adult , Age of Onset , Dihydroxyphenylalanine/analogs & derivatives , Dopamine Agonists/therapeutic use , Female , Gene Duplication , Humans , Intellectual Disability/etiology , Karyotyping , Magnetic Resonance Imaging , Positron-Emission Tomography , Radiopharmaceuticals , alpha-Synuclein/geneticsABSTRACT
CONTEXT: AIP mutations (AIPmut) give rise to a pituitary adenoma predisposition that occurs in familial isolated pituitary adenomas and less often in sporadic cases. The clinical and therapeutic features of AIPmut-associated pituitary adenomas have not been studied comprehensively. OBJECTIVE: The objective of the study was to assess clinical/therapeutic characteristics of AIPmut pituitary adenomas. DESIGN: This study was an international, multicenter, retrospective case collection/database analysis. SETTING: The study was conducted at 36 tertiary referral endocrine and clinical genetics departments. PATIENTS: Patients included 96 patients with germline AIPmut and pituitary adenomas and 232 matched AIPmut-negative acromegaly controls. RESULTS: The AIPmut population was predominantly young and male (63.5%); first symptoms occurred as children/adolescents in 50%. At diagnosis, most tumors were macroadenomas (93.3%); extension and invasion was common. Somatotropinomas comprised 78.1% of the cohort; there were also prolactinomas (n = 13), nonsecreting adenomas (n = 7), and a TSH-secreting adenoma. AIPmut somatotropinomas were larger (P = 0.00026), with higher GH levels (P = 0.00068), more frequent extension (P = 0.018) and prolactin cosecretion (P = 0.00023), and occurred 2 decades before controls (P < 0.000001). Gigantism was more common in the AIPmut group (P < 0.000001). AIPmut somatotropinoma patients underwent more surgical interventions (P = 0.00069) and had lower decreases in GH (P = 0.00037) and IGF-I (P = 0.028) and less tumor shrinkage with somatostatin analogs (P < 0.00001) vs. controls. AIPmut prolactinomas occurred generally in young males and frequently required surgery or radiotherapy. CONCLUSIONS: AIPmut pituitary adenomas have clinical features that may negatively impact treatment efficacy. Predisposition for aggressive disease in young patients, often in a familial setting, suggests that earlier diagnosis of AIPmut pituitary adenomas may have clinical utility.