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1.
J Clin Invest ; 2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31430258

RESUMO

Patients with paroxysmal nocturnal hemoglobinuria (PNH) have a clonal population of blood cells deficient in glycosylphosphatidylinositol-anchored (GPI-anchored) proteins, resulting from a mutation in the X-linked gene PIGA. Here we report on a set of patients in whom PNH results instead from biallelic mutation of PIGT on chromosome 20. These PIGT-PNH patients have clinically typical PNH, but they have in addition prominent autoinflammatory features, including recurrent attacks of aseptic meningitis. In all these patients we find a germ-line point mutation in one PIGT allele, whereas the other PIGT allele is removed by somatic deletion of a 20q region comprising maternally imprinted genes implicated in myeloproliferative syndromes. Unlike in PIGA-PNH cells, GPI is synthesized in PIGT-PNH cells and, since its attachment to proteins is blocked, free GPI is expressed on the cell surface. From studies of patients' leukocytes and of PIGT-KO THP-1 cells we show that, through increased IL-1ß secretion, activation of the lectin pathway of complement and generation of C5b-9 complexes, free GPI is the agent of autoinflammation. Eculizumab treatment abrogates not only intravascular hemolysis, but also autoinflammation. Thus, PIGT-PNH differs from PIGA-PNH both in the mechanism of clonal expansion and in clinical manifestations.

2.
Am J Hum Genet ; 105(2): 395-402, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31353022

RESUMO

The glycosylphosphatidylinositol (GPI) anchor links over 150 proteins to the cell surface and is present on every cell type. Many of these proteins play crucial roles in neuronal development and function. Mutations in 18 of the 29 genes implicated in the biosynthesis of the GPI anchor have been identified as the cause of GPI biosynthesis deficiencies (GPIBDs) in humans. GPIBDs are associated with intellectual disability and seizures as their cardinal features. An essential component of the GPI transamidase complex is PIGU, along with PIGK, PIGS, PIGT, and GPAA1, all of which link GPI-anchored proteins (GPI-APs) onto the GPI anchor in the endoplasmic reticulum (ER). Here, we report two homozygous missense mutations (c.209T>A [p.Ile70Lys] and c.1149C>A [p.Asn383Lys]) in five individuals from three unrelated families. All individuals presented with global developmental delay, severe-to-profound intellectual disability, muscular hypotonia, seizures, brain anomalies, scoliosis, and mild facial dysmorphism. Using multicolor flow cytometry, we determined a characteristic profile for GPI transamidase deficiency. On granulocytes this profile consisted of reduced cell-surface expression of fluorescein-labeled proaerolysin (FLAER), CD16, and CD24, but not of CD55 and CD59; additionally, B cells showed an increased expression of free GPI anchors determined by T5 antibody. Moreover, computer-assisted facial analysis of different GPIBDs revealed a characteristic facial gestalt shared among individuals with mutations in PIGU and GPAA1. Our findings improve our understanding of the role of the GPI transamidase complex in the development of nervous and skeletal systems and expand the clinical spectrum of disorders belonging to the group of inherited GPI-anchor deficiencies.

3.
Am J Hum Genet ; 105(2): 384-394, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31256876

RESUMO

Proteins anchored to the cell surface via glycosylphosphatidylinositol (GPI) play various key roles in the human body, particularly in development and neurogenesis. As such, many developmental disorders are caused by mutations in genes involved in the GPI biosynthesis and remodeling pathway. We describe ten unrelated families with bi-allelic mutations in PIGB, a gene that encodes phosphatidylinositol glycan class B, which transfers the third mannose to the GPI. Ten different PIGB variants were found in these individuals. Flow cytometric analysis of blood cells and fibroblasts from the affected individuals showed decreased cell surface presence of GPI-anchored proteins. Most of the affected individuals have global developmental and/or intellectual delay, all had seizures, two had polymicrogyria, and four had a peripheral neuropathy. Eight children passed away before four years old. Two of them had a clinical diagnosis of DOORS syndrome (deafness, onychodystrophy, osteodystrophy, mental retardation, and seizures), a condition that includes sensorineural deafness, shortened terminal phalanges with small finger and toenails, intellectual disability, and seizures; this condition overlaps with the severe phenotypes associated with inherited GPI deficiency. Most individuals tested showed elevated alkaline phosphatase, which is a characteristic of the inherited GPI deficiency but not DOORS syndrome. It is notable that two severely affected individuals showed 2-oxoglutaric aciduria, which can be seen in DOORS syndrome, suggesting that severe cases of inherited GPI deficiency and DOORS syndrome might share some molecular pathway disruptions.

4.
Endocr J ; 66(5): 475-483, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-30867345

RESUMO

We studied cytological specimens of conventional papillary thyroid carcinoma (PTC), follicular variant papillary thyroid carcinoma (FVPTC), and noninvasive follicular thyroid tumor with papillary-like nuclear features (NIFTP) (formerly noninvasive FVPTC) to identify useful cytological parameters for their differentiation. Cytological findings of invasive FVPTC and NIFTP were very similar to each other but differed from those of conventional PTC. Intranuclear cytoplasmic inclusions, true papillary cell clusters, monolayered cell sheets, ropy colloids, multinucleate giant cells, psammoma bodies, and cystic background were the observed characteristic features of conventional PTC. Microfollicular cell clusters and dense globules of colloids were characteristic features of invasive FVPTC and NIFTP. Scoring the eight parameters (intranuclear cytoplasmic inclusions, nuclear grooves, powdery chromatin, true papillary cell clusters, ropy colloids, multinucleate giant cells, psammoma bodies, and cystic background) readily distinguished NIFTP from conventional PTC, but could not distinguish NIFTP from invasive FVPTC. The average total score of NIFTP, invasive FVPTC, and conventional PTC were 2.60 ± 0.55, 2.63 ± 0.62, and 4.57 ± 0.99, respectively. The difference between conventional PTC and NIFTP or invasive FVPTC was statistically significant (p < 0.001, Student's t-test). Individuals with more than three of the identified parameters likely harbor conventional PTC, rather than NIFTP. In this way, 87.5% (112/128) of conventional PTCs could be differentiated from NIFTP, and definitively diagnosed as malignant by cytology.

5.
J Biol Chem ; 294(13): 5038-5049, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30728244

RESUMO

Glycosylphosphatidylinositols (GPIs) are linked to many cell-surface proteins, anchor these proteins in the membrane, and are well characterized. However, GPIs that exist in the free form on the mammalian cell surface remain largely unexplored. To investigate free GPIs in cultured cell lines and mouse tissues, here we used the T5-4E10 mAb (T5 mAb), which recognizes unlinked GPIs having an N-acetylgalactosamine (GalNAc) side chain linked to the first mannose at the nonreducing terminus. We detected free GPIs bearing the GalNAc side chain on the surface of Neuro2a and CHO, but not of HEK293, K562, and C2C12 cells. Furthermore, free GPIs were present in mouse pons, medulla oblongata, spinal cord, testis, epididymis, and kidney. Using a panel of Chinese hamster ovary cells defective in both GPI-transamidase and GPI remodeling pathway, we demonstrate that free GPIs follow the same structural remodeling pathway during passage from the endoplasmic reticulum to the plasma membrane as do protein-linked GPI. Specifically, free GPIs underwent post-GPI attachment to protein 1 (PGAP1)-mediated inositol deacylation, PGAP5-mediated removal of the ethanolamine phosphate from the second mannose, and PGAP3- and PGAP2-mediated fatty acid remodeling. Moreover, T5 mAb recognized free GPIs even if the inositol-linked acyl chain or ethanolamine-phosphate side chain linked to the second mannose is not removed. In contrast, addition of a fourth mannose by phosphatidylinositol glycan anchor biosynthesis class Z (PIGZ) inhibited T5 mAb-mediated detection of free GPIs. Our results indicate that free GPIs are normal components of the plasma membrane in some tissues and further characterize free GPIs in mammalian cells.


Assuntos
Membrana Celular/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Animais , Células CHO , Linhagem Celular , Membrana Celular/química , Cricetulus , Retículo Endoplasmático/metabolismo , Ácidos Graxos/metabolismo , Glicosilfosfatidilinositóis/análise , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL
6.
Am J Hum Genet ; 103(4): 602-611, 2018 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-30269814

RESUMO

Inherited GPI deficiencies (IGDs) are a subset of congenital disorders of glycosylation that are increasingly recognized as a result of advances in whole-exome sequencing (WES) and whole-genome sequencing (WGS). IGDs cause a series of overlapping phenotypes consisting of seizures, dysmorphic features, multiple congenital malformations, and severe intellectual disability. We present a study of six individuals from three unrelated families in which WES or WGS identified bi-allelic phosphatidylinositol glycan class S (PIGS) biosynthesis mutations. Phenotypes included severe global developmental delay, seizures (partly responding to pyridoxine), hypotonia, weakness, ataxia, and dysmorphic facial features. Two of them had compound-heterozygous variants c.108G>A (p.Trp36∗) and c.101T>C (p.Leu34Pro), and two siblings of another family were homozygous for a deletion and insertion leading to p.Thr439_Lys451delinsArgLeuLeu. The third family had two fetuses with multiple joint contractures consistent with fetal akinesia. They were compound heterozygous for c.923A>G (p.Glu308Gly) and c.468+1G>C, a splicing mutation. Flow-cytometry analyses demonstrated that the individuals with PIGS mutations show a GPI-AP deficiency profile. Expression of the p.Trp36∗ variant in PIGS-deficient HEK293 cells revealed only partial restoration of cell-surface GPI-APs. In terms of both biochemistry and phenotype, loss of function of PIGS shares features with PIGT deficiency and other IGDs. This study contributes to the understanding of the GPI-AP biosynthesis pathway by describing the consequences of PIGS disruption in humans and extending the family of IGDs.

7.
BMJ Case Rep ; 20182018 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-30262533

RESUMO

We report the case of a patient with PIGT mutations who experienced recurrent aseptic meningitis 121 times over 16 years before developing paroxysmal nocturnal haemoglobinuria (PNH). Each episode was preceded by urticaria and arthralgia. After developing PNH, haemolysis occurred prior to meningitis. Flow cytometry revealed deficiency of the glycophosphatidylinositol (GPI)-anchored complement regulatory proteins, CD59 and CD55, and he was diagnosed with PNH. All the symptoms disappeared on administering eculizumab, an anti-C5 antibody. We did not detect mutation in PIGA, which is regarded as the cause of PNH. However, we detected a germ-line mutation and a somatic microdeletion in chromosome 20q including PIGT; PIGT is essential for transferring GPI anchor to the precursors of CD59 and CD55, which play important roles in complement regulation. Loss of these proteins leads to complement overactivation, causing inflammatory symptoms, including recurrent meningitis. PIGT mutations should be considered a novel pathogenesis of recurrent meningitis of unknown aetiology.

8.
Oncol Lett ; 16(4): 4219-4222, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30214557

RESUMO

The resistance mechanisms to anaplastic lymphoma kinase (ALK) inhibitors comprise ALK gene variations, such as ALK point mutations and copy-number gains, the activation of bypass signaling through the activation of other oncogenes and small cell lung cancer (SCLC) transformation. To date, few studies have investigated whether tumor markers for SCLC correlate with the SCLC transformation in EGFR-mutant NSCLC and ALK-positive non-SCLC (NSCLC). The present case study reported a patient with SCLC transformation after alectinib treatment. The patient exhibited elevation of pro-gastrin-releasing peptide precursor and neuron-specific enolase levels, which may be predictive of SCLC transformation during the resistance to ALK-tyrosine kinase inhibitors.

9.
Clin Cancer Res ; 24(22): 5658-5672, 2018 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-30087143

RESUMO

Purpose: EGFR inhibitors (EGFRi) are effective against EGFR-mutant lung cancers. The efficacy of these drugs, however, is mitigated by the outgrowth of resistant cells, most often driven by a secondary acquired mutation in EGFR, T790M We recently demonstrated that T790M can arise de novo during treatment; it follows that one potential therapeutic strategy to thwart resistance would be identifying and eliminating these cells [referred to as drug-tolerant cells (DTC)] prior to acquiring secondary mutations like T790M Experimental Design: We have developed DTCs to EGFRi in EGFR-mutant lung cancer cell lines. Subsequent analyses of DTCs included RNA-seq, high-content microscopy, and protein translational assays. Based on these results, we tested the ability of MCL-1 BH3 mimetics to combine with EGFR inhibitors to eliminate DTCs and shrink EGFR-mutant lung cancer tumors in vivo Results: We demonstrate surviving EGFR-mutant lung cancer cells upregulate the antiapoptotic protein MCL-1 in response to short-term EGFRi treatment. Mechanistically, DTCs undergo a protein biosynthesis enrichment resulting in increased mTORC1-mediated mRNA translation of MCL-1, revealing a novel mechanism in which lung cancer cells adapt to short-term pressures of apoptosis-inducing kinase inhibitors. Moreover, MCL-1 is a key molecule governing the emergence of early EGFR-mutant DTCs to EGFRi, and we demonstrate it can be effectively cotargeted with clinically emerging MCL-1 inhibitors both in vitro and in vivo Conclusions: Altogether, these data reveal that this novel therapeutic combination may delay the acquisition of secondary mutations, therefore prolonging therapy efficacy. Clin Cancer Res; 24(22); 5658-72. ©2018 AACR.

10.
Intern Med ; 57(17): 2489-2496, 2018 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-29607953

RESUMO

An asymptomatic 70-year-old woman was referred to our hospital because of liver enzyme elevation. Enhanced abdominal computed tomography demonstrated a small, round-shaped tumor with dilation of the common bile duct and main pancreatic duct. A biopsy specimen from the papilla showed mucin-containing cells that were positive for endocrine markers on immunohistochemical staining. Endoscopic snare resection was done, and there was a positive vertical margin on pathology. Pancreaticoduodenectomy was then performed later. The final diagnosis was goblet cell carcinoid, pT2N0M0, pStage IIA [Union for International Cancer Control (UICC) 7th edition]. Ampullary goblet cell carcinoid is an extremely rare disease of which there have been no recent reports.


Assuntos
Ampola Hepatopancreática , Tumor Carcinoide/diagnóstico , Tumor Carcinoide/cirurgia , Neoplasias do Ducto Colédoco/diagnóstico , Neoplasias do Ducto Colédoco/cirurgia , Idoso , Feminino , Humanos , Ductos Pancreáticos/patologia , Pancreaticoduodenectomia , Tomografia Computadorizada por Raios X
11.
Hum Mutat ; 39(6): 822-826, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29573052

RESUMO

Defective glycosylphosphatidylinositol (GPI)-anchor biogenesis can cause a spectrum of predominantly neurological problems. For eight genes critical to this biological process, disease associations are not yet reported. Scanning exomes from 7,833 parent-child trios and 1,792 singletons from the DDD study for biallelic variants in this gene-set uncovered a rare PIGH variant in a boy with epilepsy, microcephaly, and behavioral difficulties. Although only 2/2 reads harbored this c.1A > T transversion, the presence of ∼25 Mb autozygosity at this locus implied homozygosity, which was confirmed using Sanger sequencing. A similarly-affected sister was also homozygous. FACS analysis of PIGH-deficient CHO cells indicated that cDNAs with c.1A > T could not efficiently restore expression of GPI-APs. Truncation of PIGH protein was consistent with the utilization of an in-frame start-site at codon 63. In summary, we describe siblings harboring a homozygous c.1A > T variant resulting in defective GPI-anchor biogenesis and highlight the importance of exploring low-coverage variants within autozygous regions.

12.
Epileptic Disord ; 20(1): 42-50, 2018 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-29444765

RESUMO

Inherited glycosylphosphatidylinositol anchor deficiency causes a variety of clinical symptoms, including epilepsy, however, little information is available regarding seizures as a symptom. We report three siblings with inherited glycosylphosphatidylinositol anchor deficiency with PIGL gene mutations. The phenotypes of the subjects were not consistent with CHIME syndrome or Mabry syndrome, as reported in previous studies. All shared some clinical manifestations, including transient apnoea as neonates, dysmorphic facial features, and intellectual disability. Between one week and 3 months of life, all patients developed myoclonic seizures. Myoclonic jerks were easily evoked by sudden unexpected acoustic or tactile stimuli. None showed elevation of serum alkaline phosphatase. Vitamin B6 was given to one of the three siblings, but failed to suppress seizures. The presence of early infancy-onset stimulation-induced myoclonic seizures combined with dysmorphic facial features should lead physicians to consider the possibility of inherited glycosylphosphatidylinositol anchor deficiency.


Assuntos
Epilepsias Mioclônicas/fisiopatologia , Glicosilfosfatidilinositóis/deficiência , Erros Inatos do Metabolismo/complicações , N-Acetilglucosaminiltransferases/genética , Adolescente , Idade de Início , Criança , Pré-Escolar , Epilepsias Mioclônicas/etiologia , Epilepsias Mioclônicas/genética , Feminino , Glicosilfosfatidilinositóis/genética , Humanos , Masculino , Mutação , Irmãos
13.
Nat Commun ; 9(1): 405, 2018 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-29374258

RESUMO

Many eukaryotic proteins are anchored to the cell surface via the glycolipid glycosylphosphatidylinositol (GPI). Mammalian GPIs have a conserved core but exhibit diverse N-acetylgalactosamine (GalNAc) modifications, which are added via a yet unresolved process. Here we identify the Golgi-resident GPI-GalNAc transferase PGAP4 and show by mass spectrometry that PGAP4 knockout cells lose GPI-GalNAc structures. Furthermore, we demonstrate that PGAP4, in contrast to known Golgi glycosyltransferases, is not a single-pass membrane protein but contains three transmembrane domains, including a tandem transmembrane domain insertion into its glycosyltransferase-A fold as indicated by comparative modeling. Mutational analysis reveals a catalytic site, a DXD-like motif for UDP-GalNAc donor binding, and several residues potentially involved in acceptor binding. We suggest that a juxtamembrane region of PGAP4 accommodates various GPI-anchored proteins, presenting their acceptor residue toward the catalytic center. In summary, we present insights into the structure of PGAP4 and elucidate the initial step of GPI-GalNAc biosynthesis.


Assuntos
Acetilgalactosamina/química , Glicosilfosfatidilinositóis/química , Complexo de Golgi/metabolismo , N-Acetilgalactosaminiltransferases/química , Acetilgalactosamina/biossíntese , Motivos de Aminoácidos , Animais , Células CHO , Domínio Catalítico , Cricetulus , Cristalografia por Raios X , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Complexo de Golgi/ultraestrutura , Humanos , Camundongos , Camundongos Knockout , Modelos Moleculares , Mutação , N-Acetilgalactosaminiltransferases/genética , N-Acetilgalactosaminiltransferases/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Homologia Estrutural de Proteína , Especificidade por Substrato
14.
J Cell Biol ; 217(2): 585-599, 2018 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-29255114

RESUMO

Glycosylphosphatidylinositol (GPI) anchoring of proteins is a conserved posttranslational modification in the endoplasmic reticulum (ER). Soon after GPI is attached, an acyl chain on the GPI inositol is removed by post-GPI attachment to proteins 1 (PGAP1), a GPI-inositol deacylase. This is crucial for switching GPI-anchored proteins (GPI-APs) from protein folding to transport states. We performed haploid genetic screens to identify factors regulating GPI-inositol deacylation, identifying seven genes. In particular, calnexin cycle impairment caused inefficient GPI-inositol deacylation. Calnexin was specifically associated with GPI-APs, dependent on N-glycan and GPI moieties, and assisted efficient GPI-inositol deacylation by PGAP1. Under chronic ER stress caused by misfolded GPI-APs, inositol-acylated GPI-APs were exposed on the cell surface. These results indicated that N-glycans participate in quality control and temporal ER retention of GPI-APs, ensuring their correct folding and GPI processing before exiting from the ER. Once the system is disrupted by ER stress, unprocessed GPI-APs become exposed on the cell surface.

15.
Jpn J Infect Dis ; 71(1): 79-84, 2018 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-29093317

RESUMO

We previously developed a multiplex real-time PCR assay (Rapid Foodborne Bacterial Screening 24 ver.5, [RFBS24 ver.5]) for simultaneous detection of 24 foodborne bacterial targets. Here, to overcome the discrepancy of the results from RFBS24 ver.5 and bacterial culture methods (BC), we analyzed 246 human clinical samples from 49 gastroenteritis outbreaks using RFBS24 ver.5 and evaluated the correlation between the cycle threshold (CT) value of RFBS24 ver.5 and the BC results. The results showed that the RFBS24 ver.5 was more sensitive than BC for Campylobacter jejuni and Escherichia coli harboring astA or eae, with positive predictive values (PPV) of 45.5-87.0% and a kappa coefficient (KC) of 0.60-0.92, respectively. The CTs were significantly different between BC-positive and -negative samples (p < 0.01). All RFBS24 ver.5-positive samples were BC-positive under the lower confidence interval (CI) limit of 95% or 99% for the CT of the BC-negative samples. We set the 95% or 99% CI lower limit to the determination CT (d-CT) to discriminate for assured BC-positive results (d-CTs: 27.42-30.86), and subsequently the PPVs (94.7%-100.0%) and KCs (0.89-0.95) of the 3 targets were increased. Together, we concluded that the implication of a d-CT-based approach would be a valuable tool for rapid and accurate diagnoses using the RFBS24 ver.5 system.


Assuntos
Infecções por Campylobacter/diagnóstico , Campylobacter jejuni/genética , Infecções por Escherichia coli/diagnóstico , Escherichia coli/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Infecções por Campylobacter/microbiologia , Surtos de Doenças , Infecções por Escherichia coli/microbiologia , Gastroenterite/diagnóstico , Gastroenterite/microbiologia , Humanos , Limite de Detecção , Sensibilidade e Especificidade
16.
Brain Dev ; 40(1): 53-57, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28728837

RESUMO

We report an 11-month-old boy with acetazolamide-responsive epileptic apnea and inherited glycosylphosphatidylinositol (GPI)-anchor deficiency who presented with decreased serum alkaline phosphatase associated with compound PIGT mutations. The patient exhibited congenital anomalies, severe intellectual disability, and seizures, including epileptic apnea with epileptiform discharges from bilateral temporal areas. Brain magnetic resonance imaging revealed delayed myelination and progressive atrophy of the brainstem, cerebellum, and cerebrum. Whole-exome sequencing revealed compound heterozygous mutations in PIGT (c.250G>T, p.Glu84X and c.1096G>T, p.Gly366Trp), which encodes a subunit of the GPI transamidase complex. Flow cytometry revealed decreased expression of CD16 (a GPI anchor protein) on granulocytes, supporting the putative pathogenicity of the mutations. Phenobarbital, clonazepam, and potassium bromide decreased the frequency of tonic seizure and acetazolamide decreased epileptic apnea. To our knowledge, this is the first reported case of intractable seizures accompanied by epileptic apnea associated with GPI anchor deficiency and a compound PIGT mutation.


Assuntos
Apneia/genética , Epilepsia/genética , Glicosilfosfatidilinositóis/deficiência , Anormalidades Múltiplas/genética , Aciltransferases/genética , Aciltransferases/metabolismo , Apneia/metabolismo , Atrofia , Deficiências do Desenvolvimento/genética , Epilepsia/metabolismo , Glicosilfosfatidilinositóis/genética , Glicosilfosfatidilinositóis/metabolismo , Humanos , Lactente , Deficiência Intelectual/genética , Masculino , Hipotonia Muscular/genética , Mutação , Convulsões/genética
17.
Am J Hum Genet ; 101(5): 856-865, 2017 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-29100095

RESUMO

Approximately one in every 200 mammalian proteins is anchored to the cell membrane through a glycosylphosphatidylinositol (GPI) anchor. These proteins play important roles notably in neurological development and function. To date, more than 20 genes have been implicated in the biogenesis of GPI-anchored proteins. GPAA1 (glycosylphosphatidylinositol anchor attachment 1) is an essential component of the transamidase complex along with PIGK, PIGS, PIGT, and PIGU (phosphatidylinositol-glycan biosynthesis classes K, S, T, and U, respectively). This complex orchestrates the attachment of the GPI anchor to the C terminus of precursor proteins in the endoplasmic reticulum. Here, we report bi-allelic mutations in GPAA1 in ten individuals from five families. Using whole-exome sequencing, we identified two frameshift mutations (c.981_993del [p.Gln327Hisfs∗102] and c.920delG [p.Gly307Alafs∗11]), one intronic splicing mutation (c.1164+5C>T), and six missense mutations (c.152C>T [p.Ser51Leu], c.160_161delinsAA [p.Ala54Asn], c.527G>C [p.Trp176Ser], c.869T>C [p.Leu290Pro], c.872T>C [p.Leu291Pro], and c.1165G>C [p.Ala389Pro]). Most individuals presented with global developmental delay, hypotonia, early-onset seizures, cerebellar atrophy, and osteopenia. The splicing mutation was found to decrease GPAA1 mRNA. Moreover, flow-cytometry analysis of five available individual samples showed that several GPI-anchored proteins had decreased cell-surface abundance in leukocytes (FLAER, CD16, and CD59) or fibroblasts (CD73 and CD109). Transduction of fibroblasts with a lentivirus encoding the wild-type protein partially rescued the deficiency of GPI-anchored proteins. These findings highlight the role of the transamidase complex in the development and function of the cerebellum and the skeletal system.


Assuntos
Aciltransferases/genética , Atrofia/genética , Doenças Ósseas Metabólicas/genética , Deficiências do Desenvolvimento/genética , Epilepsia/genética , Glicoproteínas de Membrana/genética , Mutação/genética , Adolescente , Adulto , Alelos , Cerebelo/patologia , Criança , Pré-Escolar , Exoma/genética , Feminino , Fibroblastos/patologia , Glicosilfosfatidilinositóis/genética , Humanos , Masculino , Hipotonia Muscular/genética , Linhagem , RNA Mensageiro/genética , Convulsões/genética
18.
Hum Mutat ; 38(7): 805-815, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28337824

RESUMO

Inherited GPI (glycosylphosphatidylinositol) deficiencies (IGDs), a recently defined group of diseases, show a broad spectrum of symptoms. Hyperphosphatasia mental retardation syndrome, also known as Mabry syndrome, is a type of IGDs. There are at least 26 genes involved in the biosynthesis and transport of GPI-anchored proteins; however, IGDs constitute a rare group of diseases, and correlations between the spectrum of symptoms and affected genes or the type of mutations have not been shown. Here, we report four newly identified and five previously described Japanese families with PIGO (phosphatidylinositol glycan anchor biosynthesis class O) deficiency. We show how the clinical severity of IGDs correlates with flow cytometric analysis of blood, functional analysis using a PIGO-deficient cell line, and the degree of hyperphosphatasia. The flow cytometric analysis and hyperphosphatasia are useful for IGD diagnosis, but the expression level of GPI-anchored proteins and the degree of hyperphosphatasia do not correlate, although functional studies do, with clinical severity. Compared with PIGA (phosphatidylinositol glycan anchor biosynthesis class A) deficiency, PIGO deficiency shows characteristic features, such as Hirschsprung disease, brachytelephalangy, and hyperphosphatasia. This report shows the precise spectrum of symptoms according to the severity of mutations and compares symptoms between different types of IGD.


Assuntos
Estudos de Associação Genética , Deficiência Intelectual/genética , Transtornos de Aprendizagem/genética , Proteínas de Membrana/genética , Adolescente , Criança , Pré-Escolar , Análise Mutacional de DNA , Exoma , Feminino , Genótipo , Células HEK293 , Humanos , Lactente , Deficiência Intelectual/patologia , Japão , Transtornos de Aprendizagem/patologia , Masculino , Proteínas de Membrana/deficiência , Mutação , Linhagem , Fenótipo , Síndrome
19.
Eur J Hum Genet ; 25(6): 669-679, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28327575

RESUMO

Over 150 different proteins attach to the plasma membrane using glycosylphosphatidylinositol (GPI) anchors. Mutations in 18 genes that encode components of GPI-anchor biogenesis result in a phenotypic spectrum that includes learning disability, epilepsy, microcephaly, congenital malformations and mild dysmorphic features. To determine the incidence of GPI-anchor defects, we analysed the exome data from 4293 parent-child trios recruited to the Deciphering Developmental Disorders (DDD) study. All probands recruited had a neurodevelopmental disorder. We searched for variants in 31 genes linked to GPI-anchor biogenesis and detected rare biallelic variants in PGAP3, PIGN, PIGT (n=2), PIGO and PIGL, providing a likely diagnosis for six families. In five families, the variants were in a compound heterozygous configuration while in a consanguineous Afghani kindred, a homozygous c.709G>C; p.(E237Q) variant in PIGT was identified within 10-12 Mb of autozygosity. Validation and segregation analysis was performed using Sanger sequencing. Across the six families, five siblings were available for testing and in all cases variants co-segregated consistent with them being causative. In four families, abnormal alkaline phosphatase results were observed in the direction expected. FACS analysis of knockout HEK293 cells that had been transfected with wild-type or mutant cDNA constructs demonstrated that the variants in PIGN, PIGT and PIGO all led to reduced activity. Splicing assays, performed using leucocyte RNA, showed that a c.336-2A>G variant in PIGL resulted in exon skipping and p.D113fs*2. Our results strengthen recently reported disease associations, suggest that defective GPI-anchor biogenesis may explain ~0.15% of individuals with developmental disorders and highlight the benefits of data sharing.


Assuntos
Aciltransferases/genética , Deficiências do Desenvolvimento/genética , Exoma , Proteínas de Membrana/genética , N-Acetilglucosaminiltransferases/genética , Fosfotransferases/genética , Polimorfismo de Nucleotídeo Único , Aciltransferases/metabolismo , Adulto , Criança , Deficiências do Desenvolvimento/patologia , Células HEK293 , Heterozigoto , Homozigoto , Humanos , Proteínas de Membrana/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Linhagem , Fosfotransferases/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo
20.
Hum Mol Genet ; 26(9): 1706-1715, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28334793

RESUMO

There are over 150 known human proteins which are tethered to the cell surface via glycosylphosphatidylinositol (GPI) anchors. These proteins play a variety of important roles in development, and particularly in neurogenesis. Not surprisingly, mutations in the GPI anchor biosynthesis and remodeling pathway cause a number of developmental disorders. This group of conditions has been termed inherited GPI deficiencies (IGDs), a subgroup of congenital disorders of glycosylation; they present with variable phenotypes, often including seizures, hypotonia and intellectual disability. Here, we report two siblings with compound heterozygous variants in the gene phosphatidylinositol glycan anchor biosynthesis, class P (PIGP) (NM_153681.2: c.74T > C;p.Met25Thr and c.456delA;p.Glu153AsnFs*34). PIGP encodes a subunit of the enzyme that catalyzes the first step of GPI anchor biosynthesis. Both children presented with early-onset refractory seizures, hypotonia, and profound global developmental delay, reminiscent of other IGD phenotypes. Functional studies with patient cells showed reduced PIGP mRNA levels, and an associated reduction of GPI-anchored cell surface proteins, which was rescued by exogenous expression of wild-type PIGP. This work associates mutations in the PIGP gene with a novel autosomal recessive IGD, and expands our knowledge of the role of PIG genes in human development.


Assuntos
Hexosiltransferases/genética , Proteínas de Membrana/genética , Espasmos Infantis/genética , Anormalidades Múltiplas/genética , Adulto , Linhagem Celular , Criança , Deficiências do Desenvolvimento/genética , Glicosilfosfatidilinositóis/deficiência , Glicosilfosfatidilinositóis/genética , Glicosilfosfatidilinositóis/metabolismo , Hemoglobinúria Paroxística/genética , Hexosiltransferases/metabolismo , Humanos , Deficiência Intelectual/genética , Proteínas de Membrana/metabolismo , Hipotonia Muscular/genética , Mutação , Linhagem , Convulsões/genética , Espasmos Infantis/metabolismo
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