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Breast cancer (BC) is the most common malignancy affecting Western women today. It is estimated that as many as 10% of BC cases can be attributed to germline variants. However, the genetic basis of the majority of familial BC cases has yet to be identified. Discovering predisposing genes contributing to familial BC is challenging due to their presumed rarity, low penetrance, and complex biological mechanisms. Here, we focused on an analysis of rare missense variants in a cohort of 12 families of Middle Eastern origins characterized by a high incidence of BC cases. We devised a novel, high-throughput, variant analysis pipeline adapted for family studies, which aims to analyze variants at the protein level by employing state-of-the-art machine learning models and three-dimensional protein structural analysis. Using our pipeline, we analyzed 1218 rare missense variants that are shared between affected family members and classified 80 genes as candidate pathogenic. Among these genes, we found significant functional enrichment in peroxisomal and mitochondrial biological pathways which segregated across seven families in the study and covered diverse ethnic groups. We present multiple evidence that peroxisomal and mitochondrial pathways play an important, yet underappreciated, role in both germline BC predisposition and BC survival.
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Neoplasias de la Mama , Aprendizaje Profundo , Predisposición Genética a la Enfermedad , Humanos , Neoplasias de la Mama/genética , Femenino , Mutación Missense , Linaje , Mutación de Línea GerminalRESUMEN
Context: Various genes have been associated with familial and sporadic primary hyperparathyroidism (PHPT), including activating mutations of the glial cells missing transcription factor 2 (GCM2) gene. Objective: The aim of this study was to assess the prevalence of the GCM2 p.Tyr394Ser variant in the Jerusalem Ashkenazi Jewish (AJ) population with PHPT, and to conclude whether routine genetic testing is justified. Methods: The blood of 40 self-reported AJ patients with PHPT and 200 AJ controls was tested for the GCM2 p.Tyr394Ser variant. Demographic and medical information was extracted from the patients' charts and evaluated accordingly. Results: Two (5%) PHPT patients and 3 (1.5%) controls were heterozygotes for the tested variant. Our patients were mostly (87.5%) sporadic cases. One of the heterozygote patients had familial PHPT; the other had 2 parathyroid adenomas, and the levels of his blood and urinary calcium were extremely high. Conclusion: Our results suggest that in AJ patients with sporadic, single-gland PHPT, the likelihood of the tested variant is low and genetic testing should be limited to those with familial PHPT or multiglandular disease.
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Deficiency of the endoplasmic reticulum transmembrane protein ARV1 leads to epileptic encephalopathy in humans and in mice. ARV1 is highly conserved, but its function in human cells is unknown. Studies of yeast arv1 null mutants indicate that it is involved in a number of biochemical processes including the synthesis of sphingolipids and glycosylphosphatidylinositol (GPI), a glycolipid anchor that is attached to the C-termini of many membrane bound proteins. GPI anchors are post-translational modifications, enabling proteins to travel from the endoplasmic reticulum (ER) through the Golgi and to attach to plasma membranes. We identified a homozygous pathogenic mutation in ARV1, p.Gly189Arg, in two brothers with infantile encephalopathy, and characterized the biochemical defect caused by this mutation. In addition to reduced expression of ARV1 transcript and protein in patients' fibroblasts, complementation tests in yeast showed that the ARV1 p.Gly189Arg mutation leads to deficient maturation of Gas1, a GPI-anchored protein, but does not affect sphingolipid synthesis. Our results suggest, that similar to mutations in other proteins in the GPI-anchoring pathway, including PIGM, PIGA, and PIGQ, ARV1 p.Gly189Arg causes a GPI anchoring defect and leads to early onset epileptic encephalopathy.
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Encefalopatías/genética , Proteínas Portadoras/genética , Glicosilfosfatidilinositoles/biosíntesis , Discapacidad Intelectual/genética , Proteínas de la Membrana/genética , Convulsiones/genética , Adolescente , Niño , Retículo Endoplásmico/metabolismo , Fibroblastos/metabolismo , Prueba de Complementación Genética , Aparato de Golgi/metabolismo , Homocigoto , Humanos , Lípidos/química , Masculino , Manosiltransferasas/genética , Mutación , Linaje , Dominios Proteicos , TemperaturaRESUMEN
OBJECTIVES: Use of thiopurines for inflammatory bowel diseases (IBDs) is declining in some parts of the world. We aimed to explore outcomes of thiopurines and predictors of response in a real-world prospective cohort of children with dose optimization. METHODS: Children with IBD treated with thiopurines without biologics were enrolled. Dosing was guided by thiopurine S-methyltransferase-activity at baseline and by clinical response and toxicity at 4 months; 1 year into the study, therapeutic drug monitoring at 4 months was also considered in the decision making. The primary outcome was steroid-free remission without treatment escalation by 12 months (SFR), using the intention-to-treat approach. RESULTS: A total of 129 children were included (74% Crohn disease [CD] and 26% ulcerative colitis [UC]). SFR was achieved in 37 (39%) CD and 13 (39%) UC patients, and SFR with normal erythrocyte sedimentation rate/C-reactive protein in 20 (21%) and 9 (27%), respectively. At 4 months, mean corpuscular volume/white blood cell ratio and Δ absolute neutrophil count weakly correlated with 6-thioguanine (râ=â0.33, Pâ=â0.02 and râ=â0.32, Pâ=â0.02, respectively). In CD, SFR was associated with 4-month median weighted Pediatric Crohn Disease Activity Index (2.5 [IQR 0-7.5] in responders vs 5 in nonresponders [0-12.5], Pâ=â0.048) and Δabsolute neutrophil count (1.7 [IQR 0.7-4.1] vs 0.05 [-2.3-0.9]; Pâ=â0.03). Mild drug-related adverse events were recorded in 30 children (22%), 3 required stopping the drug. CONCLUSIONS: In this real-life prospective cohort using dose optimization, thiopurines were safe and effective in 21% of CD and 27% of UC patients, including normalization of C-reactive protein and erythrocyte sedimentation rate. Thiopurines remain a viable option in the treatment algorithm of mild-moderate pediatric IBD, especially in girls whose risk for lymphoma is lower.
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Colitis Ulcerosa , Enfermedades Inflamatorias del Intestino , Azatioprina/uso terapéutico , Niño , Estudios de Cohortes , Colitis Ulcerosa/tratamiento farmacológico , Femenino , Humanos , Inmunosupresores/uso terapéutico , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Mercaptopurina/uso terapéutico , Estudios ProspectivosRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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OBJECTIVES: Loss-of-function mutations of BMPR1A cause juvenile polyposis syndrome (JPS), but large genomic deletions in BMPR1A are rare, reported in few families only, and data regarding the associated phenotype are limited. METHODS: We investigated clinical features and genomic data of 7 extended seemingly unrelated families with a genomic deletion of the entire coding region of BMPR1A. We defined mutation size, mutation prevalence, and tumor pathogenesis using whole-genome sequencing, targeted genotyping, and haplotype analysis. RESULTS: Patients with JPS from 7 families of Bukharin Jewish ancestry carried a deletion of 429 kb, encompassing the BMPR1A coding sequence and 8 downstream genes. Haplotype analysis and testing controls identified this as a common founder mutation occurring in 1/124 individuals of Bukharin origin. Tumor testing did not demonstrate loss of heterozygosity. Among carriers, JPS was almost fully penetrant, but clinical features varied widely, ranging from mild to very severe, including pan-enteric polyps, gastritis, and colorectal, esophageal, and testicular cancer, and carriers with phenotypes, which would not have raised suspicion of JPS. DISCUSSION: The phenotype in this large cohort was extremely variable, although all carriers shared the same variant and the same genetic background. New observations include a preponderance of adenomatous rather than juvenile polyps, possible association with testicular cancer, and unexpected upper gastrointestinal involvement.
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Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Gastritis/complicaciones , Poliposis Intestinal/congénito , Síndromes Neoplásicos Hereditarios/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Preescolar , Neoplasias Colorrectales/complicaciones , Neoplasias Colorrectales/etnología , Neoplasias Colorrectales/genética , Neoplasias Esofágicas/complicaciones , Neoplasias Esofágicas/etnología , Neoplasias Esofágicas/genética , Femenino , Gastritis/etnología , Gastritis/genética , Genoma , Heterocigoto , Humanos , Poliposis Intestinal/genética , Pólipos Intestinales/complicaciones , Pólipos Intestinales/etnología , Pólipos Intestinales/genética , Pólipos Intestinales/patología , Israel/etnología , Judíos/genética , Masculino , Persona de Mediana Edad , Linaje , Fenotipo , Eliminación de Secuencia/genética , Neoplasias Testiculares/complicaciones , Neoplasias Testiculares/etnología , Neoplasias Testiculares/genética , Adulto JovenRESUMEN
OBJECTIVE: To identify the genetic basis of a childhood-onset syndrome of variable severity characterised by progressive spinocerebellar ataxia, mental retardation, psychotic episodes and cerebellar atrophy. METHODS: Identification of the underlying mutations by whole exome and whole genome sequencing. Consequences were examined in patients' cells and in yeast. RESULTS: Two brothers from a consanguineous Palestinian family presented with progressive spinocerebellar ataxia, mental retardation and psychotic episodes. Serial brain imaging showed severe progressive cerebellar atrophy. Whole exome sequencing revealed a novel mutation: pitrilysin metallopeptidase 1 (PITRM1) c.2795C>T, p.T931M, homozygous in the affected children and resulting in 95% reduction in PITRM1 protein. Whole genome sequencing revealed a chromosome X structural rearrangement that also segregated with the disease. Independently, two siblings from a second Palestinian family presented with similar, somewhat milder symptoms and the same PITRM1 mutation on a shared haplotype. PITRM1T931M carrier frequency was 0.027 (3/110) in the village of the first family evaluated, and 0/300 among Palestinians from other locales. PITRM1 is a mitochondrial matrix enzyme that degrades 10-65 amino acid oligopeptides, including the mitochondrial fraction of amyloid-beta peptide. Analysis of peptide cleavage activity by the PITRM1T931M protein revealed a significant decrease in the degradation capacity specifically of peptides ≥40 amino acids. CONCLUSION: PITRM1T931M results in childhood-onset recessive cerebellar pathology. Severity of PITRM1-related disease may be affected by the degree of impairment in cleavage of mitochondrial long peptides. Disruption and deletion of X linked regulatory segments may also contribute to severity.
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Enfermedades Cerebelosas/genética , Cerebelo/patología , Mutación con Pérdida de Función , Metaloendopeptidasas/genética , Adolescente , Edad de Inicio , Árabes/genética , Atrofia , Enfermedades Cerebelosas/enzimología , Cerebelo/enzimología , Niño , Humanos , Masculino , Mitocondrias/enzimología , Proteínas Mitocondriales/genética , Linaje , Secuenciación del Exoma , Secuenciación Completa del Genoma , Adulto JovenRESUMEN
Hereditary mixed polyposis syndrome is a rare colon cancer predisposition syndrome caused by a duplication of a noncoding sequence near the gremlin 1, DAN family BMP antagonist gene (GREM1) originally described in Ashkenazi Jews. Few families with GREM1 duplications have been described, so there are many questions about detection and management. We report 4 extended families with the duplication near GREM1 previously found in Ashkenazi Jews; 3 families were identified at cancer genetic clinics in Israel and 1 family was identified in a cohort of patients with familial colorectal cancer. Their clinical features include extracolonic tumors, onset of polyps in adolescence, and rapid progression of some polyps to advanced adenomas. One family met diagnostic criteria for Lynch syndrome. Expansion of the hereditary mixed polyposis syndrome phenotype can inform surveillance strategies for carriers of GREM1 duplications.
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Poliposis Adenomatosa del Colon/diagnóstico , Poliposis Adenomatosa del Colon/genética , Biomarcadores de Tumor/genética , Colon/patología , Neoplasias Colorrectales Hereditarias sin Poliposis/diagnóstico , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Detección Precoz del Cáncer/métodos , Duplicación de Gen , Péptidos y Proteínas de Señalización Intercelular/genética , Poliposis Adenomatosa del Colon/etnología , Poliposis Adenomatosa del Colon/patología , Adulto , Anciano , Anciano de 80 o más Años , Colonoscopía , Neoplasias Colorrectales Hereditarias sin Poliposis/etnología , Neoplasias Colorrectales Hereditarias sin Poliposis/patología , Análisis Mutacional de ADN , Progresión de la Enfermedad , Femenino , Predisposición Genética a la Enfermedad , Herencia , Humanos , Israel , Judíos/genética , Masculino , Persona de Mediana Edad , Técnicas de Diagnóstico Molecular , Mutación , Linaje , Fenotipo , Factores de Tiempo , Adulto JovenRESUMEN
PURPOSE: Population screening of three common BRCA1/BRCA2 mutations in Ashkenazi Jews (AJ) apparently fulfills screening criteria. We compared streamlined BRCA screening via self-referral with proactive recruitment in medical settings. METHODS: Unaffected AJ, age ≥25 years without known familial mutations, were either self-referred or recruiter-enrolled. Before testing, participants received written information and self-reported family history (FH). After testing, both non-carriers with significant FH and carriers received in-person genetic counseling. Psychosocial questionnaires were self-administered 1 week and 6 months after enrollment. RESULTS: Of 1,771 participants, 58% were recruiter-enrolled and 42% were self-referred. Screening uptake was 67%. Recruited enrollees were older (mean age 54 vs. 48, P < 0.001) and had less suggestive FH (23 vs. 33%, P < 0.001). Of 32 (1.8%) carriers identified, 40% had no significant FH. Post-test counseling compliance was 100% for carriers and 89% for non-carrier women with FH. All groups expressed high satisfaction (>90%). At 6 months, carriers had significantly increased distress and anxiety, greater knowledge, and similar satisfaction; 90% of participants would recommend general AJ BRCA screening. CONCLUSION: Streamlined BRCA screening results in high uptake, very high satisfaction, and no excess psychosocial harm. Proactive recruitment captured older women less selected for FH. Further research is necessary to target younger women and assess other populations.Genet Med advance online publication 08 December 2016.
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Proteína BRCA1/genética , Proteína BRCA2/genética , Pruebas Genéticas/métodos , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/epidemiología , Femenino , Efecto Fundador , Genes BRCA1 , Genes BRCA2 , Asesoramiento Genético/métodos , Predisposición Genética a la Enfermedad , Humanos , Judíos/genética , Masculino , Tamizaje Masivo/métodos , Persona de Mediana Edad , Mutación , Derivación y Consulta , Encuestas y CuestionariosRESUMEN
New technologies are revealing genetic variants of unknown significance (VUS), raising questions about the indications that call for preimplanation genetic diagnosis (PGD). Two couples requesting PGD for VUS are presented. The first couple requested PGD for Lynch syndrome. Whole exome sequencing identified in a healthy male with a family history of Lynch-associated tumours, a MLH1 missense variant. The variant had not been reported as pathogenic, but was predicted as damaging by algorithms. The second couple had a child diagnosed with pervasive developmental disorder and intellectual disability, carrying a microduplication on chr:Xp.22.3, and a microdeletion on chr:17q21.31. The maternally inherited X linked microduplication was also present in the mother's healthy brother and daughter, whereas the chr17 microdeletion was a de-novo event. As chromosomal microarrays and whole-exome sequencing are becoming standard tests, couples are requesting PGD for these VUS. The risk of possible genetic diseases can be reduced by carrying out PGD for uncertain findings, yet will inevitably lead to the birth of affected children despite the transfer of embryos that are not carriers of the familial variants. Findings of unknown significance demand urgent discussion and guidelines for their use as a risk-reduction measure in the preimplantation setting.
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Exoma , Fertilización In Vitro , Enfermedades Genéticas Congénitas/diagnóstico , Diagnóstico Preimplantación , Femenino , Humanos , Masculino , Análisis por Micromatrices , Embarazo , Conducta de Reducción del RiesgoRESUMEN
OBJECTIVES: Thiopurines are effective for maintenance of remission in inflammatory bowel disease (IBD) in only about half of patients. Predictors of response may assist in selecting the most appropriate patients for thiopurine therapy. Thiopurines inhibit Rac1, a GTPase that exerts an antiapoptotic effect on T-lymphocytes. A genetic association was recently demonstrated between a Rac1 single nucleotide polymorphism (SNP) and poorer response to thiopurines in adult patients with Crohn disease. We aimed to determine whether Rac1 SNPs are associated with response to thiopurines in children with IBD. METHODS: Children with IBD treated with thiopurines were prospectively followed for 1 year and were genotyped for 3 Rac1 SNPs previously found to be relevant to IBD: rs10951982, rs4720672, and rs34932801. The rate of sustained steroid-free remission (SSFR) without treatment escalation by 12 months was compared between wild types (WTs) and heterozygotes. RESULTS: A total of 59 patients were studied (63% boys, 80% having Crohn disease, mean age 13â±â4.1). Nineteen of the 41 WT (46%) and 9 of the 15 (60%) heterozygotes for rs10951982 were in SSFR (Pâ=â0.55). Similarly, 21 of the 45 (47%) WT and 8 of the 12 (67%) heterozygotes for rs4720672 were in remission (Pâ=â0.33). Finally, 21 of the 45 (47%) WT and 3 of the 5 (60%) heterozygotes for rs34932801 were in remission (Pâ=â0.66). All of the 3 comparisons remained nonsignificant in a sensitivity analysis of only the patients with Crohn disease. CONCLUSIONS: We did not find an association between 3 Rac1 SNPs and thiopurine effectiveness by 12 months in a prospective study of children with IBD. Other predictors of response should be sought to optimize patient selection for thiopurine therapy.
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Azatioprina/uso terapéutico , Resistencia a Medicamentos , Inmunosupresores/uso terapéutico , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Mercaptopurina/uso terapéutico , Polimorfismo de Nucleótido Simple , Proteína de Unión al GTP rac1/genética , Adolescente , Niño , Estudios de Cohortes , Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/genética , Colitis Ulcerosa/metabolismo , Enfermedad de Crohn/tratamiento farmacológico , Enfermedad de Crohn/genética , Enfermedad de Crohn/metabolismo , Inhibidores Enzimáticos/uso terapéutico , Femenino , Estudios de Asociación Genética , Heterocigoto , Homocigoto , Humanos , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/metabolismo , Israel , Estudios Longitudinales , Masculino , Inducción de Remisión , Proteína de Unión al GTP rac1/antagonistas & inhibidores , Proteína de Unión al GTP rac1/metabolismoRESUMEN
In the Ashkenazi Jewish (AJ) population of Israel, 11% of breast cancer and 40% of ovarian cancer are due to three inherited founder mutations in the cancer predisposition genes BRCA1 and BRCA2. For carriers of these mutations, risk-reducing salpingo-oophorectomy significantly reduces morbidity and mortality. Population screening for these mutations among AJ women may be justifiable if accurate estimates of cancer risk for mutation carriers can be obtained. We therefore undertook to determine risks of breast and ovarian cancer for BRCA1 and BRCA2 mutation carriers ascertained irrespective of personal or family history of cancer. Families harboring mutations in BRCA1 or BRCA2 were ascertained by identifying mutation carriers among healthy AJ males recruited from health screening centers and outpatient clinics. Female relatives of the carriers were then enrolled and genotyped. Among the female relatives with BRCA1 or BRCA2 mutations, cumulative risk of developing either breast or ovarian cancer by age 60 and 80, respectively, were 0.60 (± 0.07) and 0.83 (± 0.07) for BRCA1 carriers and 0.33 (± 0.09) and 0.76 (± 0.13) for BRCA2 carriers. Risks were higher in recent vs. earlier birth cohorts (P = 0.006). High cancer risks in BRCA1 or BRCA2 mutation carriers identified through healthy males provide an evidence base for initiating a general screening program in the AJ population. General screening would identify many carriers who are not evaluated by genetic testing based on family history criteria. Such a program could serve as a model to investigate implementation and outcomes of population screening for genetic predisposition to cancer in other populations.
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Neoplasias de la Mama/genética , Genes BRCA1 , Genes BRCA2 , Pruebas Genéticas/métodos , Neoplasias Ováricas/genética , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/epidemiología , Femenino , Tamización de Portadores Genéticos , Predisposición Genética a la Enfermedad , Genética de Población , Humanos , Israel/epidemiología , Judíos/genética , Masculino , Persona de Mediana Edad , Neoplasias Ováricas/epidemiología , Factores de RiesgoRESUMEN
Persons with unexplained early-onset stroke have been targeted for screening surveys for Fabry disease, the most common of the three X-linked lysosomal disorders, because Fabry patients with stroke are more likely to have the life-threatening progressive cardiac and renal manifestations and would therefore most benefit from early diagnosis and intervention with enzyme replacement therapy (ERT). Among 175 Israeli patients with unexplained cryptogenic stroke screened for mutations in the Fabry α galactosidase A (GLA) gene, sequencing identified six with 2-4 GLA intronic variants, one of whose father and three sisters had the same variants. Two variants, c.640-16A>G (g.10115A>G) in intron 4 and c.1000-22C>T (g.10956C>T) in intron 6, were common to all patients. However, three males with a common four variant intronic haplotype had low residual enzyme activity and ~50% reduced mRNA expression. Transcript splice-site defects were not identified in any of the index cases and X-chromosome inactivation was not highly skewed in the six females. These data do not suggest that GLA intronic variants, per se, are pathogenic. Nonetheless, it is clear that a certain intronic haplotype in males with cryptogenic stroke is associated with reduced GLA expression and function.
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Enfermedad de Fabry/genética , Accidente Cerebrovascular/genética , alfa-Galactosidasa/genética , Adulto , Anciano , Regulación hacia Abajo/genética , Enfermedad de Fabry/epidemiología , Femenino , Regulación Enzimológica de la Expresión Génica , Predisposición Genética a la Enfermedad , Haplotipos , Humanos , Intrones , Masculino , Persona de Mediana Edad , ARN Mensajero/metabolismo , Factores Sexuales , Accidente Cerebrovascular/epidemiologíaRESUMEN
BACKGROUND: Thiopurine S-methyltransferase (TPMT) is a key enzyme that deactivates thiopurines, into their inactive metabolite, 6-methylmercaptopurine. Intermediate and low TPMT activity may lead to leukopenia following thiopurine treatment. The aim of this study was to determine TPMT activity and TPMT alleles (genotype-phenotype correlation) in Jews, aiming to develop an evidence-based pharmacogenetic assay for this population. METHODS: TPMT activity was determined in 228 Jewish volunteers by high performance liquid chromatography. Common allelic variants in the Caucasian population [TPMT*2 (G238C), TPMT *3A (G460A and A719G), TPMT* 3B (G460A) and TPMT*3C (A719G)] were tested. Phenotype-genotype correlation was examined and discordant cases were fully sequenced to identify novel genetic variants. RESULTS: Mean TPMT activity was 15.4 ± 4 U/ml red blood cells (range 1-34). Intermediate activity was found in 33/228 (14%) subjects and absent activity was found in one sample (0.4%). Only eight individuals (3.5% of the entire cohort and 24% of those with intermediate/low activity) were identified as carriers of a TPMT genetic variant, all of whom had the TPMT*3A allele. Sequencing the entire TPMT coding region and splice junctions in the remainder of the discordant cases did not reveal any novel variants. CONCLUSION: Genotyping TPMT in Jews yields a much lower rate of variants than identified in the general Caucasian population. We conclude that a biochemical assay to determine TPMT enzymatic activity should be performed in Jews before starting thiopurine treatment in order to identify low activity subjects.
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Regulación Enzimológica de la Expresión Génica/fisiología , Genotipo , Judíos/genética , Metiltransferasas/metabolismo , Adulto , Anciano , Femenino , Variación Genética , Humanos , Masculino , Metiltransferasas/genética , Persona de Mediana Edad , FarmacogenéticaRESUMEN
The aim of this study was to develop and perform a preimplantation genetic diagnosis (PGD) assay discriminating between wild-type and mutant alleles in two families with germline mosaicism. Family 1 had two children affected with severe myoclonic epilepsy (SCNA1A del exons 1-22). Family 2 had two children with tuberous sclerosis (TSC2 C1327T) and two healthy children. Neither mutation was detected in genomic DNA derived from the parents in either family. Informative microsatellite markers flanking SCNA1A and TSC2 along with the identified mutations were used to construct haplotypes. For tuberous sclerosis, single spermatozoa were analysed using a multiplex assay that included six informative markers and the TSC2 mutation. In family 1, deletion in the maternal allele was detected in the affected child. In family 2, both affected children and one healthy child shared the same paternal allele. To confirm mutant paternal transmission, single spermatozoa were analysed for the mutation along with six markers. Of 44 single spermatozoa, four showed the mutant T allele, allowing linkage between the mutation and the genetic markers. Both families delivered healthy children following IVF/PGD. In conclusion, germline mosaicism complicates allele assignment when constructing haplotypes for PGD. Sperm analysis is a useful tool for verifying allelic linkage.
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Epilepsias Mioclónicas/diagnóstico , Mutación de Línea Germinal , Mosaicismo , Diagnóstico Preimplantación/métodos , Espermatozoides/metabolismo , Esclerosis Tuberosa/diagnóstico , Alelos , Epilepsias Mioclónicas/embriología , Epilepsias Mioclónicas/genética , Epilepsias Mioclónicas/metabolismo , Exones , Salud de la Familia , Femenino , Ligamiento Genético , Marcadores Genéticos , Humanos , Masculino , Reacción en Cadena de la Polimerasa Multiplex , Mutación , Canal de Sodio Activado por Voltaje NAV1.1/genética , Canal de Sodio Activado por Voltaje NAV1.1/metabolismo , Linaje , Análisis de la Célula Individual , Esclerosis Tuberosa/embriología , Esclerosis Tuberosa/genética , Esclerosis Tuberosa/metabolismo , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
BACKGROUND: Fabry disease is one of three X-linked lysosomal disorders. Because of X-chromosome inactivation (XCI), wherein there is (random) transcriptional silencing of one of the X-chromosomes in each female cell, females are mosaic for the expression of (some) X-linked genes. Thus, based on penetrance and expression, some females heterozygous for Fabry disease are symptomatic but not to the same degree as hemizygous males. The purpose of this study was to ascertain whether skewed X-inactivation favoring the mutant α-galactosidase A allele exists in our cohort of female heterozygotes of Fabry disease. METHOD: All patients were evaluated by physical examination and ascribed disease-specific severity sub-scores for each of the four categories (cardiac, renal, neurological, general) and a total score using the Mainz Severity Score Index (MSSI). Blood samples were drawn for enzymatic activity of α-galactosidase A and for DNA extraction for analysis for α-galactosidase A mutations. XCI ratios were determined from peripheral blood leukocyte samples. The X-chromosome inactivation ratio was determined in each heterozygote. RESULTS: Of 77 samples, only 18.2% were highly skewed (80/20). Only 14.3% of samples with nonsense mutations were highly skewed. There were no correlations between the XCI ratios and age, enzymatic activity of α-galactosidase A, MSSI sub-scores or total score, or with the clinical signs of cardiac involvement, neuropathic pain, or proteinuria. CONCLUSION: These findings are comparable with others in Fabry disease, i.e., essentially the same as seen in normal non-elderly female population, raising the question of the mechanism underlying symptomatic phenotypic expression in heterozygous females with Fabry disease.
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Enfermedad de Fabry/enzimología , Enfermedad de Fabry/genética , Inactivación del Cromosoma X/genética , Adulto , Estudios de Cohortes , Femenino , Humanos , Riñón/enzimología , Persona de Mediana Edad , Mutación , Miocardio/enzimología , Sistema Nervioso/enzimología , Dolor/enzimología , Dolor/genética , Proteinuria/enzimología , Proteinuria/genética , Índice de Severidad de la Enfermedad , alfa-Galactosidasa/sangre , alfa-Galactosidasa/genéticaRESUMEN
Preimplantation genetic diagnosis (PGD) allows birth of unaffected children for couples at risk for a genetic disorder. We present the strategy and outcome of PGD for four lysosomal storage disorders (LSD): Tay-Sachs disease (TSD), Gaucher disease (GD), Fabry disease (FD), and Hunter syndrome (HS), and subsequent development of stem cell lines. For each disease, we developed a family-specific fluorescent multiplex single-cell PCR protocol that included the familial mutation and informative markers surrounding the mutation. Embryo biopsy and PGD analysis were performed on either oocytes (polar bodies one and two) or on single blastomeres from a six-cell embryo. We treated twenty families carrying mutations in these lysosomal storage disorders, including 3 couples requiring simultaneous analysis for two disorders (TSD/GD, TSD/balanced Robertsonian translocation 45XYder(21;14), and HS/oculocutaneus albinism). These analyses led to an overall pregnancy rate/embryo transfer of 38% and the birth of 20 unaffected children from 17 families. We have found that PGD for lysosomal disorders is a safe and effective method to prevent birth of affected children. In addition, by using mutant embryos for the derivation of stem cell lines, we have successfully established GD and HS hESC lines for use as valuable models in LSD research.
RESUMEN
The factors limiting the rather inefficient derivation of human embryonic stem cells (HESCs) are not fully understood. The aim of this study was to analyze the sex ratio in our 42 preimplantation genetic diagnosis (PGD)-HESC lines, in an attempt to verify its affect on the establishment of HESC lines. The ratio between male and female PGD-derived cell lines was compared. We found a significant increase in female cell lines (76%). This finding was further confirmed by a meta-analysis for combining the results of all PGD-derived HESC lines published to date (148) and all normal karyotyped HESC lines derived from spare in vitro fertilization embryos worldwide (397). Further, gender determination of embryos demonstrated that this difference originates from the actual derivation process rather than from unequal representation of male and female embryos. It can therefore be concluded that the clear-cut tendency for female preponderance is attributed to suboptimal culture conditions rather than from a true gender imbalance in embryos used for derivation of HESC lines. We propose a mechanism in which aberrant X chromosome inactivation and/or overexpression of critical metabolic X-linked genes might explain this sex dimorphism.
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Embrión de Mamíferos/fisiología , Células Madre Embrionarias/citología , Razón de Masculinidad , Biomarcadores/análisis , Línea Celular , Cromosomas Humanos/fisiología , Embrión de Mamíferos/citología , Desarrollo Embrionario , Células Madre Embrionarias/fisiología , Femenino , Genoma Humano , Humanos , Cariotipo , Masculino , Diagnóstico Preimplantación , Inactivación del Cromosoma XRESUMEN
OBJECTIVE: To develop a reliable and accurate preimplantation genetic diagnosis (PGD) method in six families with endocrine diseases: persistent hyperinsulinemic hypoglycemia of infancy (PHHI), congenital adrenal hyperplasia (CAH) salt-wasting form, Sanjat-Sakati syndrome and multiple endocrine neoplasia 2A (MEN 2A). METHODS: For each disease a battery of at least four informative markers surrounding the tested gene were identified and for each family a protocol of multiplex fluorescent markers was developed and performed on single cells. RESULTS: PGD for PHHI was performed in three families. In family 1 two healthy children were born from different cycles, in family 2 three healthy children were born from two cycles, and in family 3 a healthy boy was born. For CAH in one family a healthy girl was born. One PGD cycle for Sanjat-Sakati resulted in a clinical pregnancy that was terminated due to high nuccal translucency (46X0). For one family with MEN 2A disease, the eighth PGD cycle resulted in birth of healthy twins. In all children genetic confirmation of the healthy status was performed. CONCLUSIONS: PGD is an effective method for preventing birth of affected children with endocrine disorders. Increasing the awareness of clinicians to the availability of these methods is most important.
Asunto(s)
Enfermedades del Desarrollo Óseo/genética , Enfermedades del Desarrollo Óseo/prevención & control , Enfermedades del Sistema Endocrino/genética , Enfermedades del Sistema Endocrino/prevención & control , Enfermedades Pancreáticas/genética , Enfermedades Pancreáticas/prevención & control , Diagnóstico Preimplantación/métodos , Anomalías Múltiples/genética , Anomalías Múltiples/prevención & control , Hiperplasia Suprarrenal Congénita/genética , Hiperplasia Suprarrenal Congénita/prevención & control , Adulto , Enfermedades del Desarrollo Óseo/congénito , Hiperinsulinismo Congénito , Transferencia de Embrión , Enfermedades del Sistema Endocrino/congénito , Salud de la Familia , Femenino , Marcadores Genéticos , Trastornos del Crecimiento/congénito , Trastornos del Crecimiento/genética , Trastornos del Crecimiento/prevención & control , Humanos , Hipoparatiroidismo/congénito , Hipoparatiroidismo/genética , Hipoparatiroidismo/prevención & control , Discapacidad Intelectual/genética , Discapacidad Intelectual/prevención & control , Israel , Masculino , Neoplasia Endocrina Múltiple Tipo 2a/congénito , Neoplasia Endocrina Múltiple Tipo 2a/genética , Neoplasia Endocrina Múltiple Tipo 2a/prevención & control , Nesidioblastosis/congénito , Nesidioblastosis/genética , Nesidioblastosis/prevención & control , Osteocondrodisplasias/congénito , Osteocondrodisplasias/genética , Osteocondrodisplasias/prevención & control , Enfermedades Pancreáticas/congénito , Embarazo , Resultado del Embarazo , Convulsiones/congénito , Convulsiones/genética , Convulsiones/prevención & controlRESUMEN
BACKGROUND: Preimplantation genetic diagnosis (PGD) is a technique that enables identification of unaffected embryos prior to in vitro fertilization (IVF) transfer in couples at risk for a Mendelian disorder. Most cases involve severe genetic diseases with neurological features and/or major malformations. We present two couples in which PGD was performed for prevention of type 1 Gaucher disease, a non-neuronopathic, non-lethal disorder. MATERIALS AND METHODS: We developed a multiplex fluorescent PCR protocol, simultaneously amplifying the familial mutations and eight closely spaced, highly polymorphic informative microsatellite markers surrounding the gene, to be used for PGD analysis. RESULTS: Couple #1 mother was homozygous for the N370S mutation and the father carried the 84GG mutation; their first daughter receives specific Gaucher therapy. One PGD cycle resulted in seven embryos of which four had the paternal wild type allele; two were transferred resulting in a healthy baby boy born at term. Couple #2, each a carrier (N370S and R359Q), whose first-born child had died (age 5years) of Gaucher disease, underwent 7 PGD cycles. Only one cycle resulted in a clinical pregnancy but a miscarriage was followed at 10weeks. CONCLUSIONS: PGD is an effective and accurate method for preventing Gaucher disease type I in carrier couples. Since this disease is treatable, special ethical considerations and careful selection of couples should be performed.