Familial lecithin-cholesterol acyltransferase deficiency (FLD) is a rare recessive disorder of ch... more Familial lecithin-cholesterol acyltransferase deficiency (FLD) is a rare recessive disorder of cholesterol metabolism, caused by loss-of-function mutations in the human LCAT gene, leading to alterations in the lipid/lipoprotein profile, with extremely low HDL levels.The classical FLD phenotype is characterized by diffuse corneal opacification, haemolytic anaemia and proteinuric chronic kidney disease (CKD); an incomplete form, only affecting the corneas, has been reported in a few families worldwide.We describe an intermediate phenotype of LCAT deficiency, with CKD preceding the development of corneal clouding, in two Portuguese brothers apparently homozygous for a novel missense LCAT gene mutation. The atypical phenotype, the diagnosis of membranous nephropathy in the proband's native kidney biopsy, the late-onset and delayed recognition of the corneal opacification, the co-segregation with Gilbert syndrome and the late recurrence of the primary disease in kidney allograft all ...
MEF2C haploinsufficiency syndrome is characterized by severe intellectual disability, epilepsy, s... more MEF2C haploinsufficiency syndrome is characterized by severe intellectual disability, epilepsy, stereotypic movements, minor dysmorphisms and brain abnormalities. We report the case of a patient with a new MEF2C mutation, comparing his clinical and imaging features to those previously reported in the literature. A 10 year-old boy first came to pediatric neurology clinic at the age of 11 months because of severe psychomotor delay, without regression. He presented generalized hypotonia, poor eye contact, hand-mouth stereotypies, strabismus and minor facial dimorphisms. Epileptic seizures started at 26 months of age and were refractory. Brain MRI showed a slight increase in periventricular white matter signal and globally enlarged CSF spaces. Molecular analysis revealed a de novo, pathogenic and causative MEF2C mutation. MEF2C haploinsufficiency syndrome was recently recognized as a neurodevelopmental disorder. Severe intellectual disability with inability to speak and epilepsy are universal features in patients with MEF2C mutations, although mild cognitive and speech disorders have been reported to occur in patients with duplications. Epilepsy might be absent in patients with partial deletions. Abnormal movement patterns are very common in patients with MEF2C haploinsufficiency. Delayed myelination seems to be more commonly observed in patients with MEF2C mutations, while malformations of cortical development were only reported in patients with microdeletions. Although MEF2C haploinsufficiency prevalence is yet to be determined, it should be considered in the differential diagnosis of patients with severe intellectual disability and Rett-like features.
In the present study, we evaluated the methylation status of H19 gene DMR (Differentially Methyla... more In the present study, we evaluated the methylation status of H19 gene DMR (Differentially Methylated Region) using sodium bisulphite treatment of DNA, cloning of PCR (Polymerase Chain Reaction) products and automated DNA sequencing. We studied 69 clones from 5 sperm samples obtained from males with normal sperm counts (≥ 20x106 Sz/mL) and 31 clones from 2 sperm samples isolated from patients with severe oligozoospermia (< 5x106 Sz/mL). In sperm cells from severe oligozoospermic patients, we observed that the majority of clones (26/31, 83.9%) presented incomplete methylation, in contrast to clones obtained from sperm from normozoospermic males (33/69, 47.8%). In severe oligozoospermia, the average of unmethylated CpGs was of 4.1, ranging between 1 to 17 affected CpGs, while in normozoospermia the average was of 0.7, ranging from 1 to 3 affected CpGs, being statistically significant the difference between the two groups (p=0.001). The simultaneous demethylation of multiple CpGs occurred in 5/33 (15.2%) of the clones with incomplete methylation in the normozoospermia group, while in severe oligozoospermia the majority of the cases (19/26, 73.1%) presented associated demethylation simultaneously. Concomitantly, for each CpG position, the level of methylation deficit in normozoospermia was always inferior than 10% with the exception of CpG 6 and 9 (12 and 14%, respectively), while in severe oligozoospermia the majority of cases presented values of demethylation superior than 20% (P = 0.000-0.044). The complete demethylation of CTCF protein binding site (CpG 4-8) was only found in severe oligozoospermic cases (3/31, 9.7%). In conclusion, sperm from severe oligozoospermic patients present high rates of genomic imprinting errors, leading to the possibility of transmission of these errors to the offspring during ICSI (Intracytoplasmic Sperm Injection) treatments. In the absence of methylation at H19 DMR, CTCF protein might bind to the paternal allele and, consequently, promote the transmission of two active H19 and two inactive IGF2 alleles. This situation could negatively affect the in vitro development of preimplantation embryo. Thus, these results suggest the importance of a previous study of genomic imprinting in sperm before the ICSI procedure, in order to evaluate the existence of any risk.
Analbuminemia is a rare autosomal recessive disorder manifested by the absence or severe reductio... more Analbuminemia is a rare autosomal recessive disorder manifested by the absence or severe reduction of circulating serum albumin in homozygous or compound heterozygous subjects. It is an allelic heterogeneous defect, caused by a variety of mutations within the albumin gene. The analbuminemic condition was suspected in a Portuguese boy who presented with low albumin level (about 3.8g/L) and a significant hypercholesterolemia, but with no clinical findings. The albumin gene was screened by single strand conformational polymorphism and heteroduplex analysis and submitted to direct DNA sequencing. The proband was found to be homozygous for a previously unreported G>A change at position c.1289+1, the first base of intron 10, which inactivates the strongly conserved GT dinucleotide at the 5' splice site consensus sequence of the intron. The effect of this mutation was evaluated by examining the cDNA obtained by RT-PCR from the albumin mRNA extracted from proband's leukocytes. ...
Introdução: Efectuamos um estudo de pacientes com oligozoospermia e azoospermia para determinar s... more Introdução: Efectuamos um estudo de pacientes com oligozoospermia e azoospermia para determinar se o imprinting genómico dos espermatozóides se encontra alterado. Métodos: Analisaram-se 23 amostras de sémen de pacientes em estudo de infertilidade conjugal, 7 com ...
Familial lecithin-cholesterol acyltransferase deficiency (FLD) is a rare recessive disorder of ch... more Familial lecithin-cholesterol acyltransferase deficiency (FLD) is a rare recessive disorder of cholesterol metabolism, caused by loss-of-function mutations in the human LCAT gene, leading to alterations in the lipid/lipoprotein profile, with extremely low HDL levels.The classical FLD phenotype is characterized by diffuse corneal opacification, haemolytic anaemia and proteinuric chronic kidney disease (CKD); an incomplete form, only affecting the corneas, has been reported in a few families worldwide.We describe an intermediate phenotype of LCAT deficiency, with CKD preceding the development of corneal clouding, in two Portuguese brothers apparently homozygous for a novel missense LCAT gene mutation. The atypical phenotype, the diagnosis of membranous nephropathy in the proband's native kidney biopsy, the late-onset and delayed recognition of the corneal opacification, the co-segregation with Gilbert syndrome and the late recurrence of the primary disease in kidney allograft all ...
MEF2C haploinsufficiency syndrome is characterized by severe intellectual disability, epilepsy, s... more MEF2C haploinsufficiency syndrome is characterized by severe intellectual disability, epilepsy, stereotypic movements, minor dysmorphisms and brain abnormalities. We report the case of a patient with a new MEF2C mutation, comparing his clinical and imaging features to those previously reported in the literature. A 10 year-old boy first came to pediatric neurology clinic at the age of 11 months because of severe psychomotor delay, without regression. He presented generalized hypotonia, poor eye contact, hand-mouth stereotypies, strabismus and minor facial dimorphisms. Epileptic seizures started at 26 months of age and were refractory. Brain MRI showed a slight increase in periventricular white matter signal and globally enlarged CSF spaces. Molecular analysis revealed a de novo, pathogenic and causative MEF2C mutation. MEF2C haploinsufficiency syndrome was recently recognized as a neurodevelopmental disorder. Severe intellectual disability with inability to speak and epilepsy are universal features in patients with MEF2C mutations, although mild cognitive and speech disorders have been reported to occur in patients with duplications. Epilepsy might be absent in patients with partial deletions. Abnormal movement patterns are very common in patients with MEF2C haploinsufficiency. Delayed myelination seems to be more commonly observed in patients with MEF2C mutations, while malformations of cortical development were only reported in patients with microdeletions. Although MEF2C haploinsufficiency prevalence is yet to be determined, it should be considered in the differential diagnosis of patients with severe intellectual disability and Rett-like features.
In the present study, we evaluated the methylation status of H19 gene DMR (Differentially Methyla... more In the present study, we evaluated the methylation status of H19 gene DMR (Differentially Methylated Region) using sodium bisulphite treatment of DNA, cloning of PCR (Polymerase Chain Reaction) products and automated DNA sequencing. We studied 69 clones from 5 sperm samples obtained from males with normal sperm counts (≥ 20x106 Sz/mL) and 31 clones from 2 sperm samples isolated from patients with severe oligozoospermia (< 5x106 Sz/mL). In sperm cells from severe oligozoospermic patients, we observed that the majority of clones (26/31, 83.9%) presented incomplete methylation, in contrast to clones obtained from sperm from normozoospermic males (33/69, 47.8%). In severe oligozoospermia, the average of unmethylated CpGs was of 4.1, ranging between 1 to 17 affected CpGs, while in normozoospermia the average was of 0.7, ranging from 1 to 3 affected CpGs, being statistically significant the difference between the two groups (p=0.001). The simultaneous demethylation of multiple CpGs occurred in 5/33 (15.2%) of the clones with incomplete methylation in the normozoospermia group, while in severe oligozoospermia the majority of the cases (19/26, 73.1%) presented associated demethylation simultaneously. Concomitantly, for each CpG position, the level of methylation deficit in normozoospermia was always inferior than 10% with the exception of CpG 6 and 9 (12 and 14%, respectively), while in severe oligozoospermia the majority of cases presented values of demethylation superior than 20% (P = 0.000-0.044). The complete demethylation of CTCF protein binding site (CpG 4-8) was only found in severe oligozoospermic cases (3/31, 9.7%). In conclusion, sperm from severe oligozoospermic patients present high rates of genomic imprinting errors, leading to the possibility of transmission of these errors to the offspring during ICSI (Intracytoplasmic Sperm Injection) treatments. In the absence of methylation at H19 DMR, CTCF protein might bind to the paternal allele and, consequently, promote the transmission of two active H19 and two inactive IGF2 alleles. This situation could negatively affect the in vitro development of preimplantation embryo. Thus, these results suggest the importance of a previous study of genomic imprinting in sperm before the ICSI procedure, in order to evaluate the existence of any risk.
Analbuminemia is a rare autosomal recessive disorder manifested by the absence or severe reductio... more Analbuminemia is a rare autosomal recessive disorder manifested by the absence or severe reduction of circulating serum albumin in homozygous or compound heterozygous subjects. It is an allelic heterogeneous defect, caused by a variety of mutations within the albumin gene. The analbuminemic condition was suspected in a Portuguese boy who presented with low albumin level (about 3.8g/L) and a significant hypercholesterolemia, but with no clinical findings. The albumin gene was screened by single strand conformational polymorphism and heteroduplex analysis and submitted to direct DNA sequencing. The proband was found to be homozygous for a previously unreported G>A change at position c.1289+1, the first base of intron 10, which inactivates the strongly conserved GT dinucleotide at the 5' splice site consensus sequence of the intron. The effect of this mutation was evaluated by examining the cDNA obtained by RT-PCR from the albumin mRNA extracted from proband's leukocytes. ...
Introdução: Efectuamos um estudo de pacientes com oligozoospermia e azoospermia para determinar s... more Introdução: Efectuamos um estudo de pacientes com oligozoospermia e azoospermia para determinar se o imprinting genómico dos espermatozóides se encontra alterado. Métodos: Analisaram-se 23 amostras de sémen de pacientes em estudo de infertilidade conjugal, 7 com ...
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