Cryopreservation does not alter karyotype, multipotency, or NANOG/SOX2 gene expression of amniotic fluid mesenchymal stem cells.

Cryopreservation of mesenchymal stem cells from amniotic fluid is of clinical importance, as these cells can be harvested during the prenatal period and stored for use in treatments. We examined the behavior of mesenchymal stem cells from human amniotic fluid in culture that had been subjected to cryopreservation. We assessed chromosomal stability through karyotype analysis, determined whether multipotent capacity (differentiation into adipogenic, chondrogenic, and osteogenic cells) is maintained, and analyzed SOX2 and NANOG expression after thawing. Five amniotic fluid samples were cryopreserved for 150 days. No chromosomal aberrations were observed. The expression levels of NANOG and SOX2 also were quite similar before and after cryopreservation. Capacity for differentiation into adipogenic, chondrogenic, and osteogenic tissues also remained the same. We conclude that cryopreservation of amniotic fluid does not alter karyotype, NANOG/SOX2 gene expression, or multipotent capacity of stem cells that have been collected from amniotic fluid during pregnancy.

Frequency of the S65C mutation in the hemochromatosis gene in Brazil.

Development of hereditary hemochromatosis is associated with the C282Y, H63D or S65C mutations in the hemochromatosis gene. Though there is extensive knowledge about the former two, there is little information on the mechanism of action and the allelic frequency of the S65C mutation. We examined the prevalence of the S65C mutation of the hemochromatosis gene in Brazilians with clinical suspicion of hereditary hemochromatosis. Genotyping for this mutation was carried out in 633 individuals with clinical suspicion of hereditary hemochromatosis, using the polymerase chain reaction, followed by enzymatic digestion. The sample comprised 77.1% men and 22.9% women, giving a ratio of approximately 3:1; the mean age was 48.8 +/- 13.8 years. More than half (57.3%) of the individuals in the sample were 41 to 60 years old. The frequency of heterozygotes for this mutation was 0.016; no homozygous mutant patients were found. This is the first analysis of the S65C mutation in individuals suspected of having hereditary hemochromatosis in Brazil.

Frequency of FMR1 premutation in individuals with ataxia and/or tremor and/or parkinsonism.

A late onset neurological syndrome in carriers of premutation in FMR1 gene was recently described. The condition was named fragile-X-associated tremor/ataxia syndrome (FXTAS) and includes intentional tremor, cerebellar ataxia, parkinsonism, and cognitive deficit. We ascertained the contribution of FMR1 premutation to the phenotypes ataxia, tremor and/or parkinsonism. Sixty-six men over 45 years old presenting these symptoms, isolated or combined, were tested. Also, 74 normal men, randomly chosen in the population, formed the control group. In the patient group, no premutation carrier was found, which is in agreement with other observed frequencies reported elsewhere (0-5% variation). No significant differences were found when comparing gray zone allele frequencies among target and control groups. The FXTAS contribution in patients with phenotypic manifestations of FXTAS was 15/748 (2%). The presence of gray zone alleles is not correlated with FXTAS occurrence.

Frequency of FMR1 premutation in individuals with ataxia and/or tremor and/or parkinsonism

A late onset neurological syndrome in carriers of premutation in FMR1 gene was recently described. The condition was named fragile-X-associated tremor/ataxia syndrome (FXTAS) and includes intentional tremor, cerebellar ataxia, parkinsonism, and cognitive deficit. We ascertained the contribution of FMR1 premutation to the phenotypes ataxia, tremor and/or parkinsonism. Sixty-six men over 45 years old presenting these symptoms, isolated or combined, were tested. Also, 74 normal men, randomly chosen in the population, formed the control group. In the patient group, no premutation carrier was found, which is in agreement with other observed frequencies reported elsewhere (0-5% variation). No significant differences were found when comparing gray zone allele frequencies among target and control groups. The FXTAS contribution in patients with phenotypic manifestations of FXTAS was 15/748 (2%). The presence of gray zone alleles is not correlated with FXTAS occurrence.

Determinação do sexo do feto através de uma nova técnica não invasiva baseada em PCR / Determination of fetal sex by a new PCR method non invasive

A determinação do sexo do feto é geralmente realizada pelo procedimento de ultra-som que ocorre no segundo trimestre da gestação, sendo que, quando realizado antes da 13ª semana, esse método se mostra muito incerto.Técnicas moleculares utilizando o PCR já foram descrita, e possuem maior sensibilidade na determinação do sexo. Dentre estas técnicas existentes, as invasivas consistem em amniocentese ou coleta de amostra de vilo coriônico, seguida de PCR para determinar osexo e que representam em aumento de risco na gravidez e as técnicas não invasivas que conseguem detectar o DNA fetal no sangue periférico materno. Foi desenvolvida em nosso laboratório uma técnica capaz de detectar o sexo fetal nas primeiras semanas de gestaçãocom alto índice de acerto. A técnica consistiu em desenhar iniciadores que anelassem em regiões repetitivas espalhadas no cromossomoY e “PCR Nested” para aumentar a acurácia do exame. Os resultados demonstraram que a técnica possui sensibilidade e especificidade de 100%. Além disso, a PCR foi capaz de detectar em uma diluição seriada de uma amostra de DNA genômico masculina, previamente quantificada, a presença do cromossomo Y em amostrascontendo apenas 100 femtogramas de DNA.

The determination of fetal sex is currently carried out by ultrasound that is usually performed in the second trimester. However, it is inaccurate before 13th week of gestation. Molecular techniques such as PCR already had been described and were successful of fetal gender. Among these techniques, there are invasive methods: chorionic villus sampling and amniocentesis that are avoided because it is associated with a risk of fetal loss, and the non-invasive procedures that use of fetal DNA circulating in maternal blood. We report a new Nested PCR method with specific primers for repetitive sequences of the Y-chromossome. Our results indicated that sensitivity and specificity of the method were 100% and we can accurately detect Y-chromossome sequences in samples with only 100 femtograms of DNA template.

Founder effect of the 669insA mutation in BSCL2 gene causing Berardinelli-Seip congenital lipodystrophy in a cluster from Brazil.

Congenital generalized lipodystrophy (CGL) or Berardinelli-Seip Syndrome (BSCL) is a rare autosomal recessive disease characterized by a nearly-complete absence of adipose tissue from birth and severe metabolic alterations. The 669insA mutation in exon 4 of the BSCL2 gene was identified as the major genetic alteration leading to BSCL in a group of 22 patients from the northeastern Brazilian state of Rio Grande do Norte. Aiming to investigate the causes of the high frequency of BSCL in this region, a molecular genetic study was conducted using eight microsatelite markers located in chromosome 11. Additional investigations concerning the proportion of expected homozygous and heterozygous individuals, genetic diversity, fixation index and coefficient of endogamy were undertaken, and indicated significant differences by comparing the allelic and haplotypic frequencies observed for the BSCL affected families and the control group. It was concluded that a founder effect, genetic drift and consanguineous marriages have significantly affected the structure of this population, resulting in the highest frequency of BSCL in Brazil.

Frequency of islet cell autoantibodies (IA-2 and GAD) in young Brazilian type 1 diabetes patients.

Type 1 diabetes, as an autoimmune disease, presents several islet cell-specific autoantibodies such as islet cell antibody (ICA), anti-insulin, anti-glutamic acid decarboxylase (GAD) and the antibody (Ab) against tyrosine phosphatase (PTP)-like protein known as ICA-512 (IA-2). In order to determine the frequency of the anti-GAD and anti-IA-2 autoantibodies in Brazilian type 1 diabetes patients we studied 35 diabetes mellitus (DM) type 1 patients with recent-onset disease (12 months) who were compared to 12 children with normal fasting glucose. Anti-GAD65 and anti-IA-2 autoantibodies were detected with commercial immunoprecipitation assays. The frequency of positive results in recent-onset DM type 1 patients was 80.0% for GADAb, 62.9% for IA-2Ab and 82.9% for GADAb and/or IA-2Ab. The long-duration type 1 diabetes subjects presented frequencies of 54.1% for GADAb and IA-2Ab, and 67.5% for GAD and/or IA-2 antibodies. The control group showed no positive cases. Anti-GAD and IA-2 assays showed a high frequency of positivity in these Brazilian type 1 diabetes patients, who presented the same prevalence as a Caucasian population.

Frequency of islet cell autoantibodies (IA-2 and GAD) in young Brazilian type 1 diabetes patients

Type 1 diabetes, as an autoimmune disease, presents several islet cell-specific autoantibodies such as islet cell antibody (ICA), anti-insulin, anti-glutamic acid decarboxylase (GAD) and the antibody (Ab) against tyrosine phosphatase (PTP)-like protein known as ICA-512 (IA-2). In order to determine the frequency of the anti-GAD and anti-IA-2 autoantibodies in Brazilian type 1 diabetes patients we studied 35 diabetes mellitus (DM) type 1 patients with recent-onset disease 12 months and 37 type 1 diabetes patients with long-duration diabetes 12 months who were compared to 12 children with normal fasting glucose. Anti-GAD65 and anti-IA-2 autoantibodies were detected with commercial immunoprecipitation assays. The frequency of positive results in recent-onset DM type 1 patients was 80.0 percent for GADAb, 62.9 percent for IA-2Ab and 82.9 percent for GADAb and/or IA-2Ab. The long-duration type 1 diabetes subjects presented frequencies of 54.1 percent for GADAb and IA-2Ab, and 67.5 percent for GAD and/or IA-2 antibodies. The control group showed no positive cases. Anti-GAD and IA-2 assays showed a high frequency of positivity in these Brazilian type 1 diabetes patients, who presented the same prevalence as a Caucasian population

Fetal hemoglobin levels are related to metabolic control in diabetic subjects.

We have investigated the relationship between fetal hemoglobin (HbF) levels and metabolic control in subjects with insulin-dependent (N = 79) and non-insulin-dependent diabetes mellitus (N = 242). HbF and hemoglobin A1c (HbA1c) levels were increased in subjects with type 1 and type 2 diabetes as compared to levels in nondiabetic individuals (P < 0.0001), and were significantly higher in type 1 than in type 2 diabetes subjects. Lower levels of HbA1c and HbF were observed in type 2 diabetes subjects treated by diet, intermediate levels in those treated with oral hypoglycemic agents, and higher levels in those treated with insulin. HbF and HbA1c levels were correlated in type 1 diabetes (R2 = 0.57, P < 0.0001) and type 2 diabetes (R2 = 0.58, P < 0.0001) subjects. Following intense treatment, twelve diabetic patients showed significant improvement both in HbA1c and HbF values. We conclude that increased HbF levels reflect poor metabolic control in subjects with diabetes mellitus.

Fetal hemoglobin levels are related to metabolic control in diabetic subjects

We have investigated the relationship between fetal hemoglobin (HbF) levels and metabolic control in subjects with insulin-dependent (N = 79) and non-insulin-dependent diabetes mellitus (N = 242). HbF and hemoglobin A1c (HbA1c) levels were increased in subjects with type 1 and type 2 diabetes as compared to levels in nondiabetic individuals (P<0.0001), and were significantly higher in type 1 than in type 2 diabetes subjects. Lower levels of HbA1c and HbF were observed in type 2 diabetes subjects treated by diet, intermediate levels in those treated with oral hypoglycemic agents, and higher levels in those treated with insulin. HbF and HbA1c levels were correlated in type 1 diabetes (R2 = 0.57, P<0.0001) and type 2 diabetes (R2 = 0.58, P<0.0001) subjects. Following intense treatment, twelve diabetic patients showed significant improvement both in HbA1c and HbF values. We conclude that increased HbF levels reflect poor metabolic control in subjects with diabetes mellitus.

Specific insulin and proinsulin secretion in glucokinase-deficient individuals.

Glucokinase (GCK) is an enzyme that regulates insulin secretion, keeping glucose levels within a narrow range. Mutations in the glucokinase gene cause a rare form of diabetes called maturity-onset diabetes of the young (MODY). An early onset (less than 25 years), autosomal dominant inheritance and low insulin secretion stimulated by glucose characterize MODY patients. Specific insulin and proinsulin were measured in serum by immunofluorimetric assays (IFMA) during a 75-g oral glucose tolerance test (OGTT). Two kindreds (SA and LZ) were studied and compared to non-diabetic unrelated individuals (control group 1) matched for age and body mass index (BMI). In one kindred, some of these subjects were also obese (BMI > 26 kg/m2), and other family members also presented with obesity and/or late-onset NIDDM. The MODY patients were also compared to a group of five of their first-degree relatives with obesity and/or late-onset NIDDM. The proinsulin profile was different in members of the two MODY kindreds. Fasting proinsulin and the proinsulin/insulin ratio were similar in MODY members of kindred LZ and subjects from control group 1, but were significantly lower than in MODY members of kindred SA (P < 0.02 and P < 0.01, for proinsulin and proinsulin/insulin ratio, respectively). Moreover, MODY members of family SA had higher levels of proinsulin and proinsulin/insulin ratio, although not significantly different, when compared to their first-degree relatives and to subjects from control group 2. In conclusion, we observed variable degrees of proinsulin levels and proinsulin/insulin ratio in MODY members of two different kindreds. The higher values of these parameters found in MODY and non-MODY members of kindered SA is probably related to the obesity and late-onset NIDDM background present in this family.

Specific insulin and proinsulin secretion in glucokinase-deficient individuals

Glucokinase (GCK) is an enzyme that regulates insulin secretion, keeping glucose levels within a narrow range. Mutations in the glucokinase gene cause a rare form of diabetes called maturity-onset diabetes of the young (MODY). An early onset (less than 25 years), autosomal dominant inheritance and low insulin secretion stimulated by glucose characterize MODY patients. Specific insulin and proinsulin were measured in serum by immunofluorimetric assays (IFMA) during a 75-g oral glucose tolerance test (OGTT). Two kindreds (SA and LZ) were studied and compared to non-diabetic unrelated individuals (control group 1) matched for age and body mass index (BMI). In one kindred, some of these subjects were also obese (BMI>26 kg/m2), and other family members also presented with obesity and/or late-onset NIDDM. The MODY patients were also compared to a group of five of their first-degree relatives with obesity and/or late-onset NIDDM. The proinsulin profile was different in members of the two MODY kindreds. Fasting proinsulin and the proinsulin/insulin ratio were similar in MODY members of kindred LZ and subjects from control group 1, but were significantly lower than in MODY members of kindred SA (P<0.02 and P<0.01, for proinsulin and proinsulin/insulin ratio, respectively). Moreover, MODY members of family SA had higher levels of proinsulin and proinsulin/insulin ratio, although not significantly different, when compared to their first-degree relatives and to subjects from control group 2. In conclusion, we observed variable degrees of proinsulin levels and proinsulin/insulin ratio in MODY members of two different kindreds. The higher values of these parameters found in MODY and non-MODY members of kindred SA is probably related to the obesity and late-onset NIDDM background present in this family.

[Cutis laxa associated to cardiac failure] / Cutis Laxa associada à insuficiência cardíaca congestiva.

OBJECTIVE: To focus attention on a rare pathology of the childhood which presents premature aging of the skin and can be lethal. METHODS: The authors present a case of cutis laxa, syndrome of premature aging, in an eight year-old child, and discuss the classification, diagnosis and prognosis of the disease. RESULTS: The child presented signs of premature aging when he was four years-old. The diagnosis of cutis laxa was confirmed by skin biopsy. The patient presented heart failure, a systemic complication different from those previously described, and died at eight years of age. CONCLUSIONS: The importance of the diagnosis of cutis laxa resides in the fact that besides characteristic dermatological findings, there are frequent systemic complications that can be the focus of preventive measures, since there is no specific treatment for this disease. Genetic counseling is another important issue in this condition.

Mutation screening in 18 Caucasian families suggest the existence of other MODY genes.

Maturity-onset diabetes of the young (MODY) is a heterogeneous subtype of non-insulin-dependent diabetes mellitus characterised by early onset, autosomal dominant inheritance and a primary defect in insulin secretion. To date five MODY genes have been identified: hepatocyte nuclear factor-4 alpha (HNF-4alpha/MODY1/TCF14) on chromosome 20q, glucokinase (GCK/MODY2) on chromosome 7p, hepatocyte nuclear factor-1 alpha (HNF-1alpha/MODY3/TCF1) on chromosome 12q, insulin promoter factor-1 (IPF1/MODY4) on chromosome 13q and hepatocyte nuclear factor-1 beta (HNF-1beta/MODY5/TCF2) on chromosome 17cen-q. We have screened the HNF-4alpha, HNF-1alpha and HNF-1beta genes in members of 18 MODY kindreds who tested negative for glucokinase mutations. Five missense (G31D, R159W, A161T, R200W, R271W), one substitution at the splice donor site of intron 5 (IVS5nt + 2T-->A) and one deletion mutation (P379fsdelT) were found in the HNF-1alpha gene, but no MODY-associated mutations were found in the HNF-4alpha and HNF-1beta genes. Of 67 French MODY families that we have now studied, 42 (63%) have mutations in the glucokinase gene, 14 (21%) have mutations in the HNF-1alpha gene, and 11 (16%) have no mutations in the HNF-4alpha, IPF1 and HNF-1beta genes. Eleven families do not have mutations in the five known MODY genes suggesting that there is at least one additional locus that can cause MODY.

Leptin levels, beta-cell function, and insulin sensitivity in families with congenital and acquired generalized lipoatropic diabetes.

Lipoatropic diabetes (LD) designates a group of syndromes characterized by diabetes mellitus with marked insulin resistance and either a localized or generalized absence of adipose tissue. In this study, we evaluated plasma leptin levels in subjects with congenital generalized lipoatropic diabetes (CGLD, n = 11) or acquired generalized lipoatropic diabetes (AGLD, n = 11), and assessed correlations between leptin levels and estimations of insulin secretion and insulin sensitivity using homeostasis model assessment (HOMA). Leptin levels were 0.86 +/- 0.32, 1.76 +/- 0.78, and 6.9 +/- 4.4 ng/mL in subjects with CGLD, AGLD, and controls (n = 19), respectively (ANOVA P < 0.0001). Specific insulin levels were 154 +/- 172, 177 +/- 137 and 43 +/- 22 pmol/L, respectively (P < 0.0001). Insulin sensitivity was significantly decreased in both groups with LD (P < 0.0001), whereas HOMA beta-cell function was not significantly different when compared with controls. Leptin levels were significantly correlated with body mass index, insulin levels, and HOMA beta-cell function, and inversely correlated with insulin sensitivity in control subjects but not in subjects with generalized LD. In conclusion, decreased leptin levels were observed in subjects with generalized LD, with a trend towards lower levels in the acquired than in the congenital form (P = 0.06). The temporal relationship between the decrease in leptin levels and the development of lipoatrophy should be investigated in at-risk young relatives of subjects with the acquired forms to assess the usefulness of leptin levels as a marker of lipoatrophy.

Identification of 14 new glucokinase mutations and description of the clinical profile of 42 MODY-2 families.

Mutations in glucokinase are associated with defects in insulin secretion and hepatic glycogen synthesis resulting in mild chronic hyperglycaemia, impaired glucose tolerance or diabetes mellitus. We screened members of 35 families with features of maturity-onset diabetes of the young for mutations in the glucokinase gene and found 16 different mutations. They included 14 new mutations in the glucokinase gene: 9 missense mutations (A53S, G80A, H137R, T168P, M210T, C213R, V226M, S336L and V367M); 2 nonsense mutations (E248X and S360X); a deletion of one nucleotide resulting in a frameshift (V401del1); a substitution of a conserved nucleotide at a splice acceptor site (L122-1G-->T); and a 10 base pair deletion that removed the GT of the splice donor site and the following eight nucleotides (K161 + 2del10). In addition, we found two previously identified mutations: R186X and G261R. Study of 260 subjects with glucokinase-deficient hyperglycaemia from 42 families with 36 different GCK mutations made it possible to define the clinical profile of this subtype of non-insulin-dependent diabetes mellitus (NIDDM). Hyperglycaemia due to glucokinase deficiency is often mild (fewer than 50% of subjects have overt diabetes) and is evident during the early years of life. Despite the long duration of hyperglycaemia, glucokinase-deficient subjects have a low prevalence of micro- and macro-vascular complications of diabetes. Obesity, arterial hypertension and dyslipidaemia are also uncommon in this form of NIDDM.

Antibodies to bovine serum albumin in Brazilian children and young adults with IDDM.

OBJECTIVE: To evaluate the prevalence of IgG antibodies to bovine serum albumin (BSA) in a cohort of Brazilian children and young adults with IDDM. RESEARCH DESIGN AND METHODS: Sera from 81 subjects with < 1 year of IDDM (group 1), III subjects with > 1 year of IDDM (group 2), and 207 normoglycemic subjects were tested using an immunofluorimetric assay. A receiver-operating-characteristic curve was used to establish the threshold of anti-BSA antibody titers defining the positivity of the assay. RESULTS: The distribution of the fluorimetric index (FI) of anti-BSA antibodies did not have a gaussian profile. Rank sum of FI was significantly higher in patients than in control subjects (P < 0.0001). Average logFI values of both IDDM groups were significantly higher than that of the control group (P < 0.005 for both groups). There was a trend toward higher FI levels in group 1 than in group 2 (P = 0.06). A FI cutoff of 0.7 optimized the ratio of true-positive to false-positive of the assay, with the best equilibrium between sensitivity and specificity. The prevalence of anti-BSA antibodies was 52% in group 1, 47% in group 2, and 28% in the control group (P = 0.0001). An independent association between anti-BSA antibodies and IDDM, with an odds ratio of 3.03 (P < 0.0001), was observed in a logistic regression analysis. However anti-BSA antibodies explained only 5% of the variability of IDDM versus NIDDM. CONCLUSIONS: Our results confirm that the prevalence of anti-BSA antibodies is higher in IDDM subjects than in control subjects, even after 1 year of diabetes. However, a large overlap of antibody titers is observed in patients and control subjects, suggesting that anti-BSA antibodies are neither sensitive nor specific markers of IDDM.