Osteogenesis imperfecta in Brazilian patients.

Osteogenesis Imperfecta (OI) is a heterogeneous genetic disorder characterized by bone fragility and fracture. Mutations in 20 distinct genes can cause OI, and therefore, the genetic diagnosis of OI is frequently difficult to obtain because of the great number of genes that can be related with this disease. Studies that report the most frequently mutated genes in OI patients can help to improve molecular strategies for diagnosis of the disease. In order to characterize the mutation profile of OI in Brazilian patients, we analyzed 30 unrelated patients through SSCP screening, NGS gene panel, and/or Sanger sequencing for the 11 most frequently mutated genes in the database of mutations, including COL1A1, COL1A2, P3H1, CRTAP, PPIB, SERPINH1, SERPINF1, FKBP10, SP7, WNT1 and IFITM5. Disease-causing variants were identified in COL1A1, COL1A2, FKBP10, P3H1, and IFITM5. A total of 28 distinct mutations were identified, including seven novel changes. Our data show that the analysis of these five genes is able to detect at least 95% of causative mutations in OI disorder from Brazilian population. However, it has to be taken into considerations that distinct populations can have different frequencies of disease-causing variants. Hence, it is important to replicate this study in other groups.

Low bone turnover and low bone density in a cohort of adults with Down syndrome.

UNLABELLED: Increased incidence of osteoporosis in Down syndrome has been reported, but etiology is not established. We report low bone turnover markers and bone mineral density (BMD) in a cohort of people with Down syndrome without consistent clinical risk factors. Our results should guide future studies and treatments for this common problem. INTRODUCTION: To better understand the etiology for osteoporosis in Down syndrome (DS), we measured bone density by dual-energy X-ray absorptiometry (DXA) and circulating biochemical markers of bone formation and resorption in a cohort of 30 community-dwelling DS adults. METHODS: Seventeen males and 13 females followed in the University of Arkansas Down Syndrome Clinic were evaluated by DXA to estimate BMD and underwent phlebotomy to measure serum procollagen type-1 intact N-terminal propeptide (P1NP) to evaluate bone formation, and serum C-terminal peptide of type-I collagen (CTx) to evaluate bone resorption. RESULTS: Seven of 13 DS females and 12 of 17 DS males had low bone mass at one of measured sites (z≤-2.0). When data were grouped by age, males had apparent osteopenia earlier than females. The mean P1NP in the normal group was 19.2±5.2 ng/ml vs. 2.2±0.9 ng/ml in the DS group (P=0.002). Serum CTx levels in the normal group were 0.4±0.1 ng/ml vs. 0.3±0.1 ng/ml (P=0.369). CONCLUSIONS: Low BMD in adults with DS is correlated with a significant decrease in bone formation markers, compared to controls without DS, and is independent of gender. These data suggest that diminished osteoblastic bone formation and inadequate accrual of bone mass, with no significant differences in bone resorption, are responsible for the low bone mass in DS. These observations question the use of antiresorptive therapy in this population and focus attention on increasing bone mass by other interventions.

Alterações ósseas em lactentes com sífilis congênita / Bone changes in infants with congenital syphilis

Introdução: a sífilis causa morbidade na vida intrauterina, desfechos negativos em mais de 50% dos casos e complicações nos nascidos vivos. Lesões ósseas são frequentes na sífilis congênita (SC), ocorrendo em 70 a 100% dos casos. Objetivo: relatar casos de dez lactentes com diagnóstico de SC precoce e alterações ósseas ao exame radiológico de ossos longos, acompanhados no Serviço de Infectologia do HINSG. Relato dos casos: dez lactentes apresentaram lesões ósseas, entre crianças internadas com SC no HINSG, seis do sexo feminino e quatro do masculino. Metade era proveniente da Grande Vitória, três do interior e dois da Bahia. Dois tinham menos de 1 mês de vida. Seis das mães fizeram pré-natal, três tiveram o diagnóstico de sífilis na gestação e apenas uma foi adequadamente tratada. Duas crianças apresentaram osteomielite, sendo uma com lesão lítica na tíbia e as demais tiveram periostite. Mais de um osso foi afetado em seis lactentes. Os ossos mais afetados foram: tíbia (7/10), fêmur (6/10), úmero (5/10), rádio (2/10) e ulna (2/10). Sintoma comum entre todos foi choro frequente ao movimento de membros superiores ou inferiores, motivo de as mães terem procurado o serviço médico. Discussão: alterações radiológicas são importantes no diagnóstico de SC. Neste estudo, 60% das mães fizeram pré-natal, uma foi tratada corretamente e nenhuma das crianças foi diagnosticada e tratada logo após o nascimento, somente recebendo medicação adequada após o diagnóstico no nosso serviço. Alterações radiológicas da SC podem acontecer em tempo variado após a instalação da infecção. Podem ocorrer diferentes lesões, que incluem lesões da SC precoce: osteomielite diafisária, osteítes e periostite. Osteocondrite afeta áreas da cartilagem em crescimento. Lesões da SC tardia são severas, podem causar deformidades físicas e fratura patológica na criança, necrose de osso e osteólise no adulto e, às vezes associam-se a infecção piogênica ou difusão para tecidos adjacentes, com formações de fístulas cutâneas indolores.

Introduction: syphilis morbidity in intra-uterine life, produces negative outcomes in more than 50% of cases and complications in live births. Bone injuries are frequent in congenital syphilis (CS), occurring in 70 to 100% of the cases. Objective: to report cases of ten infants diagnosed with early SC and bone changes to the radiological examination of long bones, accompanied in the infectology service of HINSG. Case report: ten infants had bone injuries among children hospitalized with CS in HINSG, six female and four male. Half of the metropolitan region of Vitória, two from the interior and three from state of Bahia. Two were less than a month old. Six of the mothers had prenatal care, three were diagnosed with syphilis during pregnancy and only one was properly treated. Two children had osteomyelitis, and with a lytic lesion in the tibia and the other had periostitis. More than one bone was affected in six infants. Bones most affected: tibia (7/10), femur (6/10), humerus (5/10), radio (2/10) and ulna (2/10). Common symptom among all were frequent crying with movement of members. Reason why mothers have sought medical service. Discussion: changes are important in radiological diagnosis of CS. In thisstudy 60% of mothers have prenatal care, one was treated correctly and none of the children were diagnosed and treated soon after birth, only receiving proper medication after diagnosis in our service. Radiological changes of CS may happen after installation according to time of infection. Various injuries may occur, including injuries of CS early: osteomyelitis diaphyseal, osteítes and periostitis. Osteochondritis affects cartilage in areas of growth. Injury of late CS are severe, can cause physical de formities and pathological fracture in children, necrosis of bone and osteolysis in adults and sometimes linking up with pyogenic infection or spread to adjacent tissues, with formation of fistulas skin painless.

Genotyping of apolipoprotein e.

Apolipoprotein E (apo E) is a 299-amino acid plasma protein involved in cholesterol transport and is found in chylomicrons, very low density lipopro-tein, intermediate-density lipoprotein, and high-density lipoprotein (1,1).

Evaluation of chimerism in DNA samples by PCR amplification of D1S80 with detection by capillary electrophoresis.

BACKGROUND: Analysis of the relative amounts of donor and recipient DNA in bone marrow after bone marrow transplantation is frequently used to determine the status of the transplant. We studied the performance of an assay to quantify chimerism based on amplification of the D1S80 variable number tandem repeat marker by PCR with detection of PCR products by capillary electrophoresis (CE). METHODS AND RESULTS: Samples from potential bone marrow donors and recipients were analyzed separately and in mixtures to simulate various degrees of chimerism from 10% to 90% and subjected to PCR/CE analysis. There was excellent agreement between the measured and known relative proportions of DNA components in chimeric samples. The lower limit of sensitivity for detection of chimerism was 1%; between-runs coefficients of variation were <5%. CONCLUSIONS: Amplification of the D1S80 minisatellite by PCR with CE detection is a reliable method for determination of the relative contribution of different DNAs in mixed samples. This method is fast, quantitative, and extremely reproducible.

Association of the prothrombin G20210A mutation with factor V Leiden in a midwestern American population.

The prothrombin G20210A mutation has been identified as a risk factor for thrombosis. We studied the relationship between prothrombin G20210A and factor V Leiden mutations in patients with thrombophilia. The first 264 patients for whom these molecular diagnostic studies were requested at our institution were included in the study. For 116 of the 264 patients, additional coagulation test results were available in the laboratory database. The prothrombin G20210A mutation was found in 16 (6.1%) of the patients and the factor V Leiden mutation in 44 (16.7%). Of the 16 patients with the prothrombin G20210A mutation, 8 also carried factor V Leiden; this association was significant. In contrast, only 2 patients of the 116 with additional coagulation testing harbored more than 1 prothrombotic risk factor. These data support the hypothesis that thrombophilia is a multigenic disorder. Among unselected samples from a Midwestern population evaluated for thrombotic risk factors, the prevalence of factor V Leiden and prothrombin G20210A mutations are similar to those found in other populations in the Western world.

Chromosomal aneuploidy in leukemic blast crisis: a potential source of error in interpretation of bone marrow engraftment analysis by VNTR amplification.

BACKGROUND: Polymerase chain reaction (PCR) amplification of polymorphic microsatellite or minisatellite DNA markers has proven to be a fast, sensitive, and specific technique in post-transplantation monitoring of bone marrow engraftment, as well as early detection of residual disease and relapse. Deletion or amplification of chromosomal segments carrying marker loci, as can occur in leukemia and other hematologic malignancies, may result in loss or increased dosage of marker alleles. Examination of these marker alleles by PCR therefore may give aberrant results, which might lead to misinterpretation of bone marrow transplantation (BMT) engraftment studies. METHODS AND RESULTS: We report a case of chronic myelogenous leukemia treated by BMT. PCR amplification of the minisatellite at the apoB locus on chromosome 2 was used to monitor the donor bone marrow engraftment. The patient experienced relapse in blast crisis with a near-haploid karyotype with loss of recipient-specific apoB allele causing an aberrant PCR result for bone marrow engraftment that mimicked full donor engraftment. CONCLUSIONS: Loss or gain of polymorphic DNA markers because of chromosomal losses or gains in some hematologic malignancies may affect the interpretation of bone marrow engraftment studies by PCR. When choosing polymorphic markers for such studies, it is important to avoid those that will be affected by expected chromosomal alteration, if possible. In addition, any abberant post-transplantation typing should prompt further investigation to rule out the possibility of chromosomal aberration. Review of all pertinent laboratory studies is important to avoid misinterpretation of results from a single test for engraftment analysis.

Genotyping of apolipoprotein E by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The genotyping of the various isoforms of Apolipoprotein E (apo E) has been performed using matrix-assisted laser desorption/ionization (MALDI-MS). The polymerase chain reaction was used to amplify the specific apo E gene sequence followed by digestion with Cfo I (Clostridium formicoaceticum), for generating restriction fragments for rapid and accurate mass analysis. An exonuclease I digestion step was introduced to remove the unused primers after PCR, which can otherwise interfere in the mass spectral analysis. By replacing the gel electrophoresis detection step with MALDI-MS, restriction isotyping of the apo E gene was achieved. Genotyping of an unknown sample and obtained from an independent diagnostic laboratory demonstrated the validity of the MALDI-MS method for the routine analysis of apo E.