Cystic fibrosis in Afro-Brazilians: XK haplotypes analysis supports the European origin of p.F508del mutation.

Cystic fibrosis (CF) is a common autosomal recessive disorder, being the p.F508del the most frequent mutation. Also, a nearby restriction fragment length polymorphism (RFLP) named XK (KM19 and XV2C) is non-randomly associated with specific CF alleles. Our aim was to analyze the occurrence of the p.F508del mutation and XK haplotypes in Afro-Brazilians CF patients and controls, since these data is available for the other two main ethnic groups found in Brazil (Euro-Brazilians and Brazilian Amerindians), contributing for the whole comprehension of these haplotypes in the Brazilian population. A total of 103 patients and 54 controls were studied. PCR and PCR-RFLP methodologies were used to identify the presence of the p.F508del and the XK haplotype in the subjects. The combined data show that 84.2% of p.F508del mutation is associated with haplotype B and only 15.8% with haplotype A; no other haplotypes were found to be associated with this mutation. Our data suggest that the occurrence of p.F508del mutation and haplotype B in Afro-Brazilian patients occurs probably due to admixture with Euro-descendants. Therefore this mutation and haplotype could be used as a admixture marker.

Whole exome sequencing identifies multiple diagnoses in congenital glaucoma with systemic anomalies.

The genetic basis of congenital glaucoma with systemic anomalies is largely unknown. Whole exome sequencing (WES) in 10 probands with congenital glaucoma and variable systemic anomalies identified pathogenic or likely pathogenic variants in three probands; in two of these, a combination of two Mendelian disorders was found to completely explain the patients' features whereas in the third case only the ocular findings could be explained by the genetic diagnosis. The molecular diagnosis for glaucoma included two cases with compound heterozygous or homozygous pathogenic alleles in CYP1B1 and one family with a dominant pathogenic variant in FOXC1; the second genetic diagnosis for the additional systemic features included compound heterozygous mutations in NPHS1 in one family and a heterozygous 18q23 deletion in another pedigree. These findings show the power of WES in the analysis of complex conditions and emphasize the importance of CYP1B1 screening in patients with congenital glaucoma regardless of the presence/absence of other systemic anomalies.

Vaginal progesterone reduces the rate of preterm birth in women with a sonographic short cervix: a multicenter, randomized, double-blind, placebo-controlled trial.

OBJECTIVES: Women with a sonographic short cervix in the mid-trimester are at increased risk for preterm delivery. This study was undertaken to determine the efficacy and safety of using micronized vaginal progesterone gel to reduce the risk of preterm birth and associated neonatal complications in women with a sonographic short cervix. METHODS: This was a multicenter, randomized, double-blind, placebo-controlled trial that enrolled asymptomatic women with a singleton pregnancy and a sonographic short cervix (10-20 mm) at 19 + 0 to 23 + 6 weeks of gestation. Women were allocated randomly to receive vaginal progesterone gel or placebo daily starting from 20 to 23 + 6 weeks until 36 + 6 weeks, rupture of membranes or delivery, whichever occurred first. Randomization sequence was stratified by center and history of a previous preterm birth. The primary endpoint was preterm birth before 33 weeks of gestation. Analysis was by intention to treat. RESULTS: Of 465 women randomized, seven were lost to follow-up and 458 (vaginal progesterone gel, n=235; placebo, n=223) were included in the analysis. Women allocated to receive vaginal progesterone had a lower rate of preterm birth before 33 weeks than did those allocated to placebo (8.9% (n=21) vs 16.1% (n=36); relative risk (RR), 0.55; 95% CI, 0.33-0.92; P=0.02). The effect remained significant after adjustment for covariables (adjusted RR, 0.52; 95% CI, 0.31-0.91; P=0.02). Vaginal progesterone was also associated with a significant reduction in the rate of preterm birth before 28 weeks (5.1% vs 10.3%; RR, 0.50; 95% CI, 0.25-0.97; P=0.04) and 35 weeks (14.5% vs 23.3%; RR, 0.62; 95% CI, 0.42-0.92; P=0.02), respiratory distress syndrome (3.0% vs 7.6%; RR, 0.39; 95% CI, 0.17-0.92; P=0.03), any neonatal morbidity or mortality event (7.7% vs 13.5%; RR, 0.57; 95% CI, 0.33-0.99; P=0.04) and birth weight < 1500 g (6.4% (15/234) vs 13.6% (30/220); RR, 0.47; 95% CI, 0.26-0.85; P=0.01). There were no differences in the incidence of treatment-related adverse events between the groups. CONCLUSIONS: The administration of vaginal progesterone gel to women with a sonographic short cervix in the mid-trimester is associated with a 45% reduction in the rate of preterm birth before 33 weeks of gestation and with improved neonatal outcome.

Alterations in oestrogen metabolism: implications for higher penetrance of familial pulmonary arterial hypertension in females.

Mutations in bone morphogenetic protein receptor type 2 (BMPR2) cause familial pulmonary arterial hypertension (FPAH), but the penetrance is reduced and females are significantly overrepresented. In addition, gene expression data implicating the oestrogen-metabolising enzyme CYP1B1 suggests a detrimental role of oestrogens or oestrogen metabolites. We examined genetic and metabolic markers of altered oestrogen metabolism in subjects with a BMPR2 mutation. Genotypes for CYP1B1 Asn453Ser (N453S) were determined for 140 BMPR2 mutation carriers (86 females and 54 males). Nested from those subjects, a case-control study of urinary oestrogen metabolite levels (2-hydroxyoestrogen (2-OHE) and 16alpha-hydroxyoestrone (16alpha-OHE(1))) was conducted in females (five affected mutation carriers versus six unaffected mutation carriers). Among females, there was four-fold higher penetrance among subjects homozygous for the wild-type genotype (N/N) than those with N/S or S/S genotypes (p = 0.005). Consistent with this finding, the 2-OHE/16alpha-OHE(1) ratio was 2.3-fold lower in affected mutation carriers compared to unaffected mutation carriers (p = 0.006). Our findings suggest that variations in oestrogens and oestrogen metabolism modify FPAH risk. Further investigation of the role of oestrogens in this disease with profound sex bias may yield new insights and, perhaps, therapeutic interventions.

Molecular recognition of acute myeloid leukemia using aptamers.

Cell surface proteins can play important roles in cancer pathogenesis. Comprehensive understanding of the surface protein expression patterns of tumor cells and, consequently, the pathogenesis of tumor cells depends on molecular probes against these proteins. To be used effectively for tumor diagnosis, classification and therapy, such probes would be capable of specific binding to targeted tumor cells. Molecular aptamers, designer DNA-RNA probes, can address this challenge by recognizing proteins, peptides and other small molecules with high affinity and specificity. Through a process known as cell-based SELEX, we used live acute myeloid leukemia (AML) cells to select a group of DNA aptamers, which can recognize AML cells with dissociation constants (Kd's) in the nanomolar range. Interestingly, one aptamer (KH1C12) compared with two control cell lines (K562 and NB4) showed significant selectivity to the target AML cell line (HL60) and could recognize the target cells within a complex mixture of normal bone marrow aspirates. The other two aptamers KK1B10 and KK1D04 recognize targets associated with monocytic differentiation. Our studies show that the selected aptamers can be used as a molecular tool for further understanding surface protein expression patterns on tumor cells and thus providing a foundation for effective molecular analysis of leukemia and its subcategories.

Cystic fibrosis gene variability in two southern Brazilian Amerindian populations: analysis of the deltaF508 mutation and the KM19 and XV2C haplotypes.

The frequencies of the deltaF508 deletion, the most common cystic fibrosis mutation in Europeans and European-derived populations, and the XV2C and KM19 restriction fragment length polymorphisms that are tightly linked to the CFTR locus vary among populations. To determine the distribution of these extragenic markers and of the deltaF508 mutation, we analyzed 326 chromosomes of individuals from two South American Indian populations, the Guarani and the Kaingang. The allele and haplotype frequencies differed greatly between the two populations as well as among Amerindians and normal European Brazilians and European Brazilian cystic fibrosis patients. The absence of the deltaF508 mutation and the B haplotype are in agreement with the hypothesis that the deltaF508 mutation occurred after the divergence of these two populations. This finding is useful for populations containing a large Amerindian component and helps us to understand the origins of the deltaF508 deletion, the most common cystic fibrosis mutation in Europeans and European-derived populations, as well as the different incidences of cystic fibrosis in continental groups.

siRNA therapeutics: big potential from small RNAs.

RNA interference (RNAi) is now an umbrella term referring to post-transcriptional gene silencing mediated by either degradation or translation arrest of target RNA. This process is initiated by double-stranded RNA with sequence homology driving specificity. The discovery that 21-23 nucleotide RNA duplexes (small-interfering RNAs, siRNAs) mediate RNAi in mammalian cells opened the door to the therapeutic use of siRNAs. While much work remains to optimize delivery and maintain specificity, the therapeutic advantages of siRNAs for treatment of viral infection, dominant disorders, cancer, and neurological disorders show great promise.

Genetic mutations in surfactant protein C are a rare cause of sporadic cases of IPF.

BACKGROUND: While idiopathic pulmonary fibrosis (IPF) is one of the most common forms of interstitial lung disease, the aetiology of IPF is poorly understood. Familial cases of pulmonary fibrosis suggest a genetic basis for some forms of the disease. Recent reports have linked genetic mutations in surfactant protein C (SFTPC) with familial forms of pulmonary fibrosis, including one large family in which a number of family members were diagnosed with usual interstitial pneumonitis (UIP), the pathological correlate to IPF. Because of this finding in familial cases of pulmonary fibrosis, we searched for SFTPC mutations in a cohort of sporadic cases of UIP and non-specific interstitial pneumonitis (NSIP). METHODS: The gene for SFTPC was sequenced in 89 patients diagnosed with UIP, 46 patients with NSIP, and 104 normal controls. RESULTS: Ten single nucleotide polymorphisms in the SFTPC sequence were found in IPF patients and not in controls. Only one of these created an exonic change resulting in a change in amino acid sequence. In this case, a T to C substitution resulted in a change in amino acid 73 of the precursor protein from isoleucine to threonine. Of the remaining polymorphisms, one was in the 5' UTR, two were exonic without predicted amino acid sequence changes, and six were intronic. One intronic mutation suggested a potential enhancement of a splicing site. CONCLUSIONS: Mutations in SFTPC are identified infrequently in this patient population. These findings indicate that SFTPC mutations do not contribute to the pathogenesis of IPF in the majority of sporadic cases.

Identification of DNA sequences from a second pathogenicity island of uropathogenic Escherichia coli CFT073: probes specific for uropathogenic populations.

Uropathogenic Escherichia coli is the leading cause of urinary tract infection and hospital visits in North America. Cystitis and acute pyelonephritis, infection of the bladder and kidney, respectively, are the two most common syndromes encountered in patients with urinary tract infection. We sequenced and annotated 71,684 bases of a previously unidentified pathogenicity-associated island (PAI) from E. coli strain CFT073. This PAI contained 89 open-reading frames encoding a pap operon, iron-regulated genes, mobile genetic elements, and a large proportion of unknown or unidentified open-reading frames. Dot blot analysis with 11 DNA sequences from this PAI demonstrated that 7 sequences were more prevalent among uropathogens: 2 probes were more prevalent among cystitis and pyelonephritis isolates, 2 among pyelonephritis isolates only, and 3 among cystitis isolates only than among fecal isolates. These data suggest that groups of uropathogens have genetic differences that may be responsible for the different clinical outcomes.

Aminoglycosides in cystic fibrosis: a descriptive study of current practice in Australia.

AIM: To determine the diversity of clinical practice with respect to aminoglycosides in cystic fibrosis (CF) units within Australia. METHOD: In April 1999, a questionnaire on the use of aminoglycosides was sent to 30 CF units across Australia. Information was collected about drug selection, dosing, monitoring and toxicity with intravenous administration. RESULTS: Completed surveys were received from 26 of the 30 units (response rate = 86%) and all units with > 40 patients. Tobramycin was the drug of choice in all but two centres where there was equivalent use of gentamicin and tobramycin. The survey demonstrated a trend in recent years to reduce the number of doses per day with 54% of centres prescribing once daily, 23% twice daily and 23% thrice daily regimens. Initial dosing was generally based on mg/kg per day (mean 8.8, range 7.5-10 mg/kg per day). Dosing by infusion occurred in 11 of 14 units using once-daily dosing and there was equivalent use of bolus and infusion methods for multiple-daily regimens. Drug monitoring depended on dosing regimen. Units using multiple daily regimens monitored using trough +/- peak levels, whereas 50% of units using once-daily dosing used two postdose levels to alter dose. Actual toxicity, in particular nephrotoxicity, ototoxicity and vestibular toxicity was reported by 19, 27 and 12% of units, respectively. CONCLUSION: The prescribing, dosing and monitoring of aminoglycosides in CF across Australia varies greatly. This is likely to be due to a lack of definitive evidence as to the optimum use in this patient group.

Isolated growth hormone (GH) deficiency due to compound heterozygosity for two new mutations in the GH-releasing hormone receptor gene.

OBJECTIVE: Mutations in the GH releasing hormone receptor (GHRH-R) have recently been shown to cause autosomal recessive isolated GH deficiency (IGHD). Patients who are homozygous for GHRH-R mutations have a subnormal GH response to pharmacological agents that stimulate GH secretion and an appropriate response to exogenous GH therapy. We searched for mutations in the GHRH-R gene in a family in which two of three siblings were affected by IGHD. DESIGN: We sequenced the 13 coding exons, the intron-exon boundaries and 327 bases of the promoter of the GHRH-R gene from peripheral blood cell genomic DNA of an index patient. RESULTS: Both affected individuals were compound heterozygotes for two previously undescribed GHRH-R mutations: a change in codon 137 that replaces histidine with leucine (H137L), and a 5 bp deletion in exon 11 (Del 1140-1144). The patients' father was heterozygous for the H137L mutation, and the mother was heterozygous for the exon 11 deletion. We used site-directed mutagenesis to create the mutants in wild-type GHRH-R cDNA. Transient transfection of GHRH-R cDNAs in Chinese hamster ovary cells showed that cells transfected with both mutant receptors failed to increase cyclic AMP after treatment with GHRH. CONCLUSIONS: We describe a family in which two siblings with IGHD were compound heterozygotes for two new mutations in the GHRH-R gene. These results suggest that mutant alleles for GHRH-R gene may be more common than previously suspected.

Recombinant human acid alpha-glucosidase enzyme therapy for infantile glycogen storage disease type II: results of a phase I/II clinical trial.

PURPOSE: Infantile glycogen storage disease type II (GSD-II) is a fatal genetic muscle disorder caused by deficiency of acid alpha-glucosidase (GAA). The purpose of this study was to investigate the safety and efficacy of recombinant human GAA (rhGAA) enzyme therapy for this fatal disorder. METHODS: The study was designed as a phase I/II, open-label, single-dose study of rhGAA infused intravenously twice weekly in three infants with infantile GSD-II. rhGAA used in this study was purified from genetically engineered Chinese hamster ovary (CHO) cells overproducing GAA. Adverse effects and efficacy of rhGAA upon cardiac, pulmonary, neurologic, and motor functions were evaluated during 1 year of the trial period. The primary end point assessed was heart failure-free survival at 1 year of age. This was based on historical control data that virtually all patients died of cardiac failure by 1 year of age. RESULTS: The results of more than 250 infusions showed that rhGAA was generally well tolerated. Steady decreases in heart size and maintenance of normal cardiac function for more than 1 year were observed in all three infants. These infants have well passed the critical age of 1 year (currently 16, 18, and 22 months old) and continue to have normal cardiac function. Improvements of skeletal muscle functions were also noted; one patient showed marked improvement and currently has normal muscle tone and strength as well as normal neurologic and Denver developmental evaluations. Muscle biopsies confirmed that dramatic reductions in glycogen accumulation had occurred after rhGAA treatment in this patient. CONCLUSIONS: This phase I/II first study of recombinant human GAA derived from CHO cells showed that rhGAA is capable of improving cardiac and skeletal muscle functions in infantile GSD-II patients. Further study will be needed to assess the overall potential of this therapy.

Three new mutations in the gene for the growth hormone (gh)-releasing hormone receptor in familial isolated gh deficiency type ib.

Isolated GH deficiency (IGHD) is familial in 5-30% of cases. The majority of patients have the type IB form, characterized by autosomal recessive transmission, low but measurable serum concentrations of GH, and responsiveness to exogenous GH therapy. Unique mutations in the gene encoding the GHRH receptor (GHRHR) have previously been described in 2 kindreds with IGHD IB. However, the prevalence of GHRHR mutations in patients with IGHD IB is unknown. We analyzed 30 families with IGHD IB in which more than 1 member was affected. Linkage analysis was performed in 28 of the families, and in 3 families sibling pair analysis indicated linkage to the GHRHR gene locus. These 3 families as well as 2 families in which linkage analysis was not performed were screened for mutations in the 13 coding exons, the intron-exon boundaries, and 327 bases of the promoter of the GHRHR gene. We identified novel GHRHR missense mutations in 2 of the 3 kindreds with informative linkage and in 1 family in which linkage had not been performed. In 1 family affected members were homozygous for a mutation in codon 144 that replaces leucine with histidine (L144H). Affected subjects in a second family were compound heterozygotes, carrying both the L144H mutation and a second mutation in codon 242 that replaces phenylalanine with cysteine. Affected subjects in a third family were homozygous for a mutation that replaces alanine at codon 222 with glutamic acid. All 3 mutations segregated with the IGHD phenotype. All 3 mutant receptors were expressed in CHO cells, and each failed to show a cAMP response after treatment of the cells with GHRH. These results demonstrate that missense mutations in the GHRHR gene are a cause of IGHD IB, and that defects in the GHRHR gene may be a more common cause of GH deficiency than previously suspected.

A novel arginine vasopressin-neurophysin II mutation causes autosomal dominant neurohypophyseal diabetes insipidus and morphologic pituitary changes.

To determine the genetic basis of autosomal dominant neurohypophyseal diabetes insipidus (ADNDI) in a Cypriot family, we ascertained and studied a large, four-generation kindred in which all participating family members had arginine vasopressin-neurophysin II (AVP-NP-II) gene analyses done. A G to A transition was found by DNA sequence analysis at position 1773 (G1773A) of the AVP-NPII gene which is predicted to encode a substitution of tyrosine for cysteine in codon 59 (CYS59TYR). The mutation was confirmed by restriction endonuclease analysis of PCR amplification products that contain the corresponding segment of the AVP-NPII gene. To clarify the morphologic status of the pituitaries of family members, 12 affected and 3 nonaffected members had magnetic resonance imaging (MRI) studies. The bright spot of the posterior pituitary lobe was completely absent in 75% and faintly identified in 25% of the affected members who were examined with MRI. We conclude that (1) a novel G1773A transition in exon 2 of the AVP-NPII gene causes ADNDI in the large Cypriot kindred studied, (2) this mutation is predicted to encode a CYS59TYR substitution in NPII, and (3) MRI studies of the posterior pituitary lobes of affected family members show either a decreased intensity or a complete absence of the bright spot in all cases studied.

Pituitary gene mutations and the growth hormone pathway.

Hereditary forms of pituitary insufficiency not associated with anatomic defects of the central nervous system, hypothalamus, or pituitary are a heterogeneous group of disorders that result from interruptions at different points in the hypothalamic-pituitary-somatomedin-peripheral tissue axis. These different types of pituitary dwarfism can be classified on the level of the defect; mode of inheritance; whether the phenotype is isolated growth hormone deficiency (IGHD) or combined pituitary hormone deficiency (CPHD); whether the hormone is absent, deficient, or abnormal; and, in patients with GH resistance, whether insulin-like growth factor 1 (IGF1) is deficient due to GH receptor or IGF1 defects. Information on each disorder is summarized. More detailed information can be obtained through the electronic database Online Mendelian Inheritance in Man which is available at http://www3.ncbi.nlm.nih.gov/Omim/.

Distribution of a 20-mer phosphorothioate oligonucleotide, CGP69846A (ISIS 5132), into airway leukocytes and epithelial cells following intratracheal delivery to brown-norway rats.

PURPOSE: To evaluate the pulmonary distribution of CGP69846A (ISIS 5132), a phosphorothioate oligonucleotide, following intra-tracheal (i.t.) instillation into Brown-Norway rats. METHODS: The pharmacokinetic profile of [3H]-CGP69846A was investigated following i.t. instillation into both naive and inflamed airways of Brown-Norway rats. The cellular distribution was determined using autoradiography, immunohistochemistry and flow cytometry/fluorescence microscopy, in inflamed airways. RESULTS: CGP69846A displayed a dose-dependent lung retention following i.t. administration which was unaffected by local inflammation. Autoradiography and immunohistochemistry showed distribution to alveolar macrophages, eosinophils, bronchial and tracheal epithelium and alveolar cells. Studies with [FITC]-CGP69846A demonstrated a preferential association of oligonucleotide with leukocytes in bronchial lavage fluid of: macrophages > eosinophils = neutrophils >> lymphocytes. CONCLUSIONS: The dose-dependency of lung retention together with cell-specific uptake suggests that the lung can be used as a local target for antisense molecules with potentially minimal systemic effects. Furthermore, the preferential targeting of macrophages and the airway epithelium by oligonucleotides may represent rational cellular targets for antisense therapeutics.

Geographic heterogeneity of 4 common worldwide cystic fibrosis non-DF508 mutations in Brazil.

Cystic fibrosis (CF) is an autosomal recessive disease caused by at least 750 different mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The frequency of the most common mutation (DF508) in Brazilian patients of European origin is 47%. To determine the frequency of 4 other common CF mutations (G542X, G551D, R553X, and N1303K) in Brazilian patients of European origin, we used direct polymerase chain reaction (PCR) amplification of DNA obtained from dried blood spots on Guthrie cards. The DNA came from 247 non-DF508 chromosomes from 172 Brazilian CF patients ascertained from 5 different states of Brazil. The results show that the 4 mutations account for 17% of the non-DF508 alleles and only 9% of the total number of Brazilian CF alleles. Overall, the frequency of each mutation is different from northern European and North American populations but similar to southern European populations, mainly the Italian and Spanish populations. When Brazilian patients of European origin are grouped according to state of birth, the frequencies of the mutations are significantly different between southern and southeastern states of Brazil. Therefore there are serious implication for risk assessment of DNA-based tests in heterogeneous populations such as Brazilians. Further studies are needed to identify the remaining 44% of CF mutations for the different populations and regions of Brazil.

Familial growth hormone deficiency associated with MRI abnormalities.

Idiopathic growth hormone deficiency is, in most cases, a sporadic condition. In a number of these patients magnetic resonance imaging (MRI) demonstrates a small anterior pituitary, small or absent pituitary stalk, and ectopically located posterior pituitary. These findings have been attributed to a developmental defect, trauma, or ischemia at birth. We report on a case of familial isolated growth hormone deficiency with mother and son demonstrating the MRI findings described above. The son also had a Chiari type I malformation and medial deviation of the carotid arteries secondary to a narrow skull base. Testing failed to identify a mutation in either the Pit-1 gene or GH gene cluster. This case appears to be an autosomal dominant defect in early development, lending support to the hypothesis that dysgenesis, rather than birth trauma, may cause a small anterior pituitary and ectopic posterior pituitary.

Health risk influence on medical care costs and utilization among 2,898 municipal employees.

INTRODUCTION: Although worksite health promotion programs are credited with stabilizing medical benefits costs, research is needed to characterize the medical costs of cohorts with selected health risk factors. The purpose of this study was to compare medical cost outcomes in City of Birmingham, Alabama, employees who differ on selected health risk factors. METHODS: Health risk appraisal and medical claims cost data were examined in 2,898 employees participating in health screening during 1992 and 1993. Probit analysis was employed to test the null hypotheses that there are no differences in (1) probability of medical service utilization and (2) probability of medical service cost quartile (high, moderate, and low) between groups characterized by risks. Age, gender, race, education, marital status, and diabetes were included as covariates in each model examined. In addition, smoking habits was included as a covariate in models involving risk taking behavior and psychosocial risk. RESULTS: Significant differences in medical care utilization and costs were found between risk groups based on psychosocial risk, cardiovascular disease risk, and total risk. No association was found between risk-taking behavior and utilization and costs. CONCLUSION: Subjects reporting psychosocial, cardiovascular disease, and total risk factors were more likely to use medical services and to be in the high or high/moderate cost categories.