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.

Expansion of genetic services utilizing a general genetic counseling clinic.

We established a general genetic counseling clinic (GCC) to help reduce long wait times for new patient appointments and to enhance services for a subset of patients. Genetic counselors, who are licensed in Tennessee, were the primary providers and MD geneticists served as medical advisors. This article describes the clinic referral sources, reasons for referral and patient dispositions following their GCC visit(s). We obtained patients by triaging referrals made to our medical genetics division. Over 24 months, our GCC provided timely visits for 321 patients, allowing the MD geneticists to focus on patients needing a clinical exam and/or complex medical management. Following their GCC visit(s), over 80 % of patients did not need additional appointments with an MD geneticist. The GCC allowed the genetic counselor to spend more time with patients than is possible in our traditional medical genetics clinic. Patient satisfaction surveys (n = 30) were very positive overall concerning the care provided. Added benefits for the genetic counselors were increased professional responsibility, autonomy and visibility as health care providers. We conclude that genetic counselors are accepted as health care providers by patients and referring providers for a subset of clinical genetics cases. A GCC can expand genetic services, complement more traditional genetic clinic models and utilize the strengths of the genetic counselor health care provider.

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.

22q13.3 deletion syndrome: clinical and molecular analysis using array CGH.

The 22q13.3 deletion syndrome results from loss of terminal segments of varying sizes at 22qter. Few genotype-phenotype correlations have been found but all patients have mental retardation and severe delay, or absence of, expressive speech. We carried out clinical and molecular characterization of 13 patients. Developmental delay and speech abnormalities were common to all and comparable in frequency and severity to previously reported cases. Array-based comparative genomic hybridization showed the deletions to vary from 95 kb to 8.5 Mb. We also carried out high-resolution 244K array comparative genomic hybridization in 10 of 13 patients, that defined the proximal and distal breakpoints of each deletion and helped determine the size, extent, and gene content within the deletion. Two patients had a smaller 95 kb terminal deletion with breakpoints within the SHANK3 gene while three other patients had a similar 5.5 Mb deletion implying the recurrent nature of these deletions. The two largest deletions were found in patients with ring chromosome 22. No correlation could be made with deletion size and phenotype although complete/partial SHANK3 was deleted in all patients. There are very few reports on array comparative genomic hybridization analysis on patients with the 22q13.3 deletion syndrome, and we aim to accurately characterize these patients both clinically and at the molecular level, to pave the way for further genotype-phenotype correlations. (c) 2010 Wiley-Liss, Inc.

Transcripts from a novel BMPR2 termination mutation escape nonsense mediated decay by downstream translation re-initiation: implications for treating pulmonary hypertension.

Bone morphogenetic protein receptor type 2 (BMPR2) gene mutations are a major risk factor for heritable pulmonary arterial hypertension (HPAH), an autosomal dominant fatal disease. We have previously shown that BMPR2 transcripts that contain premature termination codon (PTC) mutations are rapidly and nearly completely degraded through nonsense mediated decay (NMD). Here we report a unique PTC mutation (W13X) that did not behave in the predicted manner. We found that patient-derived cultured lymphocytes (CLs) contained readily detectable levels of the PTC-containing transcript. Further analysis suggested that this transcript escaped NMD by translational re-initiation at a downstream Kozak sequence, resulting in the omission of 173 amino acids. Treatment of CLs containing the PTC with an aminoglycoside decreased the truncated protein levels, with a reciprocal increase in full-length BMPR2 protein and, importantly, BMPR-II signaling. This is the first demonstration of aminoglycoside-mediated 'repair' of a BMPR2 mutation at the protein level in patient-derived cells and has obvious implications for treatment of HPAH where no disease-specific treatment options are available. Our data also suggest the need for a more thorough characterization of mutations prior to labeling them as haploinsufficient or dominant negative based simply on sequencing data.

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.

Isolated growth hormone deficiency type II caused by a point mutation that alters both splice site strength and splicing enhancer function.

A heterozygous single base mutation in the human growth hormone (GH) gene (GH-1) was identified in a family presenting with isolated GH deficiency type II (IGHD II). Affected individuals have a guanine to adenine transition at the first nucleotide of exon 3 (E3+1 G-->A) that results in exon skipping and production of a dominant-negative 17.5-kDa isoform. We show that the mechanistic basis for exon skipping is due to the unique position of this mutation because it weakens the 3' splice site and simultaneously disrupts a splicing enhancer located within the first seven bases of exon 3. A G-->T mutation at this same position not only affects splicing but also results in a premature stop codon for those transcripts that include exon 3. Thus, mutations that alter the first nucleotide of exon 3 illustrate the various mechanisms by which changes in sequence can cause disease: splice site selection, splicing enhancer function, messenger RNA decay, missense mutations, and nonsense mutations. For IGHD II, only exon skipping leads to production of the dominant-negative isoform, with increasing skipping correlating with increasing disease severity.

Mutations of the E1beta subunit gene (PDHB) in four families with pyruvate dehydrogenase deficiency.

Pyruvate dehydrogenase complex (PDC) deficiencies are a major cause of primary lactic acidosis. Most cases result from mutations of the gene for the pyruvate dehydrogenase E1alpha subunit (PDHA1), with fewer cases resulting from mutations in genes for E3, E3-binding protein, E2, and the E1beta subunit (PDHB). We have found four cases of PDHB mutations among 83 analyzed cases of PDC deficiency. In this series, PDHB mutations were found to be about 10% as frequent as PDHA1 mutations. All cases were diagnosed by low PDC activity, with normal E2 and E3 activities. These included a 6.5-year-old male (consanguineous, homozygous R36C); a neonatal female who died soon after birth, (compound heterozygous C306R/D319V), a 26-year-old female (heterozygous I142M/W165S), and a 13month old female (consanguineous, homozygous Y132C) who is a sibling of a previously published case. Their ethnic background is diverse (Caucasian, Arab, and African American descent). All cases had lactic acidosis and developmental delay. Three cases had agenesis of the corpus callosum, seizures, and hypotonia; one died within the first year of life. These clinical findings are similar to those of PDHA1 deficiency, except that ataxia was more frequent in PDHA1 cases and consanguinity was found only in PDHB families. PDC activity in lymphocytes from six parents is normal, who all are heterozygous carriers for the respective mutations. Immunoreactivity of E1beta was markedly reduced in one case and showed a slightly larger form of E1beta in one case. Computer analysis predicts that: R36C affects the interaction of several amino acids resulting in conformational change, C306R affects interaction of the two beta subunits, D319 is in the interface of E1 and E2, I142M affects conformation around a K ion affecting stability of the beta subunit, W165S affects hydrophobic interaction between the beta subunits, and Y132C affects interaction between the beta subunits. All of these residues are conserved in E1beta across species, and Y132 is also conserved in other TPP-requiring enzymes. These observations support the conclusion that these are pathogenic mutations.

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.

Condensed-phase effects on the structural properties of C6H5CN-BF3 and (CH3)3CCN-BF3: IR spectra, crystallography, and computations.

Condensed-phase effects on the structure and bonding of C(6)H(5)CN-BF(3) and (CH(3))(3)CCN-BF(3) are illustrated by a variety of results, and these are compared to analogous data for the closely related complex CH(3)CN-BF(3). For the most part, the structural properties of C(6)H(5)CN-BF(3) and (CH(3))(3)CCN-BF(3) are quite similar, not only in the gas phase but also in the solid state and in argon matrices. However, the structures do change significantly from medium to medium, and these changes are reflected in the data presented below. Specifically, the measured crystallographic structure of C(6)H(5)CN-BF(3) (s) has a B-N distance that is 0.17 A shorter than that in the equilibrium gas-phase structure obtained via B3LYP calculations. Notable differences between calculated gas-phase frequencies and measured solid-state frequencies for both C(6)H(5)CN-BF(3) and (CH(3))(3)CCN-BF(3) were also observed, and in the case of (CH(3))(3)CCN-BF(3), these data implicate a comparable difference between solid-state and gas-phase structure, even in the absence of crystallographic results. Frequencies measured in argon matrices were found to be quite similar for both complexes and also very near those measured previously for CH(3)CN-BF(3), suggesting that all three complexes adopt similar structures in solid argon. For C(6)H(5)CN-BF(3) and (CH(3))(3)CCN-BF(3), matrix IR frequencies differ only slightly from the computed gas-phase values, but do suggest a slight compression of the B-N bond. Ultimately, it appears that the varying degree to which these systems respond to condensed phases stems from subtle differences in the gas-phase species, which are highlighted through an examination of B-N distance potentials from B3LYP calculations. The larger organic substituents appear to stabilize the potential near 1.8 A, so that the structures are more localized in that region prior to any condensed-phase interactions. As a result, the condensed-phase effects on the structural properties of C(6)H(5)CN-BF(3) and (CH(3))(3)CCN-BF(3) are much less pronounced than those for CH(3)CN-BF(3).

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.

Anatomic constraints on cognitive theories of category specificity.

Many cognitive theories of semantic organization stem from reports of patients with selective, category-specific deficits for particular classes of objects (e.g., fruit). The anatomical assumptions underlying the competing claims can be evaluated with functional neuroimaging but the findings to date have been inconsistent and insignificant when standard statistical criteria are adopted. We hypothesized that category differences in functional brain responses might be small and task dependent. To test this hypothesis, we entered data from seven PET studies into a single multifactorial design which crossed category (living vs man-made) with a range of tasks. Reliable category-specific effects were observed but only for word retrieval and semantic decision tasks. Living things activated medial aspects of the anterior temporal poles bilaterally while tools activated a left posterior middle temporal region. These category-by-task interactions provide robust evidence for an anatomical double dissociation according to category and place strong constraints on cognitive theories of the semantic system. Furthermore they reconcile some of the apparent inconsistencies between lesion studies and functional neuroimaging data.

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.

Mutation in the gene for bone morphogenetic protein receptor II as a cause of primary pulmonary hypertension in a large kindred.

BACKGROUND: Most patients with primary pulmonary hypertension are thought to have sporadic, not inherited, disease. Because clinical disease develops in only 10 to 20 percent of persons carrying the gene for familial primary pulmonary hypertension, we hypothesized that many patients with apparently sporadic primary pulmonary hypertension may actually have familial primary pulmonary hypertension. METHODS: In a study conducted over 20 years, we developed a registry of 67 families affected by familial primary pulmonary hypertension. Through patient referrals, extensive family histories, and correlation of family pedigrees, we discovered shared ancestry among five subfamilies. We established the diagnosis of primary pulmonary hypertension by direct evaluation of patients and review of autopsy material and medical records. We assessed some family members for mutations in the gene encoding bone morphogenetic protein receptor II (BMPR2), which has recently been found to cause familial primary pulmonary hypertension. RESULTS: We linked five separately identified subfamilies that included 394 known members spanning seven generations, which were traced back to a founding couple in the mid-1800s. Familial primary pulmonary hypertension has been diagnosed in 18 family members, 12 of whom were first thought to have sporadic disease. The conditions of 7 of the 18 were initially misdiagnosed as other cardiopulmonary diseases. Six members affected with familial primary pulmonary hypertension and 6 of 10 at risk for carriage have been undergone genotype analysis, and they have the same mutation in BMPR2, a transversion of thymine to guanine at position 354 in exon 3. CONCLUSIONS: Many cases of apparently sporadic primary pulmonary hypertension may be familial. Failure to detect familial primary pulmonary hypertension results from incomplete expression within families, skipped generations, and incomplete family pedigrees. The recent discovery of mutations in BMPR2 should make it possible to identify those with susceptibility to disease.