Augmented ventilatory response to exercise in pulmonary hypertension

The ventilatory response to submaximal exercise, defined as the slope of minute ventilation over carbon dioxide production (VE/VCO2), was determined in 12 normal subjects, ten patients with pulmonary hypertension before and after heart-lung transplantation, and eight patients following heart transplantation. Patients with pulmonary hypertension show an augmented ventilatory response compared to normal subjects (pulmonary hypertension [mean, 57.7 +/- 6.8 (SE) ml/ml VCO2; normal subjects, 22.3 +/- 1.4 ml/ml VCO2; p less than 0.001]). Following heart-lung transplantation, VE/VCO2 slope fell to 24.7 +/- 1.6 ml/ml VCO2, a value which is not significantly different than the value in normal subjects. Patients after heart transplantation show a mean slope value of 25.3 +/- 1.3 ml/ml VCO2, which is not significantly different than the normal value or the value found after heart-lung transplantation. The augmented ventilatory response to exercise did not correlate with the usual chemical modulators of ventilation (arterial pH, arterial carbon dioxide tension, or arterial oxygen tension). These results suggest the following: the existence of a neural system in patients with pulmonary hypertension which results in an augmentation of ventilatory drive in response to exercise; the augmented ventilatory response reflects excessive neural activity of pulmonary afferents during exercise; narrow regulation of the ventilatory response to exercise in normal subjects which is preserved in the denervated lung, indicating that pulmonary afferents are not critical to ventilatory control during exercise in the normal subject; and the possible use of measurements of the ventilatory response to exercise as a noninvasive screening test for pulmonary hypertension.     

Association between SLC11A1 (formerly NRAMP1) and the risk of sarcoidosis in Poland

Sarcoidosis (SA) is a systemic granulomatous disorder of unknown etiology characterized by T helper 1-type inflammatory responses at sites of disease with signs of B cell hyperactivity. Like rheumatoid arthritis and diabetes, an infectious etiology has frequently been postulated but no single infectious trigger definitively identified. Polymorphic alleles at SLC11A1 have previously been associated with susceptibility to both the putative infectious agents and to these autoimmune disorders. We therefore investigated its candidacy as a genetic determinant of SA in Poland in an association-based study comparing 86 SA patients with 85 tuberculosis (TB) patients and 93 control subjects. The functional promoter (GT)(n) polymorphism and four of 10 other single nucleotide or insertion/deletion polymorphisms genotyped across SLC11A1 were informative in our sample. Consistent with previous autoimmune disease studies, allele 3 at the functional (GT)(n) promoter region repeat polymorphism was significantly associated with SA when compared with healthy controls (odds ratio 1.68; 95% CI: 1.01-2.81; P=0.04) or with TB patients (odds ratio 1.69; 95% CI: 1.042-0.78; P=0.03).     

Primary hyperoxaluria in infants: medical, ethical, and economic issues

OBJECTIVES: Survey on the current medical approach to and the economic issues affecting infants with primary hyperoxaluria type 1. METHODS: Questionnaire to specialized centers worldwide. RESULTS: Seventy-eight infants were identified: 44% were of Muslim origin and 56% were not. The consanguinity rate was 76% and 0%, respectively. Thirty-three percent were treated in developing countries (group 1) and 67% in developed countries (group 2). Initial presentation (4.9 +/- 2.8 months) consisted of failure to thrive (22%), urinary tract infection (21%), and uremia (14%). Radiologic findings included nephrocalcinosis (91%), urolithiasis (44%), or both (22%). The diagnosis was based on family history, tissue biopsy, and urine oxalate level in most patients from group 1 and on urine oxalate and glycolate levels, alanine:glyoxalate aminotransferase activity, and DNA analysis in patients from group 2. Therapeutic withdrawal was the final option for 40% of children; financial reasons were given for 10 of 17 patients from group 1 and 0 of 9 from group 2. End-stage renal disease started at 3.2 +/- 6.4 years of age and was present in half of the patients at the time of diagnosis. Fifty-two percent of the patients died: 82% in group 1 versus 33% in group 2; 33% of patients who underwent transplantation died versus 71% of those who did not. CONCLUSION: The management of primary hyperoxaluria type 1 in infants is a major example of the ethical, epidemiologic, technical, and financial challenges that are raised by recessive inherited diseases with early life-threatening onset. In certain circumstances, oxalosis can be regarded as a condition for which therapeutic withdrawal may be an acceptable option.     

Heterozygous COL9A3 variants cause severe peripheral vitreoretinal degeneration and retinal detachment

The COL9A3 gene encodes one of the three alpha chains of Type IX collagen, with heterozygous variants reported to cause multiple epiphyseal dysplasia, and suggested as contributory in some cases of sensorineural hearing loss. Patients with homozygous variants have midface hypoplasia, myopia, sensorineural hearing loss, epiphyseal changes and carry a diagnosis of Stickler syndrome. Variants in COL9A3 have not previously been reported to cause vitreoretinal degeneration and/or retinal detachments. This report describes two families with autosomal dominant inheritance and predominant features of peripheral vitreoretinal lattice degeneration and retinal detachment. Genomic sequencing revealed a heterozygous splice variant in COL9A3 [{"type":"entrez-nucleotide","attrs":{"text":"NG_016353.1","term_id":"283945463"}}NG_016353.1({"type":"entrez-nucleotide","attrs":{"text":"NM_001853.4","term_id":"1432073434"}}NM_001853.4):c.1107 + 1G>C, NC_000020.10({"type":"entrez-nucleotide","attrs":{"text":"NM_001853.4","term_id":"1432073434"}}NM_001853.4):c.1107 + 1G>C, LRG1253t1] in Family 1, and a heterozygous missense variant [{"type":"entrez-nucleotide","attrs":{"text":"NG_016353.1","term_id":"283945463"}}NG_016353.1({"type":"entrez-nucleotide","attrs":{"text":"NM_001853.4","term_id":"1432073434"}}NM_001853.4):c.388G>A p.(Gly130Ser)] in Family 2, each segregating with disease. cDNA studies of the splice variant demonstrated an in-frame deletion in the COL2 domain, and the missense variant occurred in the COL3 domain, both indicating the critical role of Type IX collagen in the vitreous base of the eye.Subject terms: Genetic testing, Medical genetics, Medical genomics     

Canadian multicenter laboratory study for standardized second-line antimicrobial susceptibility testing of Mycobacterium tuberculosis

The purpose of this study was to establish a standardized protocol for second-line antimicrobial susceptibility testing of Mycobacterium tuberculosis using the Bactec MGIT 960 system in Canadian laboratories. Four Canadian public health laboratories compared the susceptibility testing results of 9 second-line antimicrobials between the Bactec 460 and Bactec MGIT 960 systems. Based on the data generated, we have established that the Bactec MGIT 960 system provides results comparable to those obtained with the previous Bactec 460 method. The critical concentrations established for the testing of the antimicrobials used are as follows: amikacin, 1 μg/ml; capreomycin, 2.5 μg/ml; ethionamide, 5 μg/ml; kanamycin, 2.5 μg/ml; linezolid, 1 μg/ml; moxifloxacin, 0.25 μg/ml; ofloxacin, 2 μg/ml; p-aminosalicylic acid, 4 μg/ml; rifabutin, 0.5 μg/ml.     

Mycobacterium tuberculosis in Ontario,Canada: Insights from IS6110 Restriction Fragment Length Polymorphism and Mycobacterial Interspersed Repetitive-Unit-Variable-Number Tandem-Repeat Genotyping

A collection of 1,308 clinical Mycobacterium tuberculosis isolates from Ontario, Canada, was genotyped by IS6110 restriction fragment length polymorphism (RFLP) and mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) analysis. RFLP or >12 MIRU-VNTR loci were necessary for resolution of Indo-Oceanic strains. The low clustering rate and high strain diversity indicate that, in Ontario, most tuberculosis results from reactivation of latent infections.Tuberculosis (TB), which is caused by pathogens of the Mycobacterium tuberculosis complex (MTBC), remains a global scourge (19). In Canada, the average incidence rate is 5.0 cases/100,000 people, but the burden of disease varies across the country. In 2007, the four Atlantic provinces accounted for only ∼1% of TB cases, whereas ∼42% of new cases occurred in the province of Ontario (11). The Public Health Laboratories (PHL) of the Ontario Agency for Health Protection and Promotion provide diagnostic testing for TB in Ontario. The TB and Mycobacteriology Laboratory at PHL-Toronto is the largest facility of its kind in Canada, processing 50,000 patient samples plus 2,000 referred acid-fast positive cultures, with 600 to 650 new cases of TB detected annually (8, 11). Historically, the PHL has employed IS6110 restriction fragment length polymorphism (RFLP) for genotyping. Despite its utility, IS6110 RFLP is labor-intensive and only performed upon request. The current turnaround time of 21 days also makes the method incompatible with the PHL goal of universal, real-time MTBC genotyping. More recently, mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing has been introduced (6, 7, 9, 15). The current PHL strategy relies upon agarose gel electrophoresis for comparison of PCR products. This method is low throughput, and gel-to-gel variability confounds comparison of samples processed at different times. Here, we describe implementation and validation of an improved MIRU-VNTR strategy and its utility for analysis of a large clinical strain collection.The semiautomated MIRU-VNTR strategy was derived from the 12-loci method of Cowan et al. (6, 7). Briefly, multiplex PCR was performed with dye-labeled primers in 96-well plates. For each reaction, 5 ng of template DNA was combined with 11 μl of a master mix (Red Taq; Sigma-Aldrich, Oakville, Canada) containing three primer pairs. PCR conditions were 95°C for 10 min and 34 cycles of 94°C for 30 s, 62°C for 30 s, and 72°C for 60 s, with a final extension at 72°C for 7 min. PCR amplification was confirmed by electrophoresis on 1% Tris-borate-EDTA agarose gels. Samples were diluted 1:20 in sequence loading solution (Beckman Coulter, Mississauga, Canada) containing a 600-bp sequencing standard (Beckman Coulter) and then subjected to fragment analysis on a CEQ8800 genetic analysis system (Beckman Coulter). To reduce the manual labor and potential for human error associated with extensive pipetting, a Biomek NX Span-8 automation workstation (Beckman Coulter) was programmed to set up the initial PCRs and dilute PCR products for fragment analysis.To validate the method, a blinded set of 99 DNA samples was provided by the Public Health Research Institute (NJ). Strain identities and MIRU-VNTR patterns were unblinded only after complete 12-digit patterns were generated for all 99 DNA samples. Concordance between PHL and Public Health Research Institute results was 100%.To evaluate the utility of MIRU-VNTR in Ontario, typing was performed on 1,308 clinical samples from the PHL strain collection for which IS6110 RFLP profiles were also available. Strains were identified as MTBC by using DNA probes (AccuProbe; Gen-Probe, San Diego, CA) and were originally isolated during 1999 to 2001. Strains identified as Mycobacterium bovis or M. bovis BCG and samples containing multiple Mycobacterium species were excluded from analysis. For cases with multiple cultures, only the first isolate was used. Genomic DNA was extracted according to standard protocols (17) in a dedicated biosafety containment facility.MIRU-VNTR and IS6110 RFLP data were analyzed with BioNumerics 5.0 (Applied Maths, St-Martin Latem, Belgium). RFLP patterns were compared using band-based Dice statistics with 1% position tolerance such that clustered strains exhibited bands of identical number and position. Strains clustered by MIRU-VNTR were identical at all 12 loci. Analysis by MIRU-VNTR plus IS6110 RFLP employed the unweighted-pair group method with arithmetic mean. MTBC lineages were assigned by comparison to the MIRU-VNTR reference strain database (1).Independently, both methods revealed a large number of unique profiles and some clustered isolates (Table ​(Table1).1). Maximum strain resolution was achieved when both methods were combined. In general, RFLP was superior for multiband IS6110 strains, whereas resolution of single-band IS6110 isolates required MIRU-VNTR. Initial typing at 12 MIRU-VNTR loci generated two large “pseudoclusters.” One contained East Asian lineage strains (pattern 223325173533; n = 110), which are known to be poorly resolved by these 12 loci (12, 16). The second was comprised of strains from the Indo-Oceanic lineage (pattern 254326223432; n = 80). This group was typed at 12 additional loci (15). Even though four of the new loci were invariant, extended typing generated 47 new patterns (Fig. ​(Fig.1).1). Thirty-five Indo-Oceanic strains had unique profiles, whereas the remaining isolates formed 12 MIRU-VNTR-defined clusters (6 clusters with 2 isolates in each cluster, 3 with 3 each, 1 with 4, and 2 with 10 each). However, 11 of these could be resolved further by RFLP.Open in a separate windowFIG. 1.Improved typing of the Indo-Oceanic pseudocluster. Whereas all strains (n = 80) shared the same, original 12-loci pattern (254326223432), typing at 12 additional loci produced 47 distinct patterns, including 35 unique profiles. The remaining strains formed 12 clusters (6× n = 2; 3× n = 3; 1× n = 4; 2× n = 10), most of which could be further resolved by RFLP. However, one pair (cluster A) was identical by both methods, and two (clusters D and G) exhibited single band shifts. Conversely, one RFLP-defined pair (cluster M) exhibited differences at two loci upon extended MIRU-VNTR typing. For each strain, IS6110 RFLP profiles and repeat values at the extended MIRU-VNTR loci are shown. Relationships between strains are indicated by the phylogenetic (unweighted-pair group method with arithmetic mean) tree, and strains in MIRU-VNTR-defined clusters are labeled (clusters A to L).

TABLE 1.

Clustering results from MIRU-VNTR and IS6110 RFLP genotyping

Novel SOX2 partner-factor domain mutation in a four-generation family

Anophthalmia (no eye), microphthalmia (small eye) and associated ocular developmental anomalies cause significant visual handicap. In most cases the underlying genetic cause is unknown, but mutations in some genes, such as SOX2, cause ocular developmental defects, particularly anophthalmia, in a subset of patients. Here, we describe a four-generation family with a p.Asp123Gly mutation in the highly conserved partner-factor interaction region of the SOX2 protein, which is important for cell-specific actions of SOX2. The proband in this family has bilateral anophthalmia and several other family members have milder ocular phenotypes, including typical optic fissure coloboma. Expression studies indicate that Sox2 is expressed in the eye at the site of closure of the optic fissure during development. The SOX2 mutation in this family implicates the partner-factor interaction region of SOX2 in contributing to the specificity of SOX2 action in optic fissure closure. Our findings indicate that investigation of SOX2 in a broad range of eye anomaly patients aids in the determination of particular functions of SOX2 in development.     

Ambiguous genitalia in the newborn: an overview and teaching tool

Ambiguous genitalia is a significant example of a disorder of sexual development, in which the external genitalia do not have the typical appearance of either sex. Although the birth of a child with ambiguous genitalia is rare, the emergent nature of the issue demands that healthcare providers have at least a familiarity with the underlying etiologies, the issues, and the initial approach to diagnosis and management. With numerous etiologies, potential difficulties with reaching a diagnosis, and many challenges with immediate and long-term care, the topic of ambiguous genitalia can be daunting. We provide a review of basic embryology, as well as a classification system for understanding the various etiological causes of ambiguous genitalia. The important clinical aspects of diagnosis and management are also highlighted, and a teaching tool has been included to help the reader (or their learners) to solidify information presented. Our overall goal is to provide practical information on ambiguous genitalia and allow the clinician to apply this information to clinically relevant scenarios.     

Alternative Recruitment Strategies Influence Saliva Sample Return Rates in Community‐Based Genetic Association Studies

Collection of saliva for DNA extraction has created new opportunities to recruit participants from the community for genetic association studies. However, sample return rates are variable. No prior study has specifically addressed how study design impacts sample return. Using data from three large‐scale genetic association studies we compared recruitment strategy and sample return rates. We found highly significant differences in sample return rates between the studies. In studies that recruited retrospectively, overall returns were much lower from families with a self‐limiting condition who provided samples at a research centre or home visit, than adult elderly individuals with a chronic disease who provided samples by post (59% vs. 84%). Prospective recruitment was associated with high agreement to participate (72%), but subsequent low return of actual saliva samples (42%). A telephone call had marginal effect on recruitment in a retrospective family study, but significantly improved returns in a prospective family study. We found no effect upon DNA yield comparing observed versus unobserved sample collection, or between male and female adult participants. Overall, study design significantly impacts upon response rates for genetic association studies recruiting from the community. Our findings will help researchers in constructing and costing a recruitment protocol.