Airway clearance techniques used by people with cystic fibrosis in the UK.

OBJECTIVES: To describe the current use of airway clearance techniques among people with cystic fibrosis (CF) in the UK, and the baseline characteristics for users of different airway clearance techniques. DESIGN: Analysis of the UK CF Registry 2011 data. SETTING AND PARTICIPANTS: All people with CF in the UK aged ≥11 years (n=6372). RESULTS: Of the 6372 people on the UK CF registry in 2011, 89% used airway clearance techniques. The most commonly used primary techniques were forced expiratory techniques (28%) and oscillating positive expiratory pressure (PEP) (23%). Postural drainage and high-frequency chest wall oscillation were used by 4% and 1% of people with CF, respectively. The male:female ratio of individuals who used exercise as their primary airway clearance technique was 2:1, compared with 1:1 for other techniques. Individuals with more severe lung disease tended to use devices such as non-invasive ventilation or high-frequency chest wall oscillation. CONCLUSIONS: Forced expiratory techniques and oscillating PEP are the most common airway clearance techniques used by people with CF in the UK, and postural drainage and high-frequency chest wall oscillation are the least common techniques. This is significant in terms of planning airway clearance technique trials, where postural drainage has been used traditionally as the comparator. The use of airway clearance techniques varies between countries, but the reasons for these differences are unknown.

Exploration of the impact of 'mild phenotypes' on median age at death in the U.K. CF registry.

BACKGROUND: The widespread availability of genetic testing allowing the identification of "milder" individuals with CF coincided with improvements in CF life expectancy but the relative contribution of case mix to that improved survival is uncertain. METHODS: Patients in the U.K. CF registry were divided into 'mild phenotype' defined as pancreatic sufficient and 'typical CF' defined as pancreatic insufficient. Distributions of age at death were compared with Mann-Whitney test. Temporal trends in incidence and prevalence were described. Jonckheere-Terpstra test was used to compare the trend for median age at death from 2007 to 2010. RESULTS: Patients with 'mild phenotype' had significantly higher age at death (32 years, interquartile range 14 years versus 27 years, interquartile range 29 years; Mann-Whitney test p-value = 0.026). The proportion of patients with 'mild phenotype' appeared to be increasing (0.128 in 2007, 0.144 in 2010). The trend for increasing age at death (from 25 years in 2007 to 29 years in 2010, Jonckheere-Terpstra test p-value = 0.012) was independent of the 'mild phenotype' patients. CONCLUSION: The impact of mild phenotypes on the improvement in the median age at death among people with CF was trivial.

The angiotensin-converting enzyme gene insertion/deletion polymorphism in Indian patients with vitiligo: a case-control study and meta-analysis.

BACKGROUND: Vitiligo is a common, acquired, idiopathic depigmenting skin disorder. Although the exact pathogenesis remains unknown, genetic susceptibility and autoimmune responses play a role in vitiligo development. Previous studies have suggested that the D allele of the insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme (ACE) gene is associated with vitiligo in Indians and Koreans. Furthermore, significantly higher serum ACE levels have been demonstrated in patients with some autoimmune and autoinflammatory disorders. OBJECTIVES: The objectives were to investigate any association between the ACE I/D polymorphism and vitiligo susceptibility in an Indian population, and to compare serum ACE levels in patients with vitiligo and healthy subjects. METHODS: The ACE I/D genotypes of 79 patients with vitiligo and 100 normal individuals were determined by polymerase chain reaction amplification. A meta-analysis was done to compare the distribution of the ACE I/D alleles and genotypes in the current and three previous studies. Serum ACE levels were evaluated by enzyme-linked immunosorbent assay. RESULTS: A significant increase in the frequency of the ACE I/D D allele was evident in patients with vitiligo in both the case-control study [P=0·005; odds ratio (OR) 1·87; 95% confidence intervals (CI) 1·22-2·85] and the meta-analysis (P=0·044; OR 1·44; 95% CI 1·01-2·06). Serum ACE levels were significantly increased in patients with vitiligo compared with healthy subjects (P<0·0001). CONCLUSIONS: In agreement with earlier reports, the ACE I/D D allele is associated with vitiligo susceptibility in the Indian population. The significantly elevated serum ACE levels in our cohort of patients with vitiligo concur with those previously found in patients with some other autoimmune diseases.

Family based studies and genetic epidemiology: theory and practice.

Family based studies have underpinned many successes in uncovering the causes of monogenic and oligogenic diseases. Now research is focussing on the identification and characterisation of genes underlying common diseases and it is widely accepted that these studies will require large population based samples. Population based family study designs have the potential to facilitate the analysis of the effects of both genes and environment. These types of studies integrate the population based approaches of classic epidemiology and the methods enabling the analysis of correlations between relatives sharing both genes and environment. The extent to which such studies are feasible will depend upon population- and disease-specific factors. To review this topic, a symposium was held to present and discuss the costs, requirements and advantages of population based family study designs. This article summarises the features of the meeting held at The University of Sheffield, August 2006.

Haplotype-specific gene expression profiles in a telomeric major histocompatibility complex gene cluster and susceptibility to autoimmune diseases.

The telomeric class III region of the major histocompatibility complex is gene dense, but apart from the three tumour necrosis factor (TNF) superfamily members (TNF, lymphotoxin alpha and lymphotoxin beta) little is known of the expression and function of the majority of the genes. Recent genetic studies in autoimmune diseases, particularly rheumatoid arthritis (RA), have suggested a human leukocyte antigen (HLA)-DR-independent disease effect in this region. To gain further insights into these associations, we used lipopolysaccharide-stimulated human macrophages to examine inducible mRNA expression and genotype-phenotype relationships for genes in this region. Following stimulation in addition to the expected induction of TNF mRNA, a 14-fold increase of ATP6V1G2 at 18 h (P<0.001) was seen, whereas B-associated transcript (BAT)2 (P<0.001) and leucocyte-specific transcript (LST)1 (P<0.001) were both downregulated. By genotyping single-nucleotide polymorphisms spanning a 70 kb interval centred on the TNF locus, we constructed haplotypes and determined associated expression profiles for 10 genes in the cluster using quantitative real-time polymerase chain reaction. Overexpression of BAT1 mRNA was associated with carriers of a haplotype containing the LST1 marker transmitted to RA cases in a family study and also DRB1(*)15 associated with susceptibility to nephritis in systemic lupus erythematosus. The implications of our findings for the understanding of genetic associations with disease susceptibility in this region are discussed.

An expectation-maximization algorithm for the analysis of allelic expression imbalance.

A significant proportion of the variation between individuals in gene expression levels is genetic, and it is likely that these differences correlate with phenotypic differences or with risk of disease. Cis-acting polymorphisms are important in determining interindividual differences in gene expression that lead to allelic expression imbalance, which is the unequal expression of homologous alleles in individuals heterozygous for such a polymorphism. This expression imbalance can be detected using a transcribed polymorphism, and, once it is established, the next step is to identify the polymorphisms that are responsible for or predictive of allelic expression levels. We present an expectation-maximization algorithm for such analyses, providing a formal statistical framework to test whether a candidate polymorphism is associated with allelic expression differences.

Clinical and genetic heterogeneity in peroneal muscular atrophy associated with vocal cord weakness.

BACKGROUND: The peroneal muscular atrophy syndrome is the most common inherited disorder of the peripheral nervous system and has extensive clinical and genetic heterogeneity. Cranial nerve involvement is rare, though there are distinct peroneal muscular atrophy syndromes in which vocal cord paralysis is a characteristic feature. Among these dHMN-VII and HMSN-IIC are clinically similar but are differentiated by sensory involvement in HMSN-IIC. The gene for dHMN-VII, designated DHMNVP, has been localised to chromosome 2q14, but the location of the gene for HMSN-IIC is currently unknown. It has been suggested that dHMN-VII and HMSN II-C are allelic disorders. OBJECTIVE: To assess the contribution of the dHMN-VII predisposition gene to peroneal muscular atrophy syndromes associated with vocal cord weakness. METHODS: Linkage analysis of microsatellite markers at chromosome 2q14 was undertaken on two families, one affected by HMSN-IIC and a second manifesting vocal cord paralysis and sensorineural deafness in addition to distal muscular atrophy. RESULTS: Two-point LOD scores at chromosome 2q14 markers encompassing the DHMNVP gene were negative in both families. CONCLUSIONS: These results suggest that at least one further gene predisposing to distal muscular weakness in association with vocal cord paralysis is likely to exist, and that dHMN-VII and HMSN-IIC are unlikely to be allelic disorders. Analyses of further HMSN-IIC families are required to confirm this.

Sampling distribution of summary linkage disequilibrium measures.

The design of association studies is critically dependent upon the extent of linkage disequilibrium (LD) across different genomic regions, often summarised in terms of the mean absolute value of summary linkage disequilibrium measures. The two most commonly used measures are D' for estimating the magnitude or extent of LD, and Delta which is directly proportional to the power of LD mapping. We studied the sampling distribution of the mean of /D'/ and /Delta/ statistics for varying sample size and major allele frequencies. When the sample size is small or one allele frequency is extreme, estimates of the magnitude of association based on the mean of /D'/ can be substantially inflated. This inflation is more marked when the haplotype frequencies have been inferred from genotype counts. The net effect of this means that smaller studies will tend to show higher levels of LD. The magnitude of this inflation can be reduced by use of a bootstrap correction, and by avoiding using markers with extreme allele frequencies. In contrast, the /Delta/ statistic is much less affected by sample size and high major allele frequencies. These effects are illustrated with real data on 36 SNPs typed in an Ashkenasi Jewish population.

Novel platelet membrane glycoprotein VI dimorphism is a risk factor for myocardial infarction.

BACKGROUND: Glycoprotein (GP) VI plays a crucial role in platelet activation and aggregation. We investigated whether polymorphic variation at the GP VI locus confers an increased risk of myocardial infarction (MI). METHODS AND RESULTS: Coding and 5' and 3' non-coding regions of the GP VI gene were analyzed by polymerase chain reaction and conformation sensitive gel electrophoresis in 21 healthy subjects. Ten dimorphisms, 5 of which predicted amino acid substitutions (T13254C, A19871G, A21908G, A22630T, C22644A), were identified. Two core haplotypes involving 7 dimorphisms (C10781A and G10873A and all those predicting amino acid substitutions) were apparent. The contribution of the T13254C dimorphism, which predicted the substitution of serine 219 by proline, to risk of MI was assessed in 525 patients with acute MI and 474 controls, all aged <75 years. The allelic odds ratio (OR) for MI associated with the 13254C allele was 1.16 (95% CI, 0.91 to 1.46; P=0.23). Compared with corresponding control subgroups, the 13254CC genotype was more common among cases who were female (OR, 4.52; 95% CI, 1.23 to 16.64; P=0.029), nonsmokers (OR, 2.50; 95% CI, 0.98 to 6.38; P=0.048), aged >/=60 years (OR, 6.48; 95% CI, 1.47 to 28.45; P=0.009) or carried the beta-fibrinogen -148T allele associated with increased fibrinogen levels (OR, 10.49; 95% CI, 1.32 to 83.42; P=0.02). In logistic regression analysis that took other cardiovascular risk factors into account, the interactions of GP VI genotype with age (P=0.005) and beta-fibrinogen genotype (P=0.035) remained significant. CONCLUSIONS: The GP VI 13254CC genotype increases the risk of MI, particularly in older individuals, and the interaction of the GP VI 13254C allele with other candidate risk alleles may accentuate this risk.

Confirmation of a gene locus for medullary cystic kidney disease (MCKD2) on chromosome 16p12.

BACKGROUND: Autosomal-dominant medullary cystic kidney disease (MCKD) is an interstitial nephropathy characterized by structural renal tubular defects that result in salt wasting and a reduction in urinary concentration. The condition has clinical and morphological similarities to autosomal-recessive juvenile nephronophthisis. Two genes predisposing to MCKD have been localized. MCKD1 on chromosome 1q21 was localized in two Cypriot families, and MCKD2 on chromosome 16p12 was localized in a single Italian family. We have evaluated a large Welsh MCKD family for linkage at these two loci. METHODS: Clinical data and DNA samples were collected from affected family members. Polymorphic microsatellite markers spanning the critical regions on chromosome 1 and chromosome 16 that encompass MCKD1 and MCKD2 were analyzed. Two-point and multipoint LOD scores were calculated. RESULTS: The family fulfilled previously published criteria for the diagnosis of MCKD, but hyperuricemia and gout were not prominent features. Twenty-one affected individuals were identified. Mean age at death or end-stage renal disease was 47 years (37 to 60). Linkage and haplotype analysis generated strongly negative results at MCKD1 on chromosome 1q21 (two-point LOD score = -5.32). Strong evidence of linkage to MCKD2 was generated with a maximum multi-point LOD score of 3.75. CONCLUSION: These results provide the first independent confirmation of a gene predisposing to MCKD on chromosome 16p12 and indicate that mutation of this gene is not restricted to a single family or population. The absence of hyperuricemia and gout in our family indicates that these are not obligatory features of MCKD2 mutations.

Localization of the gene for distal hereditary motor neuronopathy VII (dHMN-VII) to chromosome 2q14.

Distal hereditary motor neuronopathy type VII (dHMN-VII) is an autosomal dominant disorder characterized by distal muscular atrophy and vocal cord paralysis. We performed a genomewide linkage search in a large Welsh pedigree with dHMN-VII and established linkage to chromosome 2q14. Analyses of a second family with dHMN-VII confirmed the location of the gene and provided evidence for a founder mutation segregating in both pedigrees. The maximum three-point LOD score in the combined pedigree was 7.49 at D2S274. Expansion of a polyalanine tract in Engrailed-1, a transcription factor strongly expressed in the spinal cord, was excluded as the cause of dHMN-VII.

A common variant in BRCA2 is associated with both breast cancer risk and prenatal viability.

Inherited mutations in the gene BRCA2 predispose carriers to early onset breast cancer, but such mutations account for fewer than 2% of all cases in East Anglia. It is likely that low penetrance alleles explain the greater part of inherited susceptibility to breast cancer; polymorphic variants in strongly predisposing genes, such as BRCA2, are candidates for this role. BRCA2 is thought to be involved in DNA double strand break-repair. Few mice in which Brca2 is truncated survive to birth; of those that do, most are male, smaller than their normal littermates and have high cancer incidence. Here we show that a common human polymorphism (N372H) in exon 10 of BRCA2 confers an increased risk of breast cancer: the HH homozygotes have a 1.31-fold (95% CI, 1.07-1.61) greater risk than the NN group. Moreover, in normal female controls of all ages there is a significant deficiency of homozygotes compared with that expected from Hardy-Weinberg equilibrium, whereas in males there is an excess of homozygotes: the HH group has an estimated fitness of 0.82 in females and 1.38 in males. Therefore, this variant of BRCA2 appears also to affect fetal survival in a sex-dependent manner.

The extent of linkage disequilibrium in four populations with distinct demographic histories.

The design and feasibility of whole-genome-association studies are critically dependent on the extent of linkage disequilibrium (LD) between markers. Although there has been extensive theoretical discussion of this, few empirical data exist. The authors have determined the extent of LD among 38 biallelic markers with minor allele frequencies >.1, since these are most comparable to the common disease-susceptibility polymorphisms that association studies aim to detect. The markers come from three chromosomal regions-1,335 kb on chromosome 13q12-13, 380 kb on chromosome 19q13.2, and 120 kb on chromosome 22q13.3-which have been extensively mapped. These markers were examined in approximately 1,600 individuals from four populations, all of European origin but with different demographic histories; Afrikaners, Ashkenazim, Finns, and East Anglian British. There are few differences, either in allele frequencies or in LD, among the populations studied. A similar inverse relationship was found between LD and distance in each genomic region and in each population. Mean D' is.68 for marker pairs <5 kb apart and is.24 for pairs separated by 10-20 kb, and the level of LD is not different from that seen in unlinked marker pairs separated by >500 kb. However, only 50% of marker pairs at distances <5 kb display sufficient LD (delta>.3) to be useful in association studies. Results of the present study, if representative of the whole genome, suggest that a whole-genome scan searching for common disease-susceptibility alleles would require markers spaced < or = 5 kb apart.

Absence of evidence for a familial breast cancer susceptibility gene at chromosome 8p12-p22.

Several recent studies indicate that the majority of families with five or fewer cases of breast cancer and no cases of ovarian cancer are not due to BRCA1 or BRCA2. It has been proposed that a further breast cancer susceptibility gene that may account for some of these families is located on chromosome 8p12-p22. We have identified 31 site-specific breast cancer families that have a greater than 80% posterior probability of being due to genes other than BRCA1 or BRCA2. These families have been examined for linkage to 8p12-p22 using markers flanking the putative location of the gene. The overall multi-point LOD score is strongly negative across the whole 44 cM. The individual multi-point LOD score is negative in 23 families and only exceeds 0.5 in a single family (with a multi-point LOD score of 1.22). The maximum heterogeneity LOD score was 0.03 at marker D8S136 with estimated proportion linked (alpha) of 3% (95% CI 0 - 30%). These data do not lend support to the hypothesis that chromosome 8p12-p22 harbours a familial breast cancer susceptibility gene. Oncogene (2000) 19, 4170 - 4173

A systematic review of genetic polymorphisms and breast cancer risk.

Studies investigating the relationship between common genetic variants and cancer risk are being reported with rapidly increasing frequency. We have identified 46 published case-control studies that have examined the effect of common alleles of 18 different genes on breast cancer risk. Of these, 12 report statistically significant associations, none of which were reported by more than one study. However, many of the studies were small: 10 of the 46 had 80% power or greater to detect a rare allele homozygote relative risk <2.5. We therefore combined the results of individual studies to obtain more precise estimates of risk. Statistically significant differences in genotype frequencies were found in three case-control comparisons of unselected cases. These were for CYP19 (TTTA)n polymorphism [(TTTA)10 carrier odds ratio (OR) = 2.33; P = 0.002], the GSTP1 Ile105Val polymorphism (Val carrier OR = 1.60; P = 0.02), and the TP53 Arg72Pro polymorphism (Pro carrier OR = 1.27; P = 0.03). In addition, the GSTM1 gene deletion was found to be significantly associated with postmenopausal breast cancer (null homozygote OR = 1.33; P = 0.04). There was also some evidence that homozygotes for the PR PROGINS allele are protected against breast cancer, although this result was of borderline statistical significance. For polymorphisms in BRCA1, COMT, CYP17, CYP1A1, NAT1, and NAT2, the best estimate of risk either from the individual studies or the meta-analyses was sufficiently precise to exclude a relative risk of 1.5 or greater. For the polymorphisms in EDH17B2, ER, CYP2D6, CYP2E1, GSTT1, HSP70, and TNFalpha, the risk estimates, although nonsignificant, were insufficiently precise to exclude a moderate risk (>1.5). Precise estimation of the risks associated with these and other as yet untested genes, as well as investigation of more complex risks arising from gene-gene and gene-environment interactions, will require much larger studies.

A population study of mutations and LOH at breast cancer gene loci in tumours from sister pairs: two recurrent mutations seem to account for all BRCA1/BRCA2 linked breast cancer in Iceland.

The majority of breast cancer in high risk families is believed to result from a mutation in either of two genes named BRCA1 and BRCA2. A germline defect in either gene is usually followed by chromosomal deletion of the normal allele in the tumour. In Iceland two recurrent mutations have been identified, 999del5 BRCA2 and G5193A BRCA1. In this study, randomly selected pairs of sisters diagnosed with breast cancer at the age of 60 years or younger were analysed to evaluate the proportion of breast cancer resulting from BRCA1 and BRCA2. Genotypes and allele loss in tumour tissue from 42 sister pairs were compared using markers within and around the BRCA1 and BRCA2 genes. Eleven sister pairs were highly suggestive of BRCA2 linkage, and no obvious BRCA1 linkage was seen. Screening for the G5193A BRCA1 and 999del5 BRCA2 mutations showed the 999del5 mutation in the 11 BRCA2 suggestive pairs plus three pairs less indicative of linkage, and the G5193A BRCA1 mutation in one pair. When known mutation carriers are removed from the group, no indication of further linkage to BRCA1 or BRCA2 is seen. The results of our studies suggest that a large proportion of familial breast cancer in Iceland is the result of the 999del5 BRCA2 mutation, and it is unlikely that BRCA1 and BRCA2 germline mutations other than 999del5 and G5193A play a significant role in hereditary breast cancer in Iceland. Furthermore it can be concluded that most families with BRCA1 or BRCA2 linkage are easily identified by studying LOH around the defective gene in as few as two affected relatives.

Genetic heterogeneity and penetrance analysis of the BRCA1 and BRCA2 genes in breast cancer families. The Breast Cancer Linkage Consortium.

The contribution of BRCA1 and BRCA2 to inherited breast cancer was assessed by linkage and mutation analysis in 237 families, each with at least four cases of breast cancer, collected by the Breast Cancer Linkage Consortium. Families were included without regard to the occurrence of ovarian or other cancers. Overall, disease was linked to BRCA1 in an estimated 52% of families, to BRCA2 in 32% of families, and to neither gene in 16% (95% confidence interval [CI] 6%-28%), suggesting other predisposition genes. The majority (81%) of the breast-ovarian cancer families were due to BRCA1, with most others (14%) due to BRCA2. Conversely, the majority of families with male and female breast cancer were due to BRCA2 (76%). The largest proportion (67%) of families due to other genes was found in families with four or five cases of female breast cancer only. These estimates were not substantially affected either by changing the assumed penetrance model for BRCA1 or by including or excluding BRCA1 mutation data. Among those families with disease due to BRCA1 that were tested by one of the standard screening methods, mutations were detected in the coding sequence or splice sites in an estimated 63% (95% CI 51%-77%). The estimated sensitivity was identical for direct sequencing and other techniques. The penetrance of BRCA2 was estimated by maximizing the LOD score in BRCA2-mutation families, over all possible penetrance functions. The estimated cumulative risk of breast cancer reached 28% (95% CI 9%-44%) by age 50 years and 84% (95% CI 43%-95%) by age 70 years. The corresponding ovarian cancer risks were 0.4% (95% CI 0%-1%) by age 50 years and 27% (95% CI 0%-47%) by age 70 years. The lifetime risk of breast cancer appears similar to the risk in BRCA1 carriers, but there was some suggestion of a lower risk in BRCA2 carriers <50 years of age.

Analysis of genetic linkage and somatic loss of heterozygosity in affected pairs of first-degree relatives.

Recently, data on loss of constitutional heterozygosity (LOH) have been used by several groups to increase the power to detect linkage in pedigrees with an inherited cancer predisposition. This approach assumes that the predisposition is due to the inheritance of the defective copy of a tumor suppressor. In order to assess the gain of power expected from the inclusion of LOH data, we simulated segregation and somatic loss of alleles in pedigrees consisting of an affected pair of first-degree relatives. We explored the effects of pedigree structure, frequency of loss, penetrance, and recombination rate on the expected LOD score. The results indicate that, for establishment of genetic linkage, isolated parent-offspring pairs can be as informative as sib pairs and that they could represent an additional source of information in linkage studies.