Choice of Statistical Tools for Outlier Removal Causes Substantial Changes in Analyte Reference Intervals in Healthy Populations.

BACKGROUND: Reference intervals are an important aid in medical practice as they provide clinicians a guide as to whether a patient is healthy or diseased.Outlier results in population studies are removed by any of a variety of statistical measures. We have compared several methods of outlier removal and applied them to a large body of analytes from a large population of healthy persons. METHODS: We used the outlier exclusion criteria of Reed-Dixon and Tukey and calculated reference intervals using nonparametric and Harrell-Davis statistical methods and applied them to a total of 36 different analytes. RESULTS: Nine of 36 analytes had a greater than 20% difference in the upper reference limit, and for some the difference was 100% or more. CONCLUSIONS: For some analytes, great importance is attached to the reference interval. We have shown that different statistical methods for outlier removal can cause large changes to reported reference intervals. So that population studies can be readily compared, common statistical methods should be used for outlier removal.

Deleterious GRM1 mutations in schizophrenia.

We analysed a phenotypically well-characterised sample of 450 schziophrenia patients and 605 controls for rare non-synonymous single nucleotide polymorphisms (nsSNPs) in the GRM1 gene, their functional effects and family segregation. GRM1 encodes the metabotropic glutamate receptor 1 (mGluR1), whose documented role as a modulator of neuronal signalling and synaptic plasticity makes it a plausible schizophrenia candidate. In a recent study, this gene was shown to harbour a cluster of deleterious nsSNPs within a functionally important domain of the receptor, in patients with schizophrenia and bipolar disorder. Our Sanger sequencing of the GRM1 coding regions detected equal numbers of nsSNPs in cases and controls, however the two groups differed in terms of the potential effects of the variants on receptor function: 6/6 case-specific and only 1/6 control-specific nsSNPs were predicted to be deleterious. Our in-vitro experimental follow-up of the case-specific mutants showed that 4/6 led to significantly reduced inositol phosphate production, indicating impaired function of the major mGluR1 signalling pathway; 1/6 had reduced cell membrane expression; inconclusive results were obtained in 1/6. Family segregation analysis indicated that these deleterious nsSNPs were inherited. Interestingly, four of the families were affected by multiple neuropsychiatric conditions, not limited to schizophrenia, and the mutations were detected in relatives with schizophrenia, depression and anxiety, drug and alcohol dependence, and epilepsy. Our findings suggest a possible mGluR1 contribution to diverse psychiatric conditions, supporting the modulatory role of the receptor in such conditions as proposed previously on the basis of in vitro experiments and animal studies.

KCNN4 gene variant is associated with ileal Crohn's Disease in the Australian and New Zealand population.

OBJECTIVES: Crohn's disease (CD; MIM 266600) is one of the most common forms of inflammatory bowel disease (IBD), and represents a significant burden to health care in developed countries. Our aim was to determine whether a gene in the IBD linkage region on chromosome 19q13, with a role in Paneth cell secretion and T-cell activation, conferred genetic susceptibility to the development of CD. METHODS: In total, 792 CD cases and 1,244 controls of Australian origin (Caucasian) were genotyped for seven single-nucleotide polymorphisms (SNPs) in the gene encoding the intermediate conductance calcium-activated potassium channel protein (KCNN4) at 19q13.2. CD cases were phenotyped using the Montreal classification. The replication set comprised an additional 326 CD cases and 951 population-based Caucasian controls. Analysis of the KCNN4 mRNA transcript was carried out using quantitative reverse transcriptase-PCR. RESULTS: KCNN4 SNP rs2306801 was associated with CD (primary P=0.0008, odds ratio (OR) (95% confidence interval (CI)): 0.76 (0.65-0.89); replication P=0.01, OR (95% CI): 0.77 (0.61-0.97). Stratification by disease location identified the association between SNP rs2306801 and ileal CD (P=0.01). Non-inflamed ileal mucosa from CD patients carrying any of the common disease-predisposing NOD2 variants (R702W, G908R, 1007fs) had significantly reduced levels of KCNN4 mRNA expression (P=0.001). KCNN4 protein expression was detected in Paneth cells, and in T cells in inflamed lamina propria. CONCLUSIONS: Our data implicate the role of KCNN4 in ileal CD. The dual roles of KCNN4 in Paneth cell secretion and T-cell activation and also its nature as a potassium channel make it an important and practical therapeutic target.

Iminoglycinuria and hyperglycinuria are discrete human phenotypes resulting from complex mutations in proline and glycine transporters.

Iminoglycinuria (IG) is an autosomal recessive abnormality of renal transport of glycine and the imino acids proline and hydroxyproline, but the specific genetic defect(s) have not been determined. Similarly, although the related disorder hyperglycinuria (HG) without iminoaciduria has been attributed to heterozygosity of a putative defective glycine, proline, and hydroxyproline transporter, confirming the underlying genetic defect(s) has been difficult. Here we applied a candidate gene sequencing approach in 7 families first identified through newborn IG screening programs. Both inheritance and functional studies identified the gene encoding the proton amino acid transporter SLC36A2 (PAT2) as the major gene responsible for IG in these families, and its inheritance was consistent with a classical semidominant pattern in which 2 inherited nonfunctional alleles conferred the IG phenotype, while 1 nonfunctional allele was sufficient to confer the HG phenotype. Mutations in SLC36A2 that retained residual transport activity resulted in the IG phenotype when combined with mutations in the gene encoding the imino acid transporter SLC6A20 (IMINO). Additional mutations were identified in the genes encoding the putative glycine transporter SLC6A18 (XT2) and the neutral amino acid transporter SLC6A19 (B0AT1) in families with either IG or HG, suggesting that mutations in the genes encoding these transporters may also contribute to these phenotypes. In summary, although recognized as apparently simple Mendelian disorders, IG and HG exhibit complex molecular explanations depending on a major gene and accompanying modifier genes.

ATG16L1 T300A shows strong associations with disease subgroups in a large Australian IBD population: further support for significant disease heterogeneity.

OBJECTIVES: Crohn's disease (CD) and ulcerative colitis (UC) are the two most common forms of inflammatory bowel disease (IBD), representing a significant health-care burden. A variant in the autophagy gene ATG16L1 (T300A) has been newly identified as a CD susceptibility locus by genome-wide association. Our aim was to assess the contribution of T300A in determining disease susceptibility and phenotype in two independent Australian IBD cohorts and explore the relationship between T300A and known CD risk factors (NOD2[nucleotide-binding oligomerization domain containing 2] status and smoking). METHODS: In total, 669 CD and 543 UC cases, and 1,244 controls (study 1), 154 CD cases and 420 controls (study 2), and 702 unaffected parents from both groups were genotyped. We conducted case-control and family association analyses, and investigated relationships between T300A and disease subgroups and between NOD2 status and cigarette smoking (CD only). RESULTS: The strong association between CD and T300A was confirmed (P < 0.001), with a two-fold increase in disease risk associated with the GG genotype (odds ratio [OR] 1.96, 95% confidence interval [CI] 1.49-2.58), while ileal CD risk was almost three-fold (OR 2.73, CI 1.87-4.0). ATG16L1 and NOD2 were found to contribute independently to CD risk. A greater than seven-fold increased CD risk was observed for current smokers with a GG genotype (vs nonsmoking AA genotype; P < 0.001, OR 7.65, CI 4.21-13.91). A significant inverse association was found between T300A and UC (P= 0.002). This was strongest for patients with extensive, severe disease. CONCLUSIONS: We confirm the strong association between T300A and CD, specifically ileal subphenotype, and also report the first strong association of this variant with UC.

Deletion of Glu155 causes a deficiency of glutathione transferase Omega 1-1 but does not alter sensitivity to arsenic trioxide and other cytotoxic drugs.

The Omega class glutathione transferase GSTO1-1 can catalyze the reduction of pentavalent methylated arsenic species and is responsible for the biotransfomation of potentially toxic alpha-haloketones. We investigated the cause of GSTO1-1 deficiency in the T-47D breast cancer cell line and found that the cell line is hemizygous for a polymorphic allele that encodes the deletion of Glu155. Northern and Western blots show that T-47D cells contain GSTO1 mRNA but no GSTO1-1 protein suggesting that the deletion of Glu155 causes GSTO1-1 deficiency in vivo. In further support of this contention we found that lymphoblastoid cell lines from subjects who are heterozygous for the deletion of Glu155 have only 60% of normal activity with the GSTO1-1 specific substrate 4-nitrophenacyl glutathione. Pulse-chase studies showed that the deletion of Glu155 causes increased turnover of GSTO1-1 in T47-D cells. These data establish the fact that the polymorphic deletion of Glu155 can cause GSTO1-1 deficiency in vivo. GSTO1-1 expression is elevated in some cell lines that are resistant to the cytotoxic cancer drugs adriamycin, etoposide and cisplatinum but its specific contribution to multi drug resistance has not been evaluated. In this study GSTO1-1 deficient T47-D cells were used to determine if GSTO1-1 contributes directly to arsenic and drug resistance. We established stable expression of normal GSTO1-1 in T-47D cells and found that this did not alter sensitivity to arsenic trioxide, cisplatinum daunorubicin or etoposide.

Further evidence for allelic heterogeneity in Hartnup disorder.

Hartnup disorder is an autosomal recessive impairment of amino acid transport in kidney and intestine. Mutations in SLC6A19 have been shown to cosegregate with the disease in the predicted recessive manner; however, in two previous studies (Seow et al., Nat Genet 2004;36:1003-1007; Kleta et al., Nat Genet 2004;36:999-1002), not all causative alleles were identified in all affected individuals, raising the possibility that other genes may contribute to Hartnup disorder. We have now investigated six newly acquired families of Australian and Canadian (Province of Quebec) origin and resequenced the entire coding region of SLC6A19 in families with only a single disease allele identified. We also studied one American family in whom no mutations had been identified in a previous study (Kleta et al., Nat Genet 2004;36:999-1002). We have identified seven novel mutations in SLC6A19 that show functional obliteration of the protein in vitro, explaining Hartnup disorder in all reported families so far. We demonstrate that Hartnup disorder is allelically heterogeneous with two mutated SLC6A19 alleles, whether identical or not, necessary for manifestation of the characteristic aminoaciduria in affected individuals. This study resolves the previous hypothesis that other genes contribute to the Hartnup phenotype.

Persistence of the common Hartnup disease D173N allele in populations of European origin.

Hartnup disorder is an aminoaciduria that results from mutations in the recently described gene SLC6A19 on chromosome 5p15.33. The disease is inherited in a simple recessive manner and ten different mutations have been described to date. One mutation, the D173N allele, is present in 42% of Hartnup chromosomes from apparently unrelated families from both Australia and North America. We report an investigation of the origins of the D173N allele using a unique combination of variants including SNPs, microsatellites, and a VNTR across 211 Kb spanning the SLC6A19 locus. All individuals who carry the mutant allele share an identical core haplotype suggesting a single common ancestor, indicating that the elevated frequency of the D173N allele is not a result of recurrent mutation. Analyses of these data indicate that the allele is more than 1000 years old. We compare the reasons for survival of this allele with other major alleles in some other common autosomal recessive diseases occurring in European Caucasians. We postulate that survival of this allele may be a consequence of failure of the allele to completely inactivate the transport of neutral amino acids.

The clinical relevance of compound heterozygosity for the C282Y and H63D substitutions in hemochromatosis.

BACKGROUND & AIMS: Two major mutations are defined within the hemochromatosis gene, HFE. Although the effects of the C282Y substitution have been well characterized, the clinical significance of the C282Y/H63D state remains unclear. This study assessed the phenotypic expression in C282Y/H63D subjects as compared with C282Y homozygotes. METHODS: Data were obtained from 91 C282Y/H63D probands, 158 C282Y/H63D subjects identified through family screening, and 483 C282Y homozygotes. Subjects underwent clinical evaluation, genotyping, biochemical assessment, and liver biopsy examination where clinically indicated. RESULTS: C282Y/H63D probands had significantly less clinical and biochemical expression than C282Y homozygotes. Biochemical expression was higher in C282Y/H63D probands than in C282Y/H63D subjects identified through family screening (P < .001). Of the C282Y/H63D subjects with serum ferritin levels greater than 1000 mug/L, all had known comorbid factors that could have contributed to the increased ferritin level. Of the 51 C282Y/H63D subjects who underwent liver biopsy examination, significantly increased iron stores were present in 9 subjects and hepatic fibrosis was present in 13. Twelve of the 13 had evidence of hepatic steatosis, excess alcohol consumption, or diabetes. The mobilizable iron level was significantly higher in C282Y homozygous males than in compound heterozygous males (P < .001). Genetic screening of C282Y/H63D first-degree relatives detected 5 C282Y homozygotes. CONCLUSIONS: C282Y/H63D subjects referred for assessment had a high prevalence of increased iron indices but did not develop progressive clinical disease without comorbid factors such as steatosis, diabetes, or excess alcohol consumption. When fibrosis was seen, 1 or more comorbid factors almost always were present. Thus, phlebotomy therapy is warranted and cascade screening of relatives should be performed because expressing C282Y homozygotes may be detected.

Screening for hemochromatosis in asymptomatic subjects with or without a family history.

BACKGROUND: Hemochromatosis in white subjects is mostly due to homozygosity for the common C282Y substitution in HFE. Although clinical symptoms are preventable by early detection of the genetic predisposition and prophylactic treatment, population screening is not currently advocated because of the discrepancy between the common mutation prevalence and apparently lower frequency of clinical disease. This study compared screening for hemochromatosis in subjects with or without a family history. METHODS: We assessed disease expression by clinical evaluation and liver biopsy in 672 essentially asymptomatic C282Y homozygous subjects identified by either family screening or health checks. We also observed a subgroup of untreated homozygotes with normal serum ferritin levels for up to 24 years. RESULTS: Prevalence of hepatic iron overload and fibrosis were comparable between the 2 groups. Disease-related conditions were more common in male subjects identified by health checks, but they were older. Hepatic iron overload (grades 2-4) was present in 56% and 34.5% of male and female subjects, respectively; hepatic fibrosis (stages 2-4) in 18.4% and 5.4%; and cirrhosis in 5.6% and 1.9%. Hepatic fibrosis and cirrhosis correlated significantly with the hepatic iron concentration, and except in cases of cirrhosis, there was a 7.5-fold reduction in the mean fibrosis score after phlebotomy. All subjects with cirrhosis were asymptomatic. CONCLUSIONS: Screening for hemochromatosis in apparently healthy subjects homozygous for the C282Y mutation with or without a family history reveals comparable levels of hepatic iron overload and disease. Significant hepatic fibrosis is frequently found in asymptomatic subjects with hemochromatosis and, except when cirrhosis is present, is reversed by iron removal.

The molecular basis of neutral aminoacidurias.

Recent success in the molecular cloning and identification of apical neutral amino acid transporters has shed a new light on inherited neutral amino acidurias, such as Hartnup disorder and Iminoglycinuria. Hartnup disorder is caused by mutations in the neutral amino acid transporter B(0) AT1 (SLC6A19). The transporter is found in kidney and intestine, where it is involved in the resorption of all neutral amino acids. The molecular defect underlying Iminoglycinuria has not yet been identified. However, two transporters, the proton amino acid transporter PAT1 (SLC36A1) and the IMINO transporter (SLC6A20) appear to play key roles in the resorption of glycine and proline. A model is presented, involving all three transporters that can explain the phenotypic variability of iminoglycinuria.

Characterization of the monomethylarsonate reductase and dehydroascorbate reductase activities of Omega class glutathione transferase variants: implications for arsenic metabolism and the age-at-onset of Alzheimer's and Parkinson's diseases.

There are two functional Omega class glutathione transferase (GST) genes in humans. GSTO1 is polymorphic with several coding region alleles, including an A140D substitution, a potential deletion of E155 and an E208K substitution. GSTO2 is also polymorphic with an N142D substitution in the coding region. We investigated the effect of these variations on the enzyme's thioltransferase, dehydroascorbate reductase, monomethylarsonate reductase and dimethylarsonate reductase activities. Variant proteins were expressed in Escherichia coli and purified by Ni-agarose affinity chromatography. GSTO2-2 was insoluble and had to be dissolved and refolded from 8 M urea. The A140D and E208K substitutions in GSTO1-1 did not alter specific activity. The deletion of E155 caused a two- to three-fold increase in the specific activity with each substrate. This deletion also caused a significant decrease in the enzyme's heat stability. The E155 deletion has been linked to abnormal arsenic excretion patterns; however, the available data do not clearly identify the cause of this abnormality. We found that GSTO2-2 has activity with the same substrates as GSTO1-1, and the dehydroascorbate reductase activity of GSTO2-2 is approximately 70-100-fold higher than that of GSTO1-1. The polymorphic N142D substitution had no effect on the specific activity of the enzyme with any substrate. The most notable feature of GSTO2-2 was its very high dehydroascorbate reductase activity, which suggests that GSTO2-2 may significantly protect against oxidative stress by recycling ascorbate. A defect in ascorbate metabolism may provide a common mechanism by which the Omega class GSTs influence the age-at-onset of Alzheimer's and Parkinson's diseases.

Lack of association of the CD14 promoter polymorphism--159C/T with Caucasian inflammatory bowel disease.

OBJECTIVE: The inflammatory bowel diseases (IBDs), including Crohn's disease (CD) and ulcerative colitis (UC), are multifactorial diseases resulting from a complex interaction of genetic and environmental factors. The recently described CARD15 and TNF-alpha risk alleles are believed to be contributors to disease by disrupting inflammatory pathways via impaired response to bacteria. Other bacterial receptors, such as CD14, may also have a role in disease. A promoter polymorphism (-159C/T) in CD14 has been implicated in IBD in a number of studies. MATERIAL AND METHODS: We have analysed this CD14 promoter polymorphism in probands from 206 multiplex IBD families, 110 sporadic IBD individuals and 189 healthy controls from the Australian population, all of whom are Caucasian. RESULTS: We could not replicate the described association between the CD14-159T allele and CD or UC, nor did we find any evidence for an interaction between the CARD15 or TNF-alpha risk alleles and the CD14-159T allele. CONCLUSIONS: It is possible that the association seen in other studies may be due to population stratification or to the CD14 polymorphism being in linkage with the real disease-causing variant(s).

The IBD international genetics consortium provides further evidence for linkage to IBD4 and shows gene-environment interaction.

BACKGROUND AND AIMS: The inflammatory bowel diseases (IBDs) Crohn's disease (CD) and ulcerative colitis are complex disorders with an important genetic determinant. One gene associated with CD has been identified: NOD2/CARD15. Two independent genome-wide scans found significant evidence (logarithm of odds [LOD] 3.6) and suggestive evidence (LOD 2.8) for linkage on locus 14q11-12, also known as the IBD4 locus. To further characterize this locus, we assessed gene-environment interaction (IBD4 x smoking) and phenotypic heterogeneity in a large cohort of IBD-affected sibling pairs as part of an ongoing international collaborative effort. PATIENTS AND METHODS: A total of 733 IBD families, comprising 892 affected sibling pairs, were genotyped for microsatellites D14S261, D14S283, D14S972, and D14S275, spanning the IBD4 locus. Information on gender, ethnicity, age at onset, smoking at diagnosis, extraintestinal manifestations, and disease location was available. RESULTS: A significant distortion in the mean allele sharing (MAS) between affected siblings was observed for CD patients only at each of the four markers (54.6%, 52.8%, 50.4%, and 53.3%, respectively). Maximum linkage for CD was observed at marker D14S261 (multipoint nonparametric linkage score 2.36; P

Hartnup disorder is caused by mutations in the gene encoding the neutral amino acid transporter SLC6A19.

Hartnup disorder (OMIM 234500) is an autosomal recessive abnormality of renal and gastrointestinal neutral amino acid transport noted for its clinical variability. We localized a gene causing Hartnup disorder to chromosome 5p15.33 and cloned a new gene, SLC6A19, in this region. SLC6A19 is a sodium-dependent and chloride-independent neutral amino acid transporter, expressed predominately in kidney and intestine, with properties of system B(0). We identified six mutations in SLC6A19 that cosegregated with disease in the predicted recessive manner, with most affected individuals being compound heterozygotes. The disease-causing mutations that we tested reduced neutral amino acid transport function in vitro. Population frequencies for the most common mutated SLC6A19 alleles are 0.007 for 517G --> A and 0.001 for 718C --> T. Our findings indicate that SLC6A19 is the long-sought gene that is mutated in Hartnup disorder; its identification provides the opportunity to examine the inconsistent multisystemic features of this disorder.

IBD International Genetics Consortium: international cooperation making sense of complex disease.

The Inflammatory Bowel Disease International Genetic Consortium was formed in Oxford in 1997. Since then it has grown to include twelve groups from around the world that are each actively involved in identifying the genes that are involved in susceptibility to IBD. The approach of the IBDIGC is to attempt to overcome one of the major issues in complex disease analysis-that of obtaining sufficient power to analyze successfully the inheritance of IBD-by collaboratively studying large numbers of well documented families with multiple affected individuals. This strategy has been marked by considerable success with the publication of a paper authored by the IBDIGC substantiating the localization of IBD1 to chromosome 16. This publication served to encourage researchers and eventually resulted in the identification by several groups simultaneously of risk alleles in the NOD2 gene that cosegregate with disease. The IBDIGC provides a model for studies in complex disease genetics, showing that research groups both large and small can participate equally in complex disease gene identification through the formation of large international consortia.

Genetic testing for HFE hemochromatosis in Australia: the value of testing relatives of simple heterozygotes.

BACKGROUND: It is unclear whether screening of relatives of C282Y and H63D heterozygotes (other than compound heterozygotes) for hemochromatosis will detect sufficient numbers of cases to justify introduction of this screening strategy. METHODS: Conditional probabilities were determined using published Australian allele frequencies and penetrance data to determine the detection rate of hemochromatosis by testing the siblings and offspring of heterozygotes (subjects with only one HFE mutation). RESULTS: The number of individuals who are at risk of developing increased body iron stores because of HFE mutations is substantially higher (1 in 80) than previously estimated. In addition, 33% of the Australian population are heterozygous for either C282Y or H63D. Based on population estimates, the relative risk to the offspring of C282Y and H63D heterozygotes of developing increased iron stores is 4.1 and 1.5, respectively, while the relative risk to each sibling is 2.3 and 1, respectively. The risk of developing clinical features of hemochromatosis or hepatic fibrosis is likely to be substantially lower. CONCLUSIONS: Although the detection rate from testing the families of unaffected heterozygotes is low, this can be justified as a clinically useful screening strategy. At the present time this strategy should be restricted to first-degree relatives of heterozygotes. Further studies are recommended to determine if cascade genetic screening is a cost-effective alternative to general population screening.