Persisting chemosensory dysfunction in COVID-19 - a cross-sectional population-based survey.

BACKGROUND: Chemosensory dysfunction (CD) has been reported as a common symptom of SARS-CoV-2 infection, but it is not well understood whether and for how long changes of smell, taste and chemesthesis persist in infected individuals. METHODOLOGY: Unselected adult residents of the German federal state of Schleswig-Holstein with Polymerase Chain Reaction (PCR)-test-confirmed SARS-CoV-2 infection were invited to participate in this large cross-sectional study. Data on the medical history and subjective chemosensory function of participants were obtained through questionnaires and visual analogue scales (VAS). Olfactory function (OF) was objectified with the Sniffin Sticks test (SST), including threshold (T), discrimination (D) and identification (I) test as well as summarized TDI score, and compared to that in healthy controls. Gustatory function (GF) was evaluated with the suprathreshold taste strips (TS) test, and trigeminal function was tested with an ampoule containing ammonia. RESULTS: Between November 2020 and June 2021, 667 infected individuals (mean age: 48.2 years) were examined 9.1 months, on average, after positive PCR testing. Of these, 45.6% had persisting subjective olfactory dysfunction (OD), 36.2% had subjective gustatory dysfunction (GD). Tested OD, tested GD and impaired trigeminal function were observed in 34.6%, 7.3% and 1.8% of participants, respectively. The mean TDI score of participants was significantly lower compared to healthy subjects. Significant associations were observed between subjective OD and GD, and between tested OD and GD. CONCLUSION: Nine months after SARS-CoV-2 infection, OD prevalence is significantly increased among infected members of the general population. Therefore, OD should be included in the list of symptoms collectively defining Long-COVID.

Long-term health sequelae and quality of life at least 6 months after infection with SARS-CoV-2: design and rationale of the COVIDOM-study as part of the NAPKON population-based cohort platform (POP).

PURPOSE: Over the course of COVID-19 pandemic, evidence has accumulated that SARS-CoV-2 infections may affect multiple organs and have serious clinical sequelae, but on-site clinical examinations with non-hospitalized samples are rare. We, therefore, aimed to systematically assess the long-term health status of samples of hospitalized and non-hospitalized SARS-CoV-2 infected individuals from three regions in Germany. METHODS: The present paper describes the COVIDOM-study within the population-based cohort platform (POP) which has been established under the auspices of the NAPKON infrastructure (German National Pandemic Cohort Network) of the national Network University Medicine (NUM). Comprehensive health assessments among SARS-CoV-2 infected individuals are conducted at least 6 months after the acute infection at the study sites Kiel, Würzburg and Berlin. Potential participants were identified and contacted via the local public health authorities, irrespective of the severity of the initial infection. A harmonized examination protocol has been implemented, consisting of detailed assessments of medical history, physical examinations, and the collection of multiple biosamples (e.g., serum, plasma, saliva, urine) for future analyses. In addition, patient-reported perception of the impact of local pandemic-related measures and infection on quality-of-life are obtained. RESULTS: As of July 2021, in total 6813 individuals infected in 2020 have been invited into the COVIDOM-study. Of these, about 36% wished to participate and 1295 have already been examined at least once. CONCLUSION: NAPKON-POP COVIDOM-study complements other Long COVID studies assessing the long-term consequences of an infection with SARS-CoV-2 by providing detailed health data of population-based samples, including individuals with various degrees of disease severity. TRIAL REGISTRATION: Registered at the German registry for clinical studies (DRKS00023742).

Serum metabolomic profiling highlights pathways associated with liver fat content in a general population sample.

BACKGROUND/OBJECTIVES: Fatty liver disease (FLD) is an important intermediate trait along the cardiometabolic disease spectrum and strongly associates with type 2 diabetes. Knowledge of biological pathways implicated in FLD is limited. An untargeted metabolomic approach might unravel novel pathways related to FLD. SUBJECTS/METHODS: In a population-based sample (n=555) from Northern Germany, liver fat content was quantified as liver signal intensity using magnetic resonance imaging. Serum metabolites were determined using a non-targeted approach. Partial least squares regression was applied to derive a metabolomic score, explaining variation in serum metabolites and liver signal intensity. Associations of the metabolomic score with liver signal intensity and FLD were investigated in multivariable-adjusted robust linear and logistic regression models, respectively. Metabolites with a variable importance in the projection >1 were entered in in silico overrepresentation and pathway analyses. RESULTS: In univariate analysis, the metabolomics score explained 23.9% variation in liver signal intensity. A 1-unit increment in the metabolomic score was positively associated with FLD (n=219; odds ratio: 1.36; 95% confidence interval: 1.27-1.45) adjusting for age, sex, education, smoking and physical activity. A simplified score based on the 15 metabolites with highest variable importance in the projection statistic showed similar associations. Overrepresentation and pathway analyses highlighted branched-chain amino acids and derived gamma-glutamyl dipeptides as significant correlates of FLD. CONCLUSIONS: A serum metabolomic profile was associated with FLD and liver fat content. We identified a simplified metabolomics score, which should be evaluated in prospective studies.

Five years of experience with the FiLaC™ laser for fistula-in-ano management: long-term follow-up from a single institution.

BACKGROUND: There are limited data available concerning endofistular therapies for fistula-in-ano, with our group reporting the first preliminary outcomes of the use of the radial fibre Fistula laser Closing (FiLaC ™) device. METHODS: The aim of this study was to assess a cohort of anal fistulae managed with laser ablation plus definitive flap closure of the internal fistula opening over a long-term follow-up. Factors governing primary healing success and secondary healing success (i.e. success after one or two operations) were determined. RESULTS: The study analysed 117 patients over a median follow-up period of 25.4 months (range 6-60 months) with 13 patients (11.1%) having Crohn's-related fistulae. No incontinence to solid and liquid stool was reported. Minor incontinence to mucus and gas was observed in two cases (1.7%), and a late abscess treated in one case (0.8%). The primary healing rate was 75/117 (64.1%) overall, and 63.5% for cryptoglandular fistulae versus 69.2% for Crohn's fistulae, respectively. Of the 42 patients who failed FiLaC™ 31 underwent a second operation ("Re-FiLaC™", fistulectomy with sphincter reconstruction or fistulotomy). The secondary healing rate, defined as healing of the fistula at the end of the study period, was 103/117 (88.0%) overall and 85.5% for cryptoglandular fistulae versus 92.3% for Crohn's fistulae. A significantly higher primary success rate was observed for intersphincteric-type fistulae with primary and secondary outcome unaffected by age, gender, presence of Crohn's disease, number of prior surgeries and the type of flap designed to close the internal fistula opening. CONCLUSIONS: There is a moderate primary success rate using first-up FiLaC™ treatment. If FiLaC™ fails, secondary success with repeat FiLaC™ or other approaches was high. The minimally invasive FiLaC™ approach may therefore represent a sensible first-line treatment option for anal fistula repair.

DNA Commission of the International Society for Forensic Genetics: Recommendations on the validation of software programs performing biostatistical calculations for forensic genetics applications.

The use of biostatistical software programs to assist in data interpretation and calculate likelihood ratios is essential to forensic geneticists and part of the daily case work flow for both kinship and DNA identification laboratories. Previous recommendations issued by the DNA Commission of the International Society for Forensic Genetics (ISFG) covered the application of bio-statistical evaluations for STR typing results in identification and kinship cases, and this is now being expanded to provide best practices regarding validation and verification of the software required for these calculations. With larger multiplexes, more complex mixtures, and increasing requests for extended family testing, laboratories are relying more than ever on specific software solutions and sufficient validation, training and extensive documentation are of upmost importance. Here, we present recommendations for the minimum requirements to validate bio-statistical software to be used in forensic genetics. We distinguish between developmental validation and the responsibilities of the software developer or provider, and the internal validation studies to be performed by the end user. Recommendations for the software provider address, for example, the documentation of the underlying models used by the software, validation data expectations, version control, implementation and training support, as well as continuity and user notifications. For the internal validations the recommendations include: creating a validation plan, requirements for the range of samples to be tested, Standard Operating Procedure development, and internal laboratory training and education. To ensure that all laboratories have access to a wide range of samples for validation and training purposes the ISFG DNA commission encourages collaborative studies and public repositories of STR typing results.

[PopGen. A population-based biobank with prospective follow-up of a control group]. / PopGen. Eine populationsbasierte Biobank mit Langzeitverfolgung der Kontrollkohorte.

The PopGen biobank was established in 2003 for research into the genetic risk factors for complex diseases. In addition to specific patient groups, a random sample from the general population was recruited as well. This cross-sectional study was mainly used as a control sample in genetic-epidemiological research. Recently, this part of the biobank was developed further into a prospective cohort study to investigate longitudinal changes of inflammatory biomarkers. Between 2005 and 2007, 1,317 study participants (age: 19-77 years, 55% men) were recruited into the PopGen control cohort, 747 via the local population registry and 570 as blood donors. Baseline assessment comprised a questionnaire, a medical examination, sampling of blood as well as ad hoc analyses of a range of biomarkers and genetic factors. Beginning in 2010, all study participants were re-invited for a program that was expanded by a food frequency questionnaire, collection of urine and stool samples, and an offer of magnetic resonance imaging to capture body fat distribution. The current response proportion is approximately 70%. The development of the PopGen controls into a prospective cohort study was originally driven by biomarker research questions, for instance related to chronic inflammation. Future follow-up will now also enable research into the influence of lifestyle variables.

The interpretation of lineage markers in forensic DNA testing.

Mitochondrial DNA (mtDNA) and the non-recombining portion of the Y-chromosome are inherited matrilinealy and patrilinealy, respectively, and without recombination. Collectively they are termed 'lineage markers'. Lineage markers may be used in forensic testing of an item, such as a hair from a crime scene, against a hypothesised source, or in relationship testing. An estimate of the evidential weight of a match is usually provided by a count of the occurrence in some database of the mtDNA or Y-STR haplotype under consideration. When the factual statement of a count in the database is applied to a case, issues of relevance of the database and sampling uncertainty may arise. In this paper, we re-examine the issues of sampling uncertainty, the relevance of the database, and the combination of autosomal and lineage marker evidence. We also review the recent developments by C.H. Brenner.

Genetic studies of common types of obesity: a critique of the current use of phenotypes.

Recent research into the genetic basis of obesity has focused upon the study of candidate genes, both functional and positional, of genes underlying weight-related Mendelian disorders and of susceptibility loci identified in genome-wide association studies. Three large genome-wide association studies on obesity, together involving more than 150,000 individuals, were published in Nature Genetics last year. The results suggested the involvement of a large number of genetic variants in disease susceptibility. Most genetic effects upon body weight are likely to become obscured by the use of inappropriate phenotypes. In particular, clinical categories such as the body mass index and Metabolic Syndrome do not provide sufficient etiological information for them to be used sensibly in genetic studies on obesity or obesity-related disease. Alleviation of this situation will not come from new genomic research tools, sophisticated statistical algorithms or ever larger sample sizes. Instead, the above notions argue in favour of so-called 'deep phenotyping'.

Examination of the current top candidate genes for AD in a genome-wide association study.

With the advent of technologies that allow simultaneous genotyping of thousands of single-nucleotide polymorphisms (SNPs) across the genome, the genetic contributions to complex diseases can be explored at an unprecedented detail. This study is among the first to apply the genome-wide association study (GWAS) approach to Alzheimer disease (AD). We present our GWAS results from the German population for genes included in the 'Top Results' list on the AlzGene database website. In addition to the apolipoprotein E locus, we identified nominally significant association signals in six of the ten genes investigated, albeit predominantly for SNPs other than those already published as being disease associated. Further, all of the four AD genes previously identified through GWAS also showed nominally significant association signals in our data. The results of our comparative study reinforce the necessity for replication and validation, not only of GWAS but also of candidate gene case-control studies, in different populations. Furthermore, cross-platform comparison of genotyping results can also identify new association signals. Finally, our data confirm that GWAS, regardless of the platform, are valuable for the identification of genetic variants associated with AD.

A German Chlamydia trachomatis screening program employing semi-automated real-time PCR: results and perspectives.

BACKGROUND: Genital Chlamydia trachomatis infection is a worldwide public health burden. A screening program for C. trachomatis was therefore initiated by the public health insurers in Germany ("Gemeinsamer Bundesausschuss", GBA) in April 2008. OBJECTIVES: To estimate C. trachomatis prevalence from screening 115,766 asymptomatic females and 20,033 female patients with unspecific abdominal pain. STUDY DESIGN: Urine samples (pooled by five for the asymptomatic screening subjects) and cervical swabs were analyzed using semi-automated real-time PCR. Infection prevalence was determined separately in four categories of women, defined by health status (asymptomatic screening vs. non-screening with unspecified symptoms) and test material used. Comparative analyses were stratified by age and pregnancy status. RESULTS: Experimental evaluation of the assay used revealed a detection limit of 379 genome copies/ml urine. For pooled urine samples, the positive predictive value was 100% whereas the negative predictive value equaled 98.1%. The observed infection prevalence was higher for cervical swabs than for urine samples. Prevalence estimates also differed significantly between pregnant and non-pregnant adolescents (< or = 20 years), irrespective of the test material used (10.2% vs. 7.3% for cervical swabs, 10.9% vs. 6.1% for pooled urine samples). CONCLUSIONS: Our retrospective study, based upon a very large number of females from all parts of Germany, revealed a high infection prevalence in adolescents, particularly in pregnant adolescents, thereby justifying the screening directive of the German GBA.

Mhc class I haplotypes associated with survival time in simian immunodeficiency virus (SIV)-infected rhesus macaques.

In both human immunodeficiency virus-infected humans and simian immunodeficiency virus (SIV)-infected macaques, genes encoded in the major histocompatibility complex (MHC) class I region are important determinants of disease progression. However, compared to the human human lymphocyte antigen complex, the macaque MHC region encodes many more class I genes. Macaques with the same immunodominant class I genes express additional Mhc genes with the potential to influence the disease course. We therefore assessed the association between of the Mhc class I haplotypes, rather than single gene variants, and survival time in SIV-infected rhesus macaques (Macaca mulatta). DNA sequence analysis and Mhc genotyping of 245 pedigreed monkeys identified 17 Mhc class I haplotypes that constitute 10 major genotypes. Among 81 vaccination-naive, SIV-infected macaques, 71 monkeys carried at least one Mhc class I haplotype encoding only MHC antigens that were incapable of inducing an effective anti-SIV cytotoxic T lymphocytes response. Study of these macaques enabled us to relate individual Mhc class I haplotypes to slow, medium and rapid disease progression. In a post hoc analysis, classification according to disease progression was found to explain at least 48% of the observed variation of survival time.

Age and origin of major Smith-Lemli-Opitz syndrome (SLOS) mutations in European populations.

BACKGROUND: Smith-Lemli-Opitz syndrome (SLOS) (MIM 270 400) is an autosomal recessive multiple congenital anomalies/mental retardation syndrome caused by mutations in the Delta7-sterol reductase (DHCR7, E.C.1.3.1.21) gene. The prevalence of SLOS has been estimated to range between 1:15000 and 1:60000 in populations of European origin. METHODS AND RESULTS: We have analysed the frequency, origin, and age of DHCR7 mutations in European populations. In 263 SLOS patients 10 common alleles (c.964-1G>C, p.Trp151X, p.Thr93Met, p.Val326Leu, p.Arg352Trp, p.Arg404Cys, p.Phe302Leu, p.Leu157Pro, p.Gly410Ser, p.Arg445Gln) were found to constitute approximately 80% of disease-causing mutations. As reported before, the mutational spectra differed significantly between populations, and frequency peaks of common mutations were observed in North-West (c.964-1G>C), North-East (p.Trp151X, p.Val326Leu) and Southern Europe (p.Thr93Met). SLOS was virtually absent from Finland. The analysis of nearly 8000 alleles from 10 different European populations confirmed a geographical distribution of DHCR7 mutations as reported in previous studies. The common Null mutations in Northern Europe (combined ca. 1:70) occurred at a much higher frequency than expected from the reported prevalence of SLOS. In contrast the most common mutation in Mediterranean SLOS patients (p.Thr93Met) had a low population frequency. Haplotypes were constructed for SLOS chromosomes, and for wild-type chromosomes of African and European origins using eight cSNPs in the DHCR7 gene. The DHCR7 orthologue was sequenced in eight chimpanzees (Pan troglodytes) and three microsatellites were analysed in 50 of the SLOS families in order to estimate the age of the three major SLOS-causing mutations. CONCLUSIONS: The results indicate a time of first appearance of c.964-1G>C and p.Trp151X some 3000 years ago in North-West and North-East Europe, respectively. The p.Thr93Met mutations on the J haplotype has probably first arisen approximately 6000 years ago in the Eastern Mediterranean. Together, it appears that a combination of founder effects, recurrent mutations, and drift have shaped the present frequency distribution of DHCR7 mutations in Europe.

Common familial influences on clustering of metabolic syndrome traits with central obesity and insulin resistance: the Kiel obesity prevention study.

OBJECTIVE: The phenotypic heterogeneity of metabolic syndrome (MSX) suggests heterogeneity of the underlying genotype. The aim of the present study was to examine the common genetic background that contributes to the clustering between the two main features (insulin resistance, central obesity) and different MSX component traits. METHODS: In all, 492 individuals from 90 families were investigated in a three-generation family path study as part of the Kiel Obesity Prevention Study (KOPS, 162 grandparents, 66.1+/-6.7 years, 173 parents, 41.3+/-5.4 years and 157 children, 10.8+/-3.4 years). Overall heritability was estimated and common familial (genetic and environmental) influences on insulin resistance (HOMA-IR) or central obesity (elevated waist circumference, WC), respectively, and different MSX traits were compared in a bivariate cross-trait correlation model. RESULTS: Prevalence of MSX (according to NCEP criteria) was 27.2% (f) and 27.8% (m) in adults and 3.5% (f) and 8.5% (m) in children and adolescents, respectively. MSX phenotype was found to be highly variable, comprising 16 subtypes of component trait combinations. Within-trait heritability was 38.5% for HOMA-IR and 53.5% for WC, cross-trait heritability was 53.4%. As much as 6-18% and 3-10% of the shared variance between different MSX component traits (lipid profile, blood pressure) and WC or HOMA-IR, respectively, may be genetic. With the exception of HDL-C, the shared genetic variance between MSX component traits and WC was higher than the genetic variance shared with HOMA-IR. CONCLUSION: A common genetic background contributes to the clustering of different MSX component traits and central obesity or insulin resistance. Common genetic influences favour central obesity as a major characteristic linking these traits.

Consequences of group fission for the patterns of relatedness among rhesus macaques.

When mammalian social groups exceed their optimal size, they often tend to split. In view of the potential evolutionary benefits, it should be more advantageous for animals to stay with kin, rather than nonkin, during such fission events. In the present study, the spontaneous fission of two social groups, R and S, of rhesus macaques living on Cayo Santiago, Puerto Rico, provided the opportunity to compare the kinship structure of the corresponding parent and daughter groups, using information on both maternal and paternal relatedness. In both instances, maternal half-siblings and pairs of animals from the same family were significantly more prevalent in the fission products than in the parent group. During the split of group R, significantly more paternal half-siblings stayed in the remnants of the parent group than joined the seceding group. Our findings are compatible with previous behavioural studies demonstrating that female primates bias their social behaviour more to maternal than to paternal kin, but that both types of half-siblings prefer each other more than unrelated animals. It remains to be clarified by future research, however, whether the observed co-segregation of paternal half-sibs in our study reflects active choice or is a by-product of the group-specific kin structures, prior to fission.

DNA commission of the International Society of Forensic Genetics: Recommendations on the interpretation of mixtures.

The DNA commission of the International Society of Forensic Genetics (ISFG) was convened at the 21st congress of the International Society for Forensic Genetics held between 13 and 17 September in the Azores, Portugal. The purpose of the group was to agree on guidelines to encourage best practice that can be universally applied to assist with mixture interpretation. In addition the commission was tasked to provide guidance on low copy number (LCN) reporting. Our discussions have highlighted a significant need for continuing education and research into this area. We have attempted to present a consensus from experts but to be practical we do not claim to have conveyed a clear vision in every respect in this difficult subject. For this reason, we propose to allow a period of time for feedback and reflection by the scientific community. Then the DNA commission will meet again to consider further recommendations.

Iron loading and morbidity among relatives of HFE C282Y homozygotes identified either by population genetic testing or presenting as patients.

BACKGROUND AND AIMS: Although most cases of hereditary haemochromatosis are associated with homozygosity for the C282Y mutation of the HFE gene, clinical penetrance varies and other genes may modify disease expression. If so, relatives from clinically affected families, by inheriting such genes, may accumulate more iron. To seek evidence for this, we compared iron status and morbidity in unselected first degree relatives of two groups of index cases from South Wales, namely asymptomatic C282Y homozygotes identified by genetic screening of blood donors (n = 56) and C282Y homozygous haemochromatosis patients presenting clinically (n = 60). METHODS: All participating relatives had a structured interview, clinical assessment, and laboratory investigations. Health related quality of life was measured (SF-36 version 2). RESULTS: In total, 92% of 180 eligible first degree relatives were interviewed in the "screened" family group and 85% of 143 eligible relatives in the "patient" group. Of 59 relatives homozygous for C282Y, 76% of men and 32% of women had the "iron phenotype" (raised transferrin saturation and serum ferritin). Logistic regression modelling of the iron phenotype risk showed that 42% of the initial model deviance could be explained by homozygosity for C282Y, another 6% by lifestyle factors, and 6% by being male. Family group membership was not a significant risk factor. Morbidity and SF-36 scores did not differ significantly either between C282Y homozygotes and relatives lacking C282Y, or between C282Y homozygotes from the "screened" and "patient" groups. Serious morbidity (including cirrhosis) was low in both groups of relatives. CONCLUSIONS: HFE C282Y homozygosity has a high penetrance for iron accumulation but a low clinical penetrance. Lack of excess morbidity among C282Y homozygous relatives of index cases who presented clinically suggests that residual unknown genetic or environmental factors do not greatly influence clinical outcome among C282Y homozygotes.

Use of X-linked markers for forensic purposes.

In forensic science, X-chromosomal short tandem repeats (ChrX STRs) bear the potential to efficiently complement the analysis of other genetic markers (autosomal, Y-chromosomal or mitochondrial). We review the population genetic properties and forensic utility of selected ChrX markers, and discuss the problems and limitations arising with their practical use. Formulae required to assess the evidential power of individual markers in different contexts are summarised and applied to ChrX STRs of interest. Since linkage and linkage disequilibrium between markers affect the inferential interpretation of genotype data, practically relevant information regarding the co-localisation and haplotypic association of ChrX STRs is provided. Finally, two examples of complex kinship testing are presented which serve to highlight the particular importance of ChrX STRs for solving deficiency cases and cases involving blood relatives.

Linkage disequilibrium mapping provides further evidence of a gene for reading disability on chromosome 6p21.3-22.

Linkage disequilibrium (LD) mapping was used to follow up reports of linkage between reading disability (RD) and an 18 cM region of chromosome 6p21.3-22. Using a two-stage approach, we tested for association between RD and 22 microsatellite markers in two independent samples of 101 (Stage 1) and 77 (Stage 2) parent/proband trios in which RD was rigorously defined. The most significant replicated associations were observed between combinations of markers D6S109/422/1665 (Stage 1, P=0.002 (adjusted for multiple testing); Stage 2, P=0.0001) and D6S506/1029/1660 (Stage 1, P=0.02 (adjusted), Stage 2, P=0.0001). The only two-marker association observed in both samples was with D6S422/1665 (P=0.01, 0.04). No single marker showed replicated association but D6S506 produced values of P=0.01 and 0.08 which were significant when combined (P=0.02). We observed weaker and less consistent evidence of association in a region of confirmed linkage to RD in previous studies. The most consistently significant haplotypic association D6S109/422/1665, showed association with single-word reading, spelling, phonological awareness, phonological decoding, orthographic accuracy and random automised naming, but not with vocabulary or Attention Deficit Hyperactivity Disorder. Our findings strongly support the presence of a gene contributing to RD in a region of chromosome 6 between markers D6S109 and D6S1260, but do not rule out the presence of a gene between D6S1556 and MOG.

Mutation screening and LD mapping in the VCFS deleted region of chromosome 22q11 in schizophrenia using a novel DNA pooling approach.

We examined whether variation within six genes from the VCFS critical region at 22q11 (DGSC, Stk22A1, DGSI, Gscl, Slc25A1 and Znf74) confers susceptibility to schizophrenia. We screened the exons and flanking intronic sequence of each gene for mutations in 14 individuals with DSM-IV schizophrenia using DHPLC. All polymorphisms identified were characterised and genotyped in a sample of 184 schizophrenics and matched controls, using novel DNA pooling methods. Of the polymorphisms identified, 17 were located within exons, six were within coding sequence, and two were non-synonymous. Pooled genotyping revealed no differences in the allele frequencies for any polymorphism between cases and controls that met our pre-defined criterion (P < or = 0.1). In a complementary approach we also attempted to define the location of a schizophrenia susceptibility locus more precisely by performing association mapping using seven microsatellites spanning the VCFS region with an average inter-marker distance of 450 kb. Conventional chi(2) analysis of genotypes in 368 cases and 368 controls revealed that none of the markers was significantly associated (P < 0.05) with schizophrenia. However, evidence for significant association (P = 0.003) was obtained for D22S944 when alleles were combined. TDT analysis of D22S944 genotyped in a further 278 cases of schizophrenia and their parents failed to find any overall allele-wise significant transmission disequilibrium (chi(2) = 18.3, P = 0.17). However, individual analysis of the alleles revealed that allele 12 was excessively non-transmitted and that this almost reached significance when corrected for multiple alleles (chi(2) = 7.35, P = 0.006, P = 0.078 corrected for 13 alleles).