No evidence for the presence of genetic variants predisposing to psychotic disorders on the non-deleted 22q11.2 allele of VCFS patients.

The velo-cardio-facial syndrome (VCFS) is caused by hemizygous deletions on chromosome 22q11.2. The VCFS phenotype is complex and characterized by frequent occurrence of neuropsychiatric symptoms with up to 25-30% of cases suffering from psychotic disorders compared with only ~1% in the general population (odds ratio≈20-25). This makes the 22q11.2 deletion one of the most prominent risk factors for schizophrenia. However, its penetrance for neuropsychiatric phenotypes is incomplete suggesting that additional risk factors are required for disease development. These additional risk factors could lie anywhere on the genome, but by reducing the normal diploid to a haploid state, the 22q11.2 deletion could result in the unmasking of otherwise recessive alleles or functional variants on the non-deleted 22q11.2 allele. To test this hypothesis, we captured and sequenced the whole 22q11.2 non-deleted region in 88 VCFS patients with (n=40) and without (n=48) psychotic disorders to identify genetic variation that could increase the risk for schizophrenia. Single nucleotide variants (SNVs), small insertions/deletions (indels) and copy number variants were called and their distributions were compared between the two diagnostic groups using variant-, gene- and region-based association tests. None of these tests resulted in statistical evidence for the existence of a genetic variation in the non-deleted allele that would increase schizophrenia risk in VCFS patients. Power analysis showed that our study was able to achieve >80% statistical power to detect association of a risk variant with an odd ratio of ⩾22. However, it is certainly under-powered to detect risk variant of smaller effect sizes. Our study did not provide evidence that genetic variants of very large effect size located on the non-deleted 22q1.2 allele in VCFS patients increase the risk for developing psychotic disorders. Variants with smaller effects may be located in the remaining 22q11.2 allele and elsewhere in the genome. Therefore, whole exome or even genome sequencing for larger sample size would appear to be the next logical steps in the search for the genetic modifiers of the 22q11.2-deletion neuropsychiatric phenotype.

Experience of a multidisciplinary task force with exome sequencing for Mendelian disorders.

BACKGROUND: In order to optimally integrate the use of high-throughput sequencing (HTS) as a tool in clinical diagnostics of likely monogenic disorders, we have created a multidisciplinary "Genome Clinic Task Force" at the University Hospitals of Geneva, which is composed of clinical and molecular geneticists, bioinformaticians, technicians, bioethicists, and a coordinator. METHODS AND RESULTS: We have implemented whole exome sequencing (WES) with subsequent targeted bioinformatics analysis of gene lists for specific disorders. Clinical cases of heterogeneous Mendelian disorders that could potentially benefit from HTS are presented and discussed during the sessions of the task force. Debate concerning the interpretation of identified variants and the content of the final report constitutes a major part of the task force's work. Furthermore, issues related to bioethics, genetic counseling, quality control, and reimbursement are also addressed. CONCLUSIONS: This multidisciplinary task force has enabled us to create a platform for regular exchanges between all involved experts in order to deal with the multiple complex issues related to HTS in clinical practice and to continuously improve the diagnostic use of HTS. In addition, this task force was instrumental to formally approve the reimbursement of HTS for molecular diagnosis of Mendelian disorders in Switzerland.

Tunisian population data on 15 PCR-based loci.

Genotype and allele frequencies distribution for 15 PCR-based loci included in the Promega PowerPlex 16 kit were determined for a Tunisian population sample of 196 unrelated individuals.

Online reference database of European Y-chromosomal short tandem repeat (STR) haplotypes.

The reference database of highly informative Y-chromosomal short tandem repeat (STR) haplotypes (YHRD), available online at http://ystr.charite.de, represents the largest collection of male-specific genetic profiles currently available for European populations. By September 2000, YHRD contained 4688 9-locus (so-called "minimal") haplotypes, 40% of which have been extended further to include two additional loci. Establishment of YHRD has been facilitated by the joint efforts of 31 forensic and anthropological institutions. All contributing laboratories have agreed to standardize their Y-STR haplotyping protocols and to participate in a quality assurance exercise prior to the inclusion of any data. In view of its collaborative character, and in order to put YHRD to its intended use, viz. the support of forensic caseworkers in their routine decision-making process, the database has been made publicly available via the Internet in February 2000. Online searches for complete or partial Y-STR haplotypes from evidentiary or non-probative material can be performed on a non-commercial basis, and yield observed haplotype counts as well as extrapolated population frequency estimates. In addition, the YHRD website provides information about the quality control test, genotyping protocols, haplotype formats and informativity, population genetic analysis, literature references, and a list of contact addresses of the contributing laboratories.

An extensive analysis of Y-chromosomal microsatellite haplotypes in globally dispersed human populations.

The genetic variance at seven Y-chromosomal microsatellite loci (or short tandem repeats [STRs]) was studied among 986 male individuals from 20 globally dispersed human populations. A total of 598 different haplotypes were observed, of which 437 (73.1%) were each found in a single male only. Population-specific haplotype-diversity values were.86-.99. Analyses of haplotype diversity and population-specific haplotypes revealed marked population-structure differences between more-isolated indigenous populations (e.g., Central African Pygmies or Greenland Inuit) and more-admixed populations (e.g., Europeans or Surinamese). Furthermore, male individuals from isolated indigenous populations shared haplotypes mainly with male individuals from their own population. By analysis of molecular variance, we found that 76.8% of the total genetic variance present among these male individuals could be attributed to genetic differences between male individuals who were members of the same population. Haplotype sharing between populations, phi(ST) statistics, and phylogenetic analysis identified close genetic affinities among European populations and among New Guinean populations. Our data illustrate that Y-chromosomal STR haplotypes are an ideal tool for the study of the genetic affinities between groups of male subjects and for detection of population structure.

Axonemal beta heavy chain dynein DNAH9: cDNA sequence, genomic structure, and investigation of its role in primary ciliary dyskinesia.

Dyneins are multisubunit protein complexes that couple ATPase activity with conformational changes. They are involved in the cytoplasmatic movement of organelles (cytoplasmic dyneins) and the bending of cilia and flagella (axonemal dyneins). Here we present the first complete cDNA and genomic sequences of a human axonemal dynein beta heavy chain gene, DNAH9, which maps to 17p12. The 14-kb-long cDNA is divided into 69 exons spread over 390 kb. The cDNA sequence of DNAH9 was determined using a combination of methods including 5' rapid amplification of cDNA ends, RT-PCR, and cDNA library screening. RT-PCR using nasal epithelium and testis RNA revealed several alternatively spliced transcripts. The genomic structure was determined using three overlapping BACs sequenced by the Whitehead Institute/MIT Center for Genome Research. The predicted protein, of 4486 amino acids, is highly homologous to sea urchin axonemal beta heavy chain dyneins (67% identity). It consists of an N-terminal stem and a globular C-terminus containing the four P-loops that constitute the motor domain. Lack of proper ciliary and flagellar movement characterizes primary ciliary dyskinesia (PCD), a genetically heterogeneous autosomal recessive disorder with respiratory tract infections, bronchiectasis, male subfertility, and, in 50% of cases, situs inversus (Kartagener syndrome, KS). Dyneins are excellent candidate genes for PCD and KS because in over 50% of cases the ultrastructural defects of cilia are related to the dynein complex. Genotype analysis was performed in 31 PCD families with two or more affected siblings using a highly informative dinucleotide polymorphism located in intron 26 of DNAH9. Two families with concordant inheritance of DNAH9 alleles in affected individuals were observed. A mutation search was performed in these two "candidate families," but only polymorphic variants were found. In the absence of pathogenic mutations, the DNAH9 gene has been excluded as being responsible for autosomal recessive PCD in these families.

An apparently dominant bipolar affective disorder (BPAD) locus on chromosome 20p11.2-q11.2 in a large Turkish pedigree.

Bipolar affective disorder (BPAD), also known as manic-depressive illness, is a common complex, polygenic disorder characterised by recurrent cyclic episodes of mania and depression. Family, twin, and adoption studies strongly suggest a genetic predisposition/susceptibility to BPAD, but no genes have yet been identified. We studied a large Turkish pedigree, with an apparently autosomal dominant BPAD, which contained 13 affected individuals. The age of onset ranged from 15-40 with a mean of 25 years. The phenotypes consisted of recurrent manic and major depressive episodes, including suicidal attempts; there was usually full remission with lithium treatment. A genome-wide linkage analysis using a dominant mode of inheritance showed strong evidence for a BPAD susceptibility locus on chromosome 20p11.2-q11.2. The highest 2-point lod score of 4.34 at theta = 0 was obtained with markers D20S604, D20S470, D20S836 and D20S838 using a dominant model with full penetrance. Haplotype analysis enabled the mapping of the BPAD locus in this family between markers D20S186 and D20S109, to a region of approximately 42 cM.

Insertion of beta-satellite repeats identifies a transmembrane protease causing both congenital and childhood onset autosomal recessive deafness.

Approximately 50% of childhood deafness is caused by mutations in specific genes. Autosomal recessive loci account for approximately 80% of nonsyndromic genetic deafness. Here we report the identification of a new transmembrane serine protease (TMPRSS3; also known as ECHOS1) expressed in many tissues, including fetal cochlea, which is mutated in the families used to describe both the DFNB10 and DFNB8 loci. An 8-bp deletion and insertion of 18 monomeric (approximately 68-bp) beta-satellite repeat units, normally present in tandem arrays of up to several hundred kilobases on the short arms of acrocentric chromosomes, causes congenital deafness (DFNB10). A mutation in a splice-acceptor site, resulting in a 4-bp insertion in the mRNA and a frameshift, was detected in childhood onset deafness (DFNB8). This is the first description of beta-satellite insertion into an active gene resulting in a pathogenic state, and the first description of a protease involved in hearing loss.

No deleterious mutations in the FOXJ1 (alias HFH-4) gene in patients with primary ciliary dyskinesia (PCD).

The transcription factor FOXJ1 (alias HFH-4 or FKHL13) of the winged-helix/forkhead family is expressed in cells with cilia or flagella, and seems to be involved in the regulation of axonemal structural proteins. The knockout mouse Foxj1(-/-) shows abnormalities of organ situs, consistent with random determination of left-right asymmetry, and a complete absence of cilia. The human FOXJ1 gene which maps to chromosome 17q, is thus an excellent candidate gene for Kartagener Syndrome (KS), a subphenotype of Primary Ciliary Dyskinesia (PCD), characterized by bronchiectasis, chronic sinusitis and situs inversus. We have collected samples from 61 PCD families, in 31 of which there are at least two affected individuals. Two families with complete aciliogenesis, and six families, in which the affected members have microsatellite alleles concordant for a locus on distal chromosome 17q, were screened for mutations in the two exons and intron-exon junctions of the FOXJ1 gene. No sequence abnormalities were observed in the DNAs of the affected individuals of the selected families. These results demonstrate that the FOXJ1 gene is not responsible for the PCD/KS phenotype in the families examined.

Swiss allele frequencies and haplotypes of 7 Y-specific STRs.

In view of application to personal identification and paternal analysis, the allele distribution of the loci DYS 19, DYS389 I and II, DYS390, DYS391, DYS392, and DYS393 were determined in a sample of 126 unrelated males from the area of Bern (Switzerland). The 7 Y-STR loci were coamplified in a total of two multiplex reactions using fluorescently-labeled primers. PCR products were separated and detected on a capillary electrophoresis ABI Prism 310 instrument. All loci were polymorphic and the allele distributions are similar to other caucasian data.

Swiss Caucasian population data for the STR loci D2S1338 and D19S433 using the AmpFISTR SGM plus PCR amplification kit.

Allele and genotype frequencies for the ten STR loci D3S1358, VWA, D16S539, D2S1338, D8S1179, D21S11, D18S51, D19S433, TH01, FGA were determined in a Swiss Caucasian population sample (n=206) using the AmpFISTR SGM Plus Amplification kit. Electrophoresis was carried out on an ABI PRISM CE 310 Genetic Analyzer instrument. Previously, allele frequencies were published for the 13 STR loci D3S1358, VWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820, THO1, TPOX, CSF1PO and D16S539 for the same samples (n=206) amplified with the AmpFISTR Profiler Plus and Cofiler PCR Amplification kits. Since the results for the eight loci D3S1358, VWA, FGA, D8S1179, D21S11, D18S51, THO1, D16S539 shared between the AmpFISTR SGM Plus, Profiler Plus and Cofiler PCR Amplification kits already are published, only the allele frequencies for the two STR loci D2S1338 and D19S433 are reported in this paper. The two loci meet Hardy-Weinberg expectations. In addition, there is little evidence for association of alleles among the 15 loci (amplified with the Profiler, Cofiler, and SGM Plus amplification kits). The allelic frequency data can be used in forensic analyses to estimate the frequency of a multiple STR locus DNA profile in the Swiss population.

Capillary electrophoresis in clinical and forensic analysis: recent advances and breakthrough to routine applications.

This paper is a comprehensive review article on capillary electrophoresis (CE) in clinical and forensic analysis. It is based upon the literature of 1997 and 1998, presents CE examples in major fields of application, and provides an overview of the key achievements encountered, including those associated with the analysis of drugs, serum proteins, hemoglobin variants, and nucleic acids. For CE in clinical and forensic analysis, the past two years witnessed a breakthrough to routine applications. As most coauthors of this review are associated with diagnostic or forensic laboratories now using CE on a routine basis, this review also contains data from routine applications in drug, protein, and DNA analysis. With the first-hand experience of providing analytical service under stringent quality control conditions, aspects of quality assurance, assay specifications for clinical and forensic CE and the pros and cons of this maturing, cost-and pollution-controlled age technology are also discussed.

Swiss Caucasian population data for 13 STR loci using AmpFISTR profiler plus and cofiler PCR amplification kits.

Allele and genotype frequencies for the 13 core STR loci (D3S1358, VWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820, THO1, TPOX, CSF1PO, and D16S539) were determined in a Swiss Caucasian population sample (n = 206) using two commercially available multiplex PCR kits (AmpFISTR Profiler Plus and AmpFISTR Cofiler) and subsequent electrophoresis on an ABI PRISM CE 310 Genetic Analyzer instrument. All loci meet Hardy-Weinberg expectations. In addition, there is little evidence for association of alleles among the 13 loci. The allelic frequency data can be used in forensic analyses and paternity tests to estimate the frequency of a multiple STR locus DNA profile in the Swiss population.

Evaluation of prostate-specific antigen (PSA) membrane test assays for the forensic identification of seminal fluid.

Prostate specific antigen (PSA, also known as p30), a glycoprotein produced by the prostatic gland and secreted into seminal plasma, is a marker used for demonstrating the presence of seminal fluid. Methods for the detection of PSA include Ouchterlony double diffusion, crossover electrophoresis, rocket immuno-electrophoresis, radial immunodiffusion, and ELISA. The extremely sensitive ELISA technique can detect PSA in concentrations as low as approximately 4 ng/mL. However, all these techniques are cumbersome and time consuming to perform in forensic laboratories, especially when only a few samples per week are processed. Various membrane tests are currently used in clinical settings to screen a patient's serum for the presence of PSA at levels greater than 4 ng/mL. In this study we evaluated three immunochromatographic PSA membrane tests by analyzing semen stains stored at room temperature for up to 30 years, post-coital vaginal swabs taken at different time after intercourse, semen-free vaginal swabs, and various female and male body fluids, including urine. The data demonstrate that PSA membrane test assays offer the same sensitivity as ELISA-based tests and provide a rapid approach for the forensic identification of seminal fluid. Furthermore, when the supernatant from a DNA extraction is used for the assay, there is essentially no DNA consumption for determining the presence of PSA in a forensic sample.

Lack of linkage or association between schizophrenia and the polymorphic trinucleotide repeat within the KCNN3 gene on chromosome 1q21.

To determine the importance of a candidate gene KCNN3 (formerly named hSKCa3) in the susceptibility to schizophrenia, we have studied the genotypes of a (CAG)n polymorphism within this gene in the DNAs of the members of 54 multiplex families with this disease. Parametric and nonparametric linkage analysis did not provide evidence for linkage between KCNN3 (that we mapped to chromosome 1q21) and schizophrenia. Furthermore, we observed no difference in the distribution of the (CAG)n alleles between affected and normal individuals. These results do not support the hypothesis that larger KCNN3 alleles are preferentially associated with schizophrenia [Chandy et al. 1998 Mol Psychiatr 3:32-37] in individuals from multiply affected families.

Validation studies of an immunochromatographic 1-step test for the forensic identification of human blood.

An immunochromatographic 1-step test for the detection of fecal occult blood was evaluated for applicability for the forensic identification of human blood in stained material. The following experiments were conducted: 1) determination of the sensitivity and specificity of the assay; 2) evaluation of different extraction media for bloodstains (sterile water, Tris buffer pH 7.5 provided in the test kit, 5% ammonia); 3) analysis of biological samples subjected to a variety of environmental insults; and 4) evaluation of casework samples. This immunochromatographic 1-step occult blood test is specific for human (primate) hemoglobin and is at least an order of magnitude more sensitive than previous methods for detecting human hemoglobin in bloodstains. The antigen is insensitive to a variety of environmental insults, except for exposure to certain detergents and household bleaches and prolonged exposure to certain preparations of luminol. The entire assay can be conducted in field testing conditions within minutes. When in the laboratory the supernatant from a DNA extraction is used for the assay, there is essentially no consumption of DNA for determining the presence of human hemoglobin in a forensic sample. The data demonstrate that this test is robust and suitable for forensic analyses.

No evidence for linkage between schizophrenia and markers at chromosome 15q13-14.

Freedman et al. [1997: Proc Natl Acad Sci USA 94:587-592] reported linkage in nine multiplex schizophrenia families to markers on chromosome 15, using impaired neuronal inhibition to repeated auditory stimuli (P50), a neurophysiological deficit associated with schizophrenia, as the phenotype. The highest LOD score obtained (5.3 at theta = 0) was for marker D15S1360 mapped to chromosome 15q13-14, less than 120 kb from the alpha7-nicotinic receptor (CHRNA7) gene. The study also reported a small positive LOD score for D15S1360 when examined for linkage to the schizophrenia phenotype. Following these findings, we examined three polymorphic markers (D15S1360, L76630, and ACTC) on chromosome 15q13-14 near the CHRNA7 gene for linkage to schizophrenia, using 54 pedigrees from an independent study. Alleles for these three markers were genotyped and analyzed using parametric and nonparametric methods. No LOD score above 1.00 was obtained for any marker, and affected sib-pair analysis likewise showed no evidence for linkage. We conclude that in our families the region around the CHRNA7 locus does not contain a major locus for susceptibility to schizophrenia.

Loss of LKB1 kinase activity in Peutz-Jeghers syndrome, and evidence for allelic and locus heterogeneity.

Peutz-Jeghers syndrome (PJS) is an autosomal dominant disease characterized by mucocutaneous pigmentation and hamartomatous polyps. There is an increased risk of benign and malignant tumors in the gastrointestinal tract and in extraintestinal tissues. One PJS locus has been mapped to chromosome 19p13.3; a second locus is suspected on chromosome 19q13.4 in a minority of families. The PJS gene on 19p13.3 has recently been cloned, and it encodes the serine/threonine kinase LKB1. The gene, which is ubiquitously expressed, is composed of 10 exons spanning 23 kb. Several LKB1 mutations have been reported in heterozygosity in PJS patients. In this study, we screened for LKB1 mutations in nine PJS families of American, Spanish, Portuguese, French, Turkish, and Indian origin and detected seven novel mutations. These included two frameshift mutations, one four-amino-acid deletion, two amino-acid substitutions, and two splicing errors. Expression of mutant LKB1 proteins (K78I, D176N, W308C, and L67P) and assessment of their autophosphorylation activity revealed a loss of the kinase activity in all of these mutants. These results provide direct evidence that the elimination of the kinase activity of LKB1 is probably responsible for the development of the PJS phenotypes. In two Indian families, we failed to detect any LKB1 mutation; in one of these families, we previously had detected linkage to markers on 19q13.3-4, which suggests locus heterogeneity of PJS. The elucidation of the molecular etiology of PJS and the positional cloning of the second potential PJS gene will further elucidate the involvement of kinases/phosphatases in the development of cancer-predisposing syndromes.

Schizophrenia susceptibility loci on chromosomes 13q32 and 8p21.

Schizophrenia is a common disorder characterized by psychotic symptoms; diagnostic criteria have been established. Family, twin and adoption studies suggest that both genetic and environmental factors influence susceptibility (heritability is approximately 71%; ref. 2), however, little is known about the aetiology of schizophrenia. Clinical and family studies suggest aetiological heterogeneity. Previously, we reported that regions on chromosomes 22, 3 and 8 may be associated with susceptibility to schizophrenia, and collaborations provided some support for regions on chromosomes 8 and 22 (refs 9-13). We present here a genome-wide scan for schizophrenia susceptibility loci (SSL) using 452 microsatellite markers on 54 multiplex pedigrees. Non-parametric linkage (NPL) analysis provided significant evidence for an SSL on chromosome 13q32 (NPL score=4.18; P=0.00002), and suggestive evidence for another SSL on chromosome 8p21-22 (NPL=3.64; P=0.0001). Parametric linkage analysis provided additional support for these SSL. Linkage evidence at chromosome 8 is weaker than that at chromosome 13, so it is more probable that chromosome 8 may be a false positive linkage. Additional putative SSL were noted on chromosomes 14q13 (NPL=2.57; P=0.005), 7q11 (NPL=2.50, P=0.007) and 22q11 (NPL=2.42, P=0.009). Verification of suggestive SSL on chromosomes 13q and 8p was attempted in a follow-up sample of 51 multiplex pedigrees. This analysis confirmed the SSL in 13q14-q33 (NPL=2.36, P=0.007) and supported the SSL in 8p22-p21 (NPL=1.95, P=0.023).

Effects of toluidine blue and destaining reagents used in sexual assault examinations on the ability to obtain DNA profiles from postcoital vaginal swabs.

Toluidine blue is an important tool to detect and document genital and perianal injuries following sexual assault. Application of toluidine blue dye and its subsequent removal from unstained areas by means of a destaining reagent, such as diluted acetic acid or a lubricant has been shown to increase the detection rate of posterior fourchette lacerations from 16% to 40% in adult rape victims. Currently, limited information on toluidine blue positive findings in sexually active control groups imposes some limitation on the interpretation of these injuries. Because injuries could otherwise be attributed to improper handling of an examination speculum or the improper insertion of the examining finger, the toluidine blue test should be performed prior to any digital or speculum examination and thus prior to the collection of forensic evidence. For forensic DNA identity testing, it becomes pertinent to determine whether toluidine blue and the destaining reagents used in a sexual assault examination have an adverse effect on the recovery of high molecular weight DNA from postcoital vaginal swabs and thereby have an impact on restriction fragment length polymorphism (RFLP) analysis or PCR-based tests. It is known that some of the lubricants used can have a destructive effect on sperm motility. In order to investigate the potential effects, postcoital vaginal swabs were taken 6 h after sexual intercourse and exposed directly to 1% toluidine blue in aqueous solution, 1-10% acetic acid, and various surgical and vaginal lubricants. Subsequently, the DNA was isolated and DNA identity typing (RFLP and PCR-based) was performed. The results demonstrate, that these reagents have no negative effect on the ability to obtain DNA profiles, either RFLP or PCR-based, from shallow and deep vaginal swabs. The quantity and quality of extractable high molecular weight DNA obtained was comparable with that from uncontaminated postcoital vaginal swabs. RFLP patterns and PCR-based typing results on the D1S80, HUMTH01, TPOX, and CSF1PO loci were consistent with the uncontaminated control swabs and the corresponding whole blood samples of the donors. Therefore, evidentiary material inadvertently contaminated with these reagents can be successfully typed.