Evidence for genetic heterogeneity between clinical subtypes of bipolar disorder.

We performed a genome-wide association study of 6447 bipolar disorder (BD) cases and 12 639 controls from the International Cohort Collection for Bipolar Disorder (ICCBD). Meta-analysis was performed with prior results from the Psychiatric Genomics Consortium Bipolar Disorder Working Group for a combined sample of 13 902 cases and 19 279 controls. We identified eight genome-wide significant, associated regions, including a novel associated region on chromosome 10 (rs10884920; P=3.28 × 10-8) that includes the brain-enriched cytoskeleton protein adducin 3 (ADD3), a non-coding RNA, and a neuropeptide-specific aminopeptidase P (XPNPEP1). Our large sample size allowed us to test the heritability and genetic correlation of BD subtypes and investigate their genetic overlap with schizophrenia and major depressive disorder. We found a significant difference in heritability of the two most common forms of BD (BD I SNP-h2=0.35; BD II SNP-h2=0.25; P=0.02). The genetic correlation between BD I and BD II was 0.78, whereas the genetic correlation was 0.97 when BD cohorts containing both types were compared. In addition, we demonstrated a significantly greater load of polygenic risk alleles for schizophrenia and BD in patients with BD I compared with patients with BD II, and a greater load of schizophrenia risk alleles in patients with the bipolar type of schizoaffective disorder compared with patients with either BD I or BD II. These results point to a partial difference in the genetic architecture of BD subtypes as currently defined.

The incidence of venous thromboembolism among Factor V Leiden carriers: a community-based cohort study.

While Factor V (FV) Leiden is a risk factor for venous thromboembolism (VTE), the incidence of VTE among FV Leiden carriers is uncertain. The objective of the study was to estimate the overall age-specific and pregnancy-related VTE incidence and the relative risk among FV Leiden carriers. In a community-based sample of 3424 south-eastern Minnesota residents, 230 (6.7%) were genotyped as FV Leiden carriers; 220 carriers (mean age = 68 years) could be matched to a non-carrier on age, gender, ethnicity and length of medical history. We performed a retrospective cohort study of carriers and non-carriers by reviewing the complete medical records in the community for demographic and baseline characteristics, pregnancies and live births, and first lifetime VTE. Over 14 722 person-years, 24 (10.9%) carriers developed VTE [overall incidence = 163 (95% CI 104, 242) per 100,000 person-years]. VTE incidence rates for ages 15-29, 30-44, 45-59 and > or = 60 years were 0, 61, 244 and 764 per 100,000 person-years, respectively (cumulative VTE incidence at age 65 years = 6.3%). VTE incidence for carriers did not differ significantly from that for non-carriers except for those > or = 60 years old (relative risk = 3.6; 95% CI 2.0, 6.0). There were 311 live births among 130 women carriers; no VTE events occurred during pregnancy or postpartum [incidence = 0 (95% CI 0, 1186) per 100,000 women-years]. Most FV Leiden carriers do not develop VTE. Among all carriers, those > or = 60 years old are at the highest risk for VTE. The incidence of VTE among asymptomatic women carriers during pregnancy is low and insufficient to warrant prophylaxis.

Application of DNA-based diagnosis to patient care: the example of hemophilia A.

DNA-based carrier testing and prenatal diagnosis are rapidly expanding medical applications of recombinant-DNA technology. The ultimate goal of DNA-based diagnosis is the determination of the causative mutation, but, in general, this is possible only for large deletions, insertions, or certain nonsense mutation that, in most diseases, involve only a few percent of affected families. If direct diagnosis of the carrier state or fetal disease state is not feasible, indirect diagnosis can be performed by following the segregation of linked polymorphisms through the family pedigree. For such indirect diagnosis, DNA from multiple family members must be analyzed. Although this procedure is highly accurate in many families, errors can potentially occur because of meiotic recombination, genetic heterogeneity, new mutations, and nonpaternity. In this review, a general introduction to DNA-based diagnosis of mendelian diseases is presented and the methods and strategy are outlined. The use of these techniques for the diagnosis of hemophilia A is then described to illustrate the principles of diagnosis and to highlight some of the complexities encountered. DNA-based diagnosis is in its infancy and has the potential to revolutionize preventive medicine.

A novel method for detecting point mutations or polymorphisms and its application to population screening for carriers of phenylketonuria.

We describe a method termed PCR (polymerase chain reaction) amplification of specific alleles (PASA), a generally applicable technique for detection of point mutations or polymorphisms. The ease and technical simplicity of PASA will make genetic analyses more accessible to the general medical community. In addition, PASA shows promise for population screening because the technique is rapid, highly reproducible, inexpensive, nonisotopic, and amenable to automation. PASA is a modification of PCR that depends on the synthesis of a PCR oligonucleotide primer that precisely matches with one of the alleles but mismatches with the other. When the mismatch occurs near the 3' end of the PCR primer, amplification is inefficient. Therefore, preferential amplification of the perfectly matched allele is obtained. We demonstrate the applicability of PASA by performing carrier detection in the family of a patient with phenylketonuria (PKU) and by screening a population of unrelated subjects for the presence of the two mutations most commonly associated with PKU. Multiple persons were screened simultaneously for the mutant alleles because a mutation could be detected in the presence of at least a 40-fold excess of the normal allele. The two PKU mutations could be detected concurrently by using a mixture of only three PCR primers, an indication that simultaneous screening of multiple mutations can be done even if three or more mutations are closely clustered. In addition to the detection of mutations, PASA can be used to detect polymorphic alleles rapidly and to distinguish pseudogenes or repetitive sequences that differ by as little as one base.

Novel association approach for determining the genetic predisposition to schizophrenia: case-control resource and testing of a candidate gene.

We have developed a two-tiered approach to elucidating the genetic predisposition to schizophrenia. The approach first involves the examination of candidate genes in a subset of schizophrenic individuals to identify DNA sequence variations of likely functional significance, i.e., that produce either structural alterations in the protein or affect the level of gene expression. Once identified, the prevalence of the aberrant allele is examined in a large group of unrelated schizophrenic cases and controls to assess whether a true disease association exists. Herein, we describe the establishment of a DNA bank on nearly 200 unrelated schizophrenic cases defined by DSM-III-R criteria and on over 300 unrelated, ethnically similar controls. Characteristics of the study sample are described. The study approach then is illustrated by testing known mutations in the phenylalanine hydroxylase gene, responsible for the autosomal recessive disease of phenylketonuria, in the case-control sample to determine if carriership of a mutant allele is associated with an increased risk of schizophrenia. Using PCR amplification of specific alleles (PASA), we screened 190 schizophrenic cases and 336 controls for two common point mutations in the phenylalanine hydroxylase gene. Two carriers were found among the controls, while none of the cases was shown to carry a mutant allele. Thus, carriership of either of two common mutations in the phenylalanine hydroxylase gene does not appear to be associated with an increased risk of schizophrenia. As additional candidate genes are tested in this case-control resource, adjustment for multiple comparisons will become crucial in order to reduce the chance of false positive findings.(ABSTRACT TRUNCATED AT 250 WORDS)

Screening the dopamine D1 receptor gene in 131 schizophrenics and eight alcoholics: identification of polymorphisms but lack of functionally significant sequence changes.

To determine whether mutations in the D1 dopamine receptor (D1 DR) gene are associated with schizophrenia, the coding sequence was examined in 106 Caucasian, 11 African-American, 8 Asian, and 6 Native American patients. Approximately 350 kb of genomic sequence was screened by dideoxy fingerprinting, a method related to single strand conformational polymorphism (SSCP) analysis that detects virtually 100% of sequence changes [Sarkar et al., 1992: Genomics 13:441-443; Liu and Sommer, 1994: PCR Methods and Applications 4:97-108]. One polymorphism was identified in Asians and one in Caucasians, but neither altered the amino acid sequence (Leu66, and Ser421, respectively). In addition, a previously reported polymorphism in the 5' untranslated region of exon 2 at bp -48 was found to be common, with an allele frequency of approximately 40% in Caucasians of Western European descent. Based on the fact that no sequence changes of likely functional significance were identified, these data suggest that mutations affecting the structure of the D1 dopamine receptor protein are uncommon and are unlikely to contribute significantly to the genetic predisposition to schizophrenia. The D1 DR gene also was examined in eight alcoholics, including 3 African-Americans and 1 Native American, but no sequence changes were identified.

Dopamine D4 receptor variants in unrelated schizophrenic cases and controls.

The D4 dopamine receptor (D4DR) exists in multiple allelic forms (Van Tol et al.: Nature 358:149-152, 1992) which involve different numbers of a 48 basepair repeat sequence in the putative third cytoplasmic loop. Different binding properties have been reported for at least three of the alleles in cDNA binding assays with clozapine, an atypical neuroleptic, and spiperone (Van Tol et al., 1992). We have examined 115 unrelated schizophrenic cases defined by DSM-III-R criteria and 115 controls of similar ethnicity to determine the frequency of seven different D4 alleles in these groups. No statistically significant difference in the distribution of the alleles existed between cases and controls, although a trend towards a greater prevalence of homozygotes for the 4-repeat allele was observed in schizophrenics.

Screening the monoamine oxidase B gene in 100 male patients with schizophrenia: a cluster of polymorphisms in African-Americans but lack of functionally significant sequence changes.

The monoamine oxidase B (MAO-B) gene was examined in 100 alleles derived from 80 Caucasian, 10 African-American, 5 Asian, and 5 Native American male patients with schizophrenia to identify sequence changes that might be associated with the disease. Approximately 235 kb of genomic sequence, primarily in coding regions, were screened by dideoxy fingerprinting, a modification of single-strand conformational polymorphism (SSCP) analysis that detects virtually 100% of sequence changes [Sarkar et al. (1992): Genomics 13:441-443; Liu and Sommer (1994): PCR Methods Appl 4:97-108]. No sequence changes of likely functional significance were identified, suggesting that mutations affecting the structure of the MAO-B protein are uncommon in the general population and are unlikely to contribute significantly to the genetic predisposition to schizophrenia. Eight polymorphisms were identified in African-Americans and Native Americans, but none were identified among Caucasians. Of the eight observed polymorphisms, a set of five transitions and one microdeletion was identified within approximately 17 kb of genomic sequence in the same 3 African-American individuals, while the remaining 7 African-Americans had a sequence identical to that in Caucasians. The presence of two such haplotypes, without intermediates, is compatible with the hypothesis that germline mutations can occur in clusters, as also suggested by other recent findings.

Identification of a missense mutation and several polymorphisms in the proenkephalin A gene of schizophrenic patients.

Schizophrenia is a complex and severe disorder of unknown cause and pathophysiology. In this study, we examined the opioid hypothesis for schizophrenia at the molecular level, focusing on the dopamine-regulated proenkephalin A gene (chromosome 8q11.23-q12). We have screened 150 schizophrenic patients for sequence variations within the promoter region, entire coding sequence, and 3'-untranslated region. We find one sequence change in a conserved amino acid that may be of functional significance. This mutation was found in a single schizophrenia patient but not in controls. Although several new, race-specific polymorphisms were identified, all other sequence changes appeared to be common polymorphisms, unlikely to contribute to the etiology of schizophrenia.

Screening the dystrophin gene suggests a high rate of polymorphism in general but no exonic deletions in schizophrenics.

The dystrophin gene, located at chromosome Xp21, was evaluated as a candidate gene in chronic schizophrenia in response to the report of a large family in which schizophrenia cosegregated with Becker muscular dystrophy [Zatz et al., 1991: Am J Hum Genet 49: A364; 1992: J Med Genet 30(2):131-134]. Genomic DNA from 94 men with chronic schizophrenia was evaluated by Southern blot analysis using cDNA probes that span exons 1-59. No exonic deletions were identified. An unexpectedly high rate of polymorphism was calculated in this study and two novel polymorphisms were found, demonstrating the usefulness of the candidate gene approach even when results of the original study are negative.

Scanning of the dopamine D1 and D5 receptor genes by REF in neuropsychiatric patients reveals a novel missense change at a highly conserved amino acid.

In previous analyses of schizophrenic patients, multiple missense changes and one nonsense change were identified in the D5 dopamine receptor (DRD5) gene, but no sequence changes of likely functional significance were identified in the D1 dopamine receptor (DRD1) gene. In the present study, we examined these genes in patients with certain other neuropsychiatric disorders that may be related to dopaminergic dysregulation. The coding regions of the DRD1 and DRD5 genes were examined in 25 and 25 autistic patients, 25 and 28 attention deficit hyperactivity disorder patients, and 51 and 43 alcoholic patients, respectively. In addition, the DRD5 gene was examined in 75 schizophrenic patients to search for additional variants affecting protein structure or expression (VAPSEs). These patients were analyzed with REF (restriction endonuclease fingerprinting), a hybrid between SSCP and restriction endonuclease digestion, which allows the entire coding sequence to be screened in one lane of a gel. Approximately 800 kb of genomic sequence were examined. No sequence changes were identified in the DRD1 gene among the 101 patient samples analyzed. Two sequence changes were identified in the DRD5 gene among the 171 patient samples. These included one previously identified silent polymorphism at base pair 978 (P326P). The change was identified in patients from all disease categories and from different ethnic backgrounds. One novel missense change, L88F, occurred in transmembrane domain II at a highly conserved amino acid in all dopamine receptors as well as in alpha1- and beta-adrenergic receptors. The mutation was identified in a Caucasian male patient with autism. Further analysis is necessary to determine if this missense change is associated with a particular neuropsychiatric phenotype.

Genotype-to-phenotype analysis: search for clinical characteristics of a missense change in the GABAA-beta 1 receptor gene.

Genotype-to-phenotype analysis reverses the classical approach to genetic disease in which an unknown genotype is sought for a known phenotype. This paper provides an example of genotype-to-phenotype analysis for the possible psychiatric effects of a missense mutation (H396Q) at a highly conserved residue of the beta 1 subunit gene of the gamma aminobutyric acid type A receptor. DNA samples from 1,507 Caucasians of Western European descent were screened, and 10 heterozygotes for H396Q were identified. These individuals were matched to homozygous normal individuals by age, gender, and length of available medical records. The complete medical records of these 20 individuals were reviewed blindly by two psychiatrists (D.C.S., L.L.H.) to assess psychiatric symptomatology, with an emphasis on anxiety and related disorders. However, no association was found between this missense change at a conserved amino acid and a dominant neuropsychiatric disease phenotype. Thus, this missense change may be neutral or only mildly deleterious, may only cause recessive disease in rare individuals, or may interact epistatically with some other gene(s).

Histamine N-methyltransferase functional polymorphism: lack of association with schizophrenia.

Histamine is a central nervous system (CNS) neurotransmitter that has been implicated in the pathophysiology of schizophrenia. Histamine N-methyltransferase (HNMT) terminates the neurotransmitter actions of histamine in the mammalian CNS, and levels of HNMT activity in human tissues are controlled, in part, by inheritance. A common C314T polymorphism in the HNMT gene causes a Thr105Ile change in encoded amino acid. The T314 allele results in decreased levels of both HNMT enzyme activity and immunoreactive protein. There is also a polymorphic CA repeat in intron 5 of the HNMT gene. The frequencies of alleles for the functional C314T polymorphism and the polymorphic CA repeat were compared between 171 schizophrenia cases and 171 ethnically matched controls to test for possible disease association. No significant difference was found between the two groups in the frequency of the T314 allele in patients with schizophrenia and controls (0.068 vs. 0.078, respectively). Allele frequencies for the polymorphic HNMT CA repeat also failed to show significant differences between cases and matched controls.

Variants in the alpha2A AR adrenergic receptor gene in psychiatric patients.

In various studies of psychiatric patients, alterations in adrenergic receptor (AR) expression or function have been suggested. Herein, the alpha2A AR gene was screened in 206 patients with schizophrenia, attention deficit hyperactivity disorder (ADHD), autism, alcohol dependence, or cocaine dependence. The entire coding region was examined for single base pair changes, using restriction endonuclease fingerprinting (REF), a screening method that can detect virtually 100% of mutations in 2-kb DNA segments. In the approximately 600 kb of screened sequence, six novel nucleotide changes were identified. The changes resulted in four missense changes (A25G, N251K, R368L, and K370N), and a sequence in the 3' untranslated region. In addition, a silent change (G363G) was found at high frequency in Asians and Native Americans. Of the four missense changes, two found in patients with alcohol/drug dependence occur in highly conserved amino acids, suggesting that these are of likely functional significance. As the alpha2A ARs are widely distributed both pre- and postsynaptically, and as many pharmacological agents with multiple effects target these receptors, the novel missense changes described herein may be candidates for involvement in alcohol/drug dependence, in other clinical disorders or traits, or in differential response to pharmacotherapy.

Gly(247)-->Asp proenkephalin A mutation is rare in schizophrenia populations.

Schizophrenia is a complex and severe disorder of unknown cause and pathophysiology. In previous work examining an opioid hypothesis for schizophrenia, we identified a missense mutation (Gly(247)-->Asp) in the proenkephalin A gene of one African-American patient. In the current study involving an extended set of African-American and other patients, we sought to identify additional mutant alleles and to determine the distribution of these alleles among several racial groups. However, the Gly(247)-->Asp allele was not detected in any of 116 African-American (67 cases, 49 controls), 659 Caucasian, 1 Hispanic, 4 Asian, and 7 Native American individuals. Therefore, it appears that this mutation is a rare event of unknown clinical significance.

From molecular variant to disease: initial steps in evaluating the association of transthyretin M119 with disease.

Traditionally, clinical research has sought to determine the molecular basis of clinical signs and symptoms. Increasingly, the traditional process will be reversed, as many structural protein variants are elucidated as a result of powerful PCR-based methods. Herein we describe a variant of transthyretin (TTR) found by direct genomic sequencing and illustrate the utility of PASA (PCR amplification of specific alleles) in the initial characterization of such variants. TTR is an intriguing protein of unknown function, but deposition of mutant TTR produces familial amyloidotic polyneuropathy (FAP). We identify a carrier of a variant TTR in which threonine119 is changed to methionine (T119----M). T119 is invariant in five mammalian species, suggesting that this residue is important for normal protein function. To determine the frequency of the M119 variant, individuals of northern- and western-European descent were rapidly screened by generating a PASA assay for the sequence change. Four additional individuals were found to be heterozygous for the mutation, for a total of five M119 alleles in 1,666 genes (1/333). Clinical records, initial clinical interviews, and family history of these patients hint at a high frequency of early-onset venous insufficiency and perhaps mild renal dysfunction. Haplotype analysis on the heterozygotes could be performed, despite the absence of samples from relatives, by performing "double PASA." The haplotype data suggest that the M119 variant derives from a common ancestor. The putative functional deficiency caused by TTR M119 should be most marked in the homozygotes, who can be calculated to occur in 1/100,000 conceptions. If viable, these individuals may provide important clues about the physiological role of TTR. Although the nature (if any) of disease caused by TTR M119 remains to be defined, the genetic and clinical data indicate that this mutation does not cause FAP. Future family studies can determine whether the heterozygous state for TTR M119 cosegregates with a disease or trait.

Multiple missense mutations in the diazepam binding inhibitor (DBI) gene identified in schizophrenia but lack of disease association.

The diazepam binding inhibitor (DBI), alternatively known as the acyl-CoA binding protein (ACBP), is involved in multiple biological actions. The polypeptide binds to the peripheral, or mitochondrial, benzodiazepine receptor and facilitates transport of cholesterol to the inner membrane to stimulate steroid synthesis. Through this action, DBI indirectly modulates gamma-aminobutyric acid (GABA)-mediated inhibitory neurotransmission. DBI can be postulated as a candidate gene for psychiatric phenotypes including anxiety, mood, and psychotic disorders. In an examination of the DBI gene among 112 individuals with schizophrenia, our laboratory has identified 18 novel single nucleotide polymorphisms (SNPs), including three missense changes in conserved amino acids, a coding region microdeletion, and multiple SNPs in the putative promoter region. Case-control association analyses were performed for the missense changes, but none was found to be significantly associated with disease.

Identification of single nucleotide polymorphisms (SNPs) and other sequence changes and estimation of nucleotide diversity in coding and flanking regions of the NMDAR1 receptor gene in schizophrenic patients.

Glutamatergic dysregulation has been hypothesized to play a role in schizophrenia. The N-methyl-D-aspartate (NMDA) type of glutamate receptor especially is of interest because, in addition to binding sites for glutamate and glycine, a necessary co-agonist, this receptor also contains noncompetitive binding sites for the psychotomimetics phencyclidine (PCP), MK-801, and ketamine. PCP-induced psychosis has been a useful disease model in that both the positive as well as the negative symptomatologies seen in schizophrenia are observed. Recently, a mouse deficient in expression of the NR1 subunit gene (NMDAR1) of the heteromeric receptor has been developed and shown to display aberrant behaviors, with reduced social and sexual interactions as well as increased stereotypic motor activity. In an extensive examination of the NMDAR1 gene in our laboratory in approximately 100 chronic schizophrenic patients, 28 unique sequence changes were identified, including eight single nucleotide polymorphisms (SNPs) in the 5' untranslated region (5'UTR), six SNPs in coding regions (cSNPs), eleven intronic SNPs, two intronic deletions of 7 and 30 bp, and an intronic microinsertion/deletion. With the exception of one previously reported cSNP, all of the identified changes were novel. The frequency of polymorphisms differed significantly by ethnicity and several appeared to be in linkage disequilibrium. None of the changes appeared likely to be of functional significance, thus suggesting that changes in the genomic NMDAR1 are unlikely to contribute to the etiology of schizophrenia. Estimates of nucleotide diversity are comparable to those observed in studies of other genes.

Trends in the incidence of polycythemia vera among Olmsted County, Minnesota residents, 1935-1989.

To investigate the suggestion that the incidence of polycythemia vera has increased in recent decades, we ascertained secular trends in the incidence of polycythemia vera in Olmsted County, Minnesota, over the 55-year period, 1935-1989. The inpatient and outpatient medical records of all potential cases of polycythemia vera in Olmsted County residents were reviewed and the diagnostic criteria of the Polycythemia Vera Study Group were applied. We found no indication of an increase in the age- and sex-adjusted incidence of polycythemia vera, which averaged 1.9 per 100,000 person-years (95% C.I., 1.4-2.5) over the study period. Incidence rates increased with age, and age-adjusted incidence rates were greater for men (2.8 per 100,000 person-years; 95% C.I., 1.8-3.9) than for women (1.3 per 100,000 person-years; 95% C.I., 0.7-1.9), with the highest incidence rate (23.5 per 100,000 person-years) among men aged 70-79 years. Survival was reduced in this inception cohort of 50 cases, compared to that expected for individuals of like age and sex (P < 0.0001); median survival following diagnosis was 7.2 years.