The association of polygenic risk for schizophrenia, bipolar disorder, and depression with neural connectivity in adolescents and young adults: examining developmental and sex differences.

Neurodevelopmental abnormalities in neural connectivity have been long implicated in the etiology of schizophrenia (SCZ); however, it remains unclear whether these neural connectivity patterns are associated with genetic risk for SCZ in unaffected individuals (i.e., an absence of clinical features of SCZ or a family history of SCZ). We examine whether polygenic risk scores (PRS) for SCZ are associated with functional neural connectivity in adolescents and young adults without SCZ, whether this association is moderated by sex and age, and if similar associations are observed for genetically related neuropsychiatric PRS. One-thousand four-hundred twenty-six offspring from 913 families, unaffected with SCZ, were drawn from the Collaborative Study of the Genetics of Alcoholism (COGA) prospective cohort (median age at first interview = 15.6 (12-26), 51.6% female, 98.1% European American, 41% with a family history of alcohol dependence). Participants were followed longitudinally with resting-state EEG connectivity (i.e., coherence) assessed every two years. Higher SCZ PRS were associated with elevated theta (3-7 Hz) and alpha (7-12 Hz) EEG coherence. Associations differed by sex and age; the most robust associations were observed between PRS and parietal-occipital, central-parietal, and frontal-parietal alpha coherence among males between ages 15-19 (B: 0.15-0.21, p < 10-4). Significant associations among EEG coherence and Bipolar and Depression PRS were observed, but differed from SCZ PRS in terms of sex, age, and topography. Findings reveal that polygenic risk for SCZ is robustly associated with increased functional neural connectivity among young adults without a SCZ diagnosis. Striking differences were observed between men and women throughout development, mapping onto key periods of risk for the onset of psychotic illness and underlining the critical importance of examining sex differences in associations with neuropsychiatric PRS across development.

Genome-wide association study identifies a novel locus for cannabis dependence.

Despite moderate heritability, only one study has identified genome-wide significant loci for cannabis-related phenotypes. We conducted meta-analyses of genome-wide association study data on 2080 cannabis-dependent cases and 6435 cannabis-exposed controls of European descent. A cluster of correlated single-nucleotide polymorphisms (SNPs) in a novel region on chromosome 10 was genome-wide significant (lowest P=1.3E-8). Among the SNPs, rs1409568 showed enrichment for H3K4me1 and H3K427ac marks, suggesting its role as an enhancer in addiction-relevant brain regions, such as the dorsolateral prefrontal cortex and the angular and cingulate gyri. This SNP is also predicted to modify binding scores for several transcription factors. We found modest evidence for replication for rs1409568 in an independent cohort of African American (896 cases and 1591 controls; P=0.03) but not European American (EA; 781 cases and 1905 controls) participants. The combined meta-analysis (3757 cases and 9931 controls) indicated trend-level significance for rs1409568 (P=2.85E-7). No genome-wide significant loci emerged for cannabis dependence criterion count (n=8050). There was also evidence that the minor allele of rs1409568 was associated with a 2.1% increase in right hippocampal volume in an independent sample of 430 EA college students (fwe-P=0.008). The identification and characterization of genome-wide significant loci for cannabis dependence is among the first steps toward understanding the biological contributions to the etiology of this psychiatric disorder, which appears to be rising in some developed nations.

An endophenotype approach to the genetics of alcohol dependence: a genome wide association study of fast beta EEG in families of African ancestry.

Fast beta (20-28 Hz) electroencephalogram (EEG) oscillatory activity may be a useful endophenotype for studying the genetics of disorders characterized by neural hyperexcitability, including substance use disorders (SUDs). However, the genetic underpinnings of fast beta EEG have not previously been studied in a population of African-American ancestry (AA). In a sample of 2382 AA individuals from 482 families drawn from the Collaborative Study on the Genetics of Alcoholism (COGA), we performed a genome-wide association study (GWAS) on resting-state fast beta EEG power. To further characterize our genetic findings, we examined the functional and clinical/behavioral significance of GWAS variants. Ten correlated single-nucleotide polymorphisms (SNPs) (r2>0.9) located in an intergenic region on chromosome 3q26 were associated with fast beta EEG power at P<5 × 10-8. The most significantly associated SNP, rs11720469 (ß: -0.124; P<4.5 × 10-9), is also an expression quantitative trait locus for BCHE (butyrylcholinesterase), expressed in thalamus tissue. Four of the genome-wide SNPs were also associated with Diagnostic and Statistical Manual of Mental Disorders Alcohol Dependence in COGA AA families, and two (rs13093097, rs7428372) were replicated in an independent AA sample (Gelernter et al.). Analyses in the AA adolescent/young adult (offspring from COGA families) subsample indicated association of rs11720469 with heavy episodic drinking (frequency of consuming 5+ drinks within 24 h). Converging findings presented in this study provide support for the role of genetic variants within 3q26 in neural and behavioral disinhibition. These novel genetic findings highlight the importance of including AA populations in genetics research on SUDs and the utility of the endophenotype approach in enhancing our understanding of mechanisms underlying addiction susceptibility.

Common biological networks underlie genetic risk for alcoholism in African- and European-American populations.

Alcohol dependence (AD) is a heritable substance addiction with adverse physical and psychological consequences, representing a major health and economic burden on societies worldwide. Genes thus far implicated via linkage, candidate gene and genome-wide association studies (GWAS) account for only a small fraction of its overall risk, with effects varying across ethnic groups. Here we investigate the genetic architecture of alcoholism and report on the extent to which common, genome-wide SNPs collectively account for risk of AD in two US populations, African-Americans (AAs) and European-Americans (EAs). Analyzing GWAS data for two independent case-control sample sets, we compute polymarker scores that are significantly associated with alcoholism (P = 1.64 × 10(-3) and 2.08 × 10(-4) for EAs and AAs, respectively), reflecting the small individual effects of thousands of variants derived from patterns of allelic architecture that are population specific. Simulations show that disease models based on rare and uncommon causal variants (MAF < 0.05) best fit the observed distribution of polymarker signals. When scoring bins were annotated for gene location and examined for constituent biological networks, gene enrichment is observed for several cellular processes and functions in both EA and AA populations, transcending their underlying allelic differences. Our results reveal key insights into the complex etiology of AD, raising the possibility of an important role for rare and uncommon variants, and identify polygenic mechanisms that encompass a spectrum of disease liability, with some, such as chloride transporters and glycine metabolism genes, displaying subtle, modifying effects that are likely to escape detection in most GWAS designs.

A genome-wide association study of alcohol-dependence symptom counts in extended pedigrees identifies C15orf53.

Several studies have identified genes associated with alcohol-use disorders (AUDs), but the variation in each of these genes explains only a small portion of the genetic vulnerability. The goal of the present study was to perform a genome-wide association study (GWAS) in extended families from the Collaborative Study on the Genetics of Alcoholism to identify novel genes affecting risk for alcohol dependence (AD). To maximize the power of the extended family design, we used a quantitative endophenotype, measured in all individuals: number of alcohol-dependence symptoms endorsed (symptom count (SC)). Secondary analyses were performed to determine if the single nucleotide polymorphisms (SNPs) associated with SC were also associated with the dichotomous phenotype, DSM-IV AD. This family-based GWAS identified SNPs in C15orf53 that are strongly associated with DSM-IV alcohol-dependence symptom counts (P=4.5 × 10(-8), inflation-corrected P=9.4 × 10(-7)). Results with DSM-IV AD in the regions of interest support our findings with SC, although the associations were less significant. Attempted replications of the most promising association results were conducted in two independent samples: nonoverlapping subjects from the Study of Addiction: Genes and Environment (SAGE) and the Australian Twin Family Study of AUDs (OZALC). Nominal association of C15orf53 with SC was observed in SAGE. The variant that showed strongest association with SC, rs12912251 and its highly correlated variants (D'=1, r(2) 0.95), have previously been associated with risk for bipolar disorder.

Family-based genome-wide association study of frontal θ oscillations identifies potassium channel gene KCNJ6.

Event-related oscillations (EROs) represent highly heritable neuroelectric correlates of cognitive processes that manifest deficits in alcoholics and in offspring at high risk to develop alcoholism. Theta ERO to targets in the visual oddball task has been shown to be an endophenotype for alcoholism. A family-based genome-wide association study was performed for the frontal theta ERO phenotype using 634 583 autosomal single nucleotide polymorphisms (SNPs) genotyped in 1560 family members from 117 families densely affected by alcohol use disorders, recruited in the Collaborative Study on the Genetics of Alcoholism. Genome-wide significant association was found with several SNPs on chromosome 21 in KCNJ6 (a potassium inward rectifier channel; KIR3.2/GIRK2), with the most significant SNP at P = 4.7 × 10(-10)). The same SNPs were also associated with EROs from central and parietal electrodes, but with less significance, suggesting that the association is frontally focused. One imputed synonymous SNP in exon four, highly correlated with our top three SNPs, was significantly associated with the frontal theta ERO phenotype. These results suggest KCNJ6 or its product GIRK2 account for some of the variations in frontal theta band oscillations. GIRK2 receptor activation contributes to slow inhibitory postsynaptic potentials that modulate neuronal excitability, and therefore influence neuronal networks.

ADH1B is associated with alcohol dependence and alcohol consumption in populations of European and African ancestry.

A coding variant in alcohol dehydrogenase 1B (ADH1B) (rs1229984) that leads to the replacement of Arg48 with His48 is common in Asian populations and reduces their risk for alcoholism, but because of very low allele frequencies the effects in European or African populations have been difficult to detect. We genotyped and analyzed this variant in three large European and African-American case-control studies in which alcohol dependence was defined by the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) criteria, and demonstrated a strong protective effect of the His48 variant (odds ratio (OR) 0.34, 95% confidence interval (CI) 0.24, 0.48) on alcohol dependence, with genome-wide significance (6.6 × 10(-10)). The hypothesized mechanism of action involves an increased aversive reaction to alcohol; in keeping with this hypothesis, the same allele is strongly associated with a lower maximum number of drinks in a 24-hour period (lifetime), with P=3 × 10(-13). We also tested the effects of this allele on the development of alcoholism in adolescents and young adults, and demonstrated a significantly protective effect. This variant has the strongest effect on risk for alcohol dependence compared with any other tested variant in European populations.

Neurocognitive deficits in male alcoholics: an ERP/sLORETA analysis of the N2 component in an equal probability Go/NoGo task.

In alcoholism research, studies concerning time-locked electrophysiological aspects of response inhibition have concentrated mainly on the P3 component of the event-related potential (ERP). The objective of the present study was to investigate the N2 component of the ERP to elucidate possible brain dysfunction related to the motor response and its inhibition using a Go/NoGo task in alcoholics. The sample consisted of 78 abstinent alcoholic males and 58 healthy male controls. The N2 peak was compared across group and task conditions. Alcoholics showed significantly reduced N2 peak amplitudes compared to normal controls for Go as well as NoGo task conditions. Control subjects showed significantly larger NoGo than Go N2 amplitudes at frontal regions, whereas alcoholics did not show any differences between task conditions at frontal regions. Standardized low resolution electromagnetic tomography analysis (sLORETA) indicated that alcoholics had significantly lower current density at the source than control subjects for the NoGo condition at bilateral anterior prefrontal regions, whereas the differences between groups during the Go trials were not statistically significant. Furthermore, NoGo current density across both groups revealed significantly more activation in bilateral anterior cingulate cortical (ACC) areas, with the maximum activation in the right cingulate regions. However, the magnitude of this difference was much less in alcoholics compared to control subjects. These findings suggest that alcoholics may have deficits in effortful processing during the motor response and its inhibition, suggestive of possible frontal lobe dysfunction.

Genome-wide association study of conduct disorder symptomatology.

Conduct disorder (CD) is one of the most prevalent childhood psychiatric conditions, and is associated with a number of serious concomitant and future problems. CD symptomatology is known to have a considerable genetic component, with heritability estimates in the range of 50%. Despite this, there is a relative paucity of studies aimed at identifying genes involved in the susceptibility to CD. In this study, we report results from a genome-wide association study of CD symptoms. CD symptoms were retrospectively reported by a psychiatric interview among a sample of cases and controls, in which cases met the criteria for alcohol dependence. Our primary phenotype was the natural log transformation of the number of CD symptoms that were endorsed, with data available for 3963 individuals who were genotyped on the Illumina Human 1M beadchip array. Secondary analyses are presented for case versus control status, in which caseness was established as endorsing three or more CD symptoms (N = 872 with CD and N = 3091 without CD). We find four markers that meet the criteria for genome-wide significance (P<5 × 10(-8)) with the CD symptom count, two of which are located in the gene C1QTNF7 (C1q and tumor necrosis factor-related protein 7). There were six additional SNPs in the gene that yielded converging evidence of association. These data provide the first evidence of a specific gene that is associated with CD symptomatology. None of the top signals resided in traditional candidate genes, underscoring the importance of a genome-wide approach for identifying novel variants involved in this serious childhood disorder.

Genetic variation in the CHRNA5 gene affects mRNA levels and is associated with risk for alcohol dependence.

Alcohol dependence frequently co-occurs with cigarette smoking, another common addictive behavior. Evidence from genetic studies demonstrates that alcohol dependence and smoking cluster in families and have shared genetic vulnerability. Recently a candidate gene study in nicotine dependent cases and nondependent smoking controls reported strong associations between a missense mutation (rs16969968) in exon 5 of the CHRNA5 gene and a variant in the 3'-UTR of the CHRNA3 gene and nicotine dependence. In this study we performed a comprehensive association analysis of the CHRNA5-CHRNA3-CHRNB4 gene cluster in the Collaborative Study on the Genetics of Alcoholism (COGA) families to investigate the role of genetic variants in risk for alcohol dependence. Using the family-based association test, we observed that a different group of polymorphisms, spanning CHRNA5-CHRNA3, demonstrate association with alcohol dependence defined by Diagnostic and Statistical Manual of Mental Disorders, 4th edn (DSM-IV) criteria. Using logistic regression we replicated this finding in an independent case-control series from the family study of cocaine dependence. These variants show low linkage disequilibrium with the SNPs previously reported to be associated with nicotine dependence and therefore represent an independent observation. Functional studies in human brain reveal that the variants associated with alcohol dependence are also associated with altered steady-state levels of CHRNA5 mRNA.

Association of the kappa-opioid system with alcohol dependence.

Opioid receptors and their endogenous peptide ligands play important roles in the reward and reinforcement of drugs such as heroin, cocaine, and alcohol. The binding of dynorphins to the kappa-opioid receptor has been shown to produce aversive states, which may prevent the development of reinforcement. We genotyped SNPs throughout OPRK1, encoding the kappa-opioid receptor, and PDYN, which encodes its ligand prodynorphin, in a group of 1860 European American individuals from 219 multiplex alcohol dependent families. Family-based analyses demonstrated associations between alcohol dependence and multiple SNPs in the promoter and 3' end of PDYN, and in intron 2 of OPRK1. Haplotype analyses further supported the association of PDYN. Thus, variations in the genes encoding both the kappa-opioid receptor and its ligand, OPRK1 and PDYN, are associated with the risk for alcohol dependence; this makes biological sense as variations in either should affect signaling through the kappa-opioid system.

A sex-adjusted and age-adjusted genome screen for nested alcohol dependence diagnoses.

Alcohol dependence is a complex disorder with a substantial genetic contribution to susceptibility. The Collaborative Study on the Genetics of Alcoholism is a multi-site study whose purpose is to detect, localize, and characterize genes contributing to this susceptibility. Previous linkage analyses of the trait of alcohol dependence in Collaborative Study on the Genetics of Alcoholism have used affected sib-pair methods with a dichotomous phenotype definition. In contrast, the analysis in this paper uses a sex-adjusted and age-adjusted multiple threshold liability model. The use of such a model, in that it includes unaffected as well as as affected subjects and in that it utilizes the differential severity of a diagnosis scale, should heuristically be more powerful than a straight affected sib-pair analysis. Three regions of interest are found on chromosome 1 (lod 5.17), chromosome 4 (lod 3.46), and chromosome 8 (lod 4.31). The region on chromosome 1 near the marker D1S532 is in the region previously reported as linked to alcohol dependence and correlated phenotypes in this dataset. The region on chromosome 4 near the alcohol dehydrogenase gene cluster has been reported to be linked to alcohol dependence in other studies, as well as to the alcohol consumption phenotype 'Maximum Number of Drinks in a 24-Hour Period' in this dataset. The region on chromosome 8 near the marker D8S1988 is homologous to a section of rat chromosome 5 to which an alcohol consumption phenotype has been linked.

A genome-wide screen for genes influencing conduct disorder.

While behavioral genetic studies have suggested that childhood conduct disorder is under genetic influence, studies aimed at gene identification are lacking. This study represents the first genome-wide linkage analysis directed toward identifying genes contributing to conduct disorder. Genome screens of retrospectively reported childhood conduct disorder and conduct disorder symptomatology were carried out in the genetically informative adult sample collected as part of the Collaborative Study on the Genetics of Alcoholism (COGA). The results suggest that regions on chromosomes 19 and 2 may contain genes conferring risk to conduct disorder. Interestingly, the same region on chromosome 2 has also been linked to alcohol dependence in this sample. Childhood conduct disorder is known to be associated with the susceptibility for future alcohol problems. Taken together, these findings suggest that some of the genes contributing to alcohol dependence in adulthood may also contribute to conduct disorder in childhood.

Warp-averaging event-related potentials.

OBJECTIVE: To align the repeated single trials of the event-related potential (ERP) in order to get an improved estimate of the ERP. METHODS: A new implementation of the dynamic time warping is applied to compute a warp-average of the single trials. The trilinear modeling method is applied to filter the single trials prior to alignment. Alignment is based on normalized signals and their estimated derivatives. These features reduce the misalignment due to aligning the random alpha waves, explaining amplitude differences in latency differences, or the seemingly small amplitudes of some components. RESULTS: Simulations and applications to visually evoked potentials show significant improvement over some commonly used methods. CONCLUSIONS: The new implementation of the dynamic time warping can be used to align the major components (P1, N1, P2, N2, P3) of the repeated single trials. The average of the aligned single trials is an improved estimate of the ERP. This could lead to more accurate results in subsequent analysis.

Visual p3 in female alcoholics.

BACKGROUND: The P300 (P3) component of the event related potential has been established as a sensitive risk marker of vulnerability to alcoholism. Most alcoholism studies have focused on men; recent studies indicate that women are equally vulnerable to developing alcoholism. METHODS: Visual P3 recorded from 31 electrode positions was evaluated in 44 alcoholic and 60 control women, 24-50 years of age. P3 amplitudes and latencies of the two groups were statistically compared using Analysis of Variance; source localization of surface amplitude values from each group were plotted using a low-resolution brain electromagnetic tomography. RESULTS: The results indicated that alcoholic women had significantly smaller P3 amplitudes in the frontal and central regions compared with controls. Source localization showed lowered activation in alcoholic women in right dorso-lateral prefrontal cortex and the ventro-medial fronto-central regions. CONCLUSIONS: The results suggest that P3 is an equally sensitive endophenotypic marker of vulnerability to alcoholism in women. The findings are discussed in terms of functional and physiologic significance of the P3 amplitude in alcoholic women and its relationship to drinking behaviors.

Auditory P3a deficits in male subjects at high risk for alcoholism.

BACKGROUND: Substantial evidence indicates that alcoholism is biologically mediated by a genetic predisposition. As the decreased P300 (P3b) event-related brain potential component does not recover with prolonged abstinence, it is unlikely to be related to drinking history but is more likely to be genetically influenced. This is supported by findings that P3b amplitudes are reduced in subjects at high-risk compared to low-risk for alcoholism. Although there are few studies of P3a in HR subjects, lower P3a amplitudes have been reported with a novel nontarget stimulus paradigm, as well as with a difficult three-stimulus visual paradigm. Using a similar three-tone auditory paradigm in which the discriminability between the target and standard tone is difficult, the P3a component can also be reliably elicited with a rare nontarget perceptually distinct stimulus. This technique was employed in young adult subjects at low-risk and high-risk for alcoholism. METHODS: A total of 17 low-risk and 24 high-risk male subjects were employed as subjects in an auditory paradigm that yielded a large amplitude P3a with a centro-frontal maximum to the nontarget and a robust low amplitude prolonged P3b with a parietal maximum amplitude to the target stimulus. Current source density maps were derived to assess topographic differences between low-risk and high-risk subjects. RESULTS: The high-risk group manifested significantly lower P3a amplitudes than the low-risk group at the frontal electrodes to rare nontarget stimuli. High-risk subjects also demonstrated a more disorganized current source density map for P3a compared to low-risk subjects. CONCLUSIONS: The reduction of P3a in the high-risk group may be due to cortical dysfunction including the frontal and prefrontal cortex. The lower P3a amplitude coupled with more disorganized current source density maps suggest inefficient brain functioning in high-risk subjects.

Mismatch negativity in subjects at high risk for alcoholism.

BACKGROUND: Evidence from P300 studies in both alcohol-dependent and high-risk (HR) individuals suggests that the reduced P300 amplitudes that often characterize these individuals may reflect a deficit in inhibition (hyperexcitability) in the central nervous system. In this context, the mismatch negativity (MMN) was investigated in the male and female HR offspring of alcohol-dependent fathers and a mixed-sex, low-risk (LR) control group. METHODS: As subjects read popular materials, they received a random sequence of 500 binaurally presented tones of 600 Hz and 1600 Hz. The designation of the rare stimulus (n = 60 trials) and frequent stimulus (n = 440 trials) was alternated across subjects. Recordings of MMN were made from 61 electrodes; risk group comparisons were restricted to the five frontal midline electrodes: Fpz, Afz, Fz, Fcz, and Cz. The MMN was obtained by calculating the integral of the area under the curve for both the frequent and rare waveforms over an interval from 100 to 190 msec and then subtracting the former from the latter. RESULTS: The primary observation was that MMN responses in the HR group were significantly larger than those in the LR group. In addition, both LR and HR individuals manifested differential responses to the rare and frequent stimuli, and MMN responses in both groups were largest at Fcz and smallest at Fpz. DISCUSSION: The results indicate that individuals at high risk for alcoholism differ electrophysiologically from LR controls. These differences were manifested as larger magnitudes of the MMN. The findings suggest the possibility that as measured by the MMN, individuals at high risk for alcoholism may be characterized by a deficit in inhibition (excessive neural excitation). The presence of these preexisting central nervous system states may lead to ethanol use for self-medication, which then may facilitate the development of both tolerance to and dependence on ethanol.

P300 event-related potential amplitude as an endophenotype of alcoholism--evidence from the collaborative study on the genetics of alcoholism.

There is substantial information supporting the role of genetic factors in the susceptibility for alcohol dependence. However, the identification of specific genes that contribute to this predisposition has proven elusive, although several theoretically relevant candidates, e.g. DRD2 or 5-HT(1B), have been considered. The difficulty in identifying specific genes may be related to the clinical heterogeneity of the disorder resulting in a poorly defined phenotype for genetic analysis. An alternative approach to the use of a diagnostic phenotype for identifying alcoholism susceptibility genes may lie in the examination of the neurobiological correlates of the disorder, the so-called endophenotypes. One possible endophenotype of alcohol dependence may be related to the P300 waveform of the event-related brain potential (ERP). Using data obtained from the Collaborative Study on the Genetics of Alcoholism (COGA), a multi-site family-based study, the utility of P300 amplitude as an endophentype was examined. Differences in P300 amplitude were found between alcoholics and nonalcoholics, between unaffected relatives of alcoholics and relatives of controls, as well as between unaffected offspring of alcoholic fathers and offspring of controls. A genetic analysis indicated that attributes of the P(3) ERP waveform are heritable, and a quantitative trait locus analysis found linkage to several chromosomal regions. These data provide significant support for P300 as an endophenotype for alcohol dependence.

Genetics of event-related brain potentials in response to a semantic priming paradigm in families with a history of alcoholism.

Event-related brain potentials (ERPs) are altered in patients with a variety of psychiatric disorders and may represent quantitative correlates of disease liability that are more amenable to genetic analysis than disease status itself. Results of a genomewide linkage screen are presented for amplitude of the N4 and P3 components of the ERP, measured at 19 scalp locations in response to a semantic priming task for 604 individuals in 100 pedigrees ascertained as part of the Collaborative Study on the Genetics of Alcoholism. N4 and P3 amplitudes in response to three stimuli (nonwords, primed words [i.e., antonyms], and unprimed words) all showed significant heritabilities, the highest being.54. Both N4 and P3 showed significant genetic correlations across stimulus type at a given lead and across leads within a stimulus, indicating shared genetic influences among the traits. There were also substantial genetic correlations between the N4 and P3 amplitudes for a given lead, even across stimulus type. N4 amplitudes showed suggestive evidence of linkage in several chromosomal regions, and P3 amplitudes showed significant evidence of linkage to chromosome 5 and suggestive evidence of linkage to chromosome 4.