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High Dimensional Endophenotype Ranking in the Search for Major Depression Risk Genes

      Background

      Despite overwhelming evidence that major depression is highly heritable, recent studies have localized only a single depression-related locus reaching genome-wide significance and have yet to identify a causal gene. Focusing on family-based studies of quantitative intermediate phenotypes or endophenotypes, in tandem with studies of unrelated individuals using categorical diagnoses, should improve the likelihood of identifying major depression genes. However, there is currently no empirically derived statistically rigorous method for selecting optimal endophentypes for mental illnesses. Here, we describe the endophenotype ranking value, a new objective index of the genetic utility of endophenotypes for any heritable illness.

      Methods

      Applying endophenotype ranking value analysis to a high-dimensional set of over 11,000 traits drawn from behavioral/neurocognitive, neuroanatomic, and transcriptomic phenotypic domains, we identified a set of objective endophenotypes for recurrent major depression in a sample of Mexican American individuals (n = 1122) from large randomly selected extended pedigrees.

      Results

      Top-ranked endophenotypes included the Beck Depression Inventory, bilateral ventral diencephalon volume, and expression levels of the RNF123 transcript. To illustrate the utility of endophentypes in this context, each of these traits were utlized along with disease status in bivariate linkage analysis. A genome-wide significant quantitative trait locus was localized on chromsome 4p15 (logarithm of odds = 3.5) exhibiting pleiotropic effects on both the endophenotype (lymphocyte-derived expression levels of the RNF123 gene) and disease risk.

      Conclusions

      The wider use of quantitative endophenotypes, combined with unbiased methods for selecting among these measures, should spur new insights into the biological mechanisms that influence mental illnesses like major depression.

      Key Words

      Major depression is a clinically heterogeneous and common mental illness (
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      Major depressive disorder.
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      ). Despite overwhelming evidence that major depression is heritable (
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      Genetic epidemiology of major depression: Review and meta-analysis.
      ), recent case-control association studies have failed to identify a locus reaching genome-wide significance (
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      ), leading some to conclude that common genetic variants with substantial odds ratios are unlikely to exist for the disease (
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      • Kong X.
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      Genome-wide association study of recurrent major depressive disorder in two European case-control cohorts.
      ). In contrast, recent family-based linkage studies of major depression identified a significant quantitative trait locus (QTL) on chromosome 3p25-26 (logarithm of odds [LOD] = 4.0) in a large sample of affected sibling pairs (
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      A genome-wide significant linkage for severe depression on chromosome 3: The Depression Network Study.
      ). This effect replicated in a smaller sample of individuals ascertained for heavy smoking (
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      A 3p26-3p25 genetic linkage finding for DSM-IV major depression in heavy smoking families.
      ). However, the causal gene(s) for this QTL remain to be identified. Given our slow pace of discovery, new approaches may be necessary to improve understanding of specific causal genes influencing risk of mental illness. One possible approach to speed gene localization/identification is the use of informative quantitative intermediate phenotypes or endophenotypes in families (
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      Novel family-based approaches to genetic risk in thrombosis.
      ,
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      ). Such an approach has strategic benefits (e.g., simultaneous identification of endophenotypes, increased power to identify genes, increased power to detect rare functional variants) over the more common paradigm that has focused on collections of unrelated individuals and relied solely on categorical diagnoses. Endophenotype exploitation should improve the likelihood of identifying major depression genes (
      • Blangero J.
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      Novel family-based approaches to genetic risk in thrombosis.
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      Discovering endophenotypes for major depression.
      ).
      Although the application of allied phenotypes has been successful in other complex illnesses (
      • Gottesman I.I.
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      The endophenotype concept in psychiatry: Etymology and strategic intentions.
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      Common variants at 30 loci contribute to polygenic dyslipidemia.
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      A major quantitative trait locus determining serum leptin levels and fat mass is located on human chromosome 2.
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      A large-scale genome-wide association study of Asian populations uncovers genetic factors influencing eight quantitative traits.
      ), difficulties choosing appropriate endophenotypes for mental disorders have limited their use in psychiatry, where relatively less is known about the biological mechanisms that predispose illness than in other areas of medicine. While the endophenotype concept is widely espoused in psychiatric genetics (
      • Gottesman I.I.
      • Gould T.D.
      The endophenotype concept in psychiatry: Etymology and strategic intentions.
      ,
      • Insel T.
      • Cuthbert B.
      Endophenotypes: Bridging genomic complexity and disorder heterogeneity.
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      • Ritsner M.
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      ), a formal or standardized approach for the identification of endophenotypes is lacking. Most studies employ purely phenotypic correlations between disease risk and a quantitative risk factor to define putative endophenotypes. However, the endophenotype concept fundamentally depends on the existence of joint genetic determination of both endophenotype and disease risk (
      • Blangero J.
      • Williams J.T.
      • Almasy L.
      Novel family-based approaches to genetic risk in thrombosis.
      ,
      • Gottesman I.I.
      • Gould T.D.
      The endophenotype concept in psychiatry: Etymology and strategic intentions.
      ). This obligatory pleiotropy is most efficiently tested using family-based observations to assess both the heritability of the endophenotype and its genetic correlation with disease liability. To facilitate the identification of optimal endophenotypes, we developed the endophenotype ranking value (ERV), a novel objective index of the genetic utility of endophenotypes for an illness. The ERV provides an unbiased and empirically derived method for choosing appropriate endophenotypes in a manner that balances the strength of the genetic signal for the endophenotype and the strength of its relation to the disorder of interest. It is defined using the square root of the heritability of the illness (hi2), the square root of the heritability of the endophenotype (he2), and their genetic correlation (ρg) and is expressed in the following formula:
      ERVie=|hi2he2ρg|


      Endophenotype ranking values vary between 0 and 1, where higher values indicate that the endophenotype and the illness are more strongly influenced by shared genetic factors. This method necessitates that endophenotypes be heritable and have some level of pleiotropy with the studied illness, reducing the heterogeneity of the disease and focusing on the proportion of shared genetic factors influencing both the endophenotype and the illness. An advantage of the ERV approach is that very large numbers of potential endophenotypes can be efficiently assessed before conducting molecular genetic analyses, analogous to high-throughput screening methods developed for drug discovery. Furthermore, the ERV approach is applicable to any heritable disease and any set of potentially relevant traits.
      Applying ERV analysis to a high-dimensional set of traits, we identified a set of significant endophenotypes for recurrent major depression (recurrent major depressive disorder [rMDD]). We focused on recurrent depression to reduce the clinical heterogeneity of the disorder and potentially increase the genetic control over the illness (
      • McGuffin P.
      • Katz R.
      • Watkins S.
      • Rutherford J.
      A hospital-based twin register of the heritability of DSM-IV unipolar depression.
      ,
      • Shi J.
      • Potash J.
      • Knowles J.
      • Weissman M.
      • Coryell W.
      • Scheftner W.
      • et al.
      Genome-wide association study of recurrent early-onset major depressive disorder.
      ). We performed an automated high-dimensional search for endophenotypes via the ranking of 37 behavioral/neurocognitive, 85 neuroanatomic, and 11,337 lymphocyte-based transcriptional candidate endophenotypes for rMDD using data acquired from 1122 Mexican American individuals from large randomly ascertained extended pedigrees who participated in the Genetics of Brain Structure and Function study. Finally, we employed the top-ranked endophentypes in bivariate linkage analysis, localizing a significant QTL exhibiting pleiotropic effects on both endophenotype and disease risk.

      Methods and Materials

       Participants

      A total of 1,122 Mexican American individuals from extended pedigrees (71 families, average size 14.9 [1–87] people) were included in the analysis. Participants were 64% female and ranged in age from 18 to 97 (mean ± SD 47.11 ± 14.2) years. Individuals in this cohort have actively participated in research for over 18 years and were randomly selected from the community with the constraints that they are of Mexican American ancestry, part of a large family, and live within the San Antonio region (see [21] for recruitment details). No other inclusion or exclusion criteria were imposed in the initial study. However, individuals were excluded from scanning for magnetic resonance imaging contraindications. In addition, individuals were excluded from scanning and neurocognitive evaluation for history of neurological illnesses, stroke, or other major neurological event. Reported pedigree relationships were empirically verified with autosomal markers and intrafamilial relationships were edited if necessary (see Table 1 for familial relationships). All participants provided written informed consent on forms approved by the Institutional Review Boards at the University of Texas Health Science Center San Antonio and at Yale University.
      Table 1Pair-Wise Pedigree Relationships
      RelationshipNumber of Pairs
      Parent-Offspring689
      Siblings784
      Grandparent-Grandchild122
      Avuncular1248
      Half Siblings135
      1st Cousins1602
      3rd Degree2128
      4th Degree2235
      5th Degree1341
      6th Degree584
      7th Degree309
      8th Degree36

       Diagnostic Assessment

      All participants received the Mini-International Neuropsychiatric Interview (MINI) (
      • Sheehan D.V.
      • Lecrubier Y.
      • Sheehan K.H.
      • Amorim P.
      • Janavs J.
      • Weiller E.
      • et al.
      The Mini-International Neuropsychiatric Interview (M.I.N.I.): The development and validation of a structured diagnostic psychiatric interview for DSM-IV and ICD-10.
      ), a semistructured interview augmented to include items on lifetime diagnostic history. Masters- and doctorate-level research staff, with established reliability (κ ≥ .85) for affective disorders, conducted all interviews. All subjects with possible psychopathology were discussed in case conferences that included licensed psychologists or psychiatrists. Lifetime consensus diagnoses were determined based on available medical records, the MINI interview, and the interviewer's narrative. Recurrent major depression was defined as two or more distinct episodes of depression meeting DSM-IV criteria.

       Behavioral and Neurocognitive Assessment

      Each participant received a 90-minute neuropsychological evaluation consisting of standard and computerized measures (
      • Glahn D.
      • Almasy L.
      • Blangero J.
      • Burk G.
      • Estrada J.
      • Peralta J.
      • et al.
      Adjudicating neurocognitive endophenotypes for schizophrenia.
      ,
      • Glahn D.
      • Almasy L.
      • Barguil M.
      • Hare E.
      • Peralta J.
      • Kent J.J.
      • et al.
      Neurocognitive endophenotypes for bipolar disorder identified in multiplex multigenerational families.
      ). Thirty-five neurocognitive variables were derived from 17 separate neuropsychological tests, including measures of attention/concentration, executive processing, working memory, declarative memory, language processing, intelligence, and emotional processing. In addition, participants completed two questionnaires indexing depressive mood: the Beck Depression Inventory-II (BDI-II) (
      • Beck A.T.
      • Steer R.A.
      • GK B.
      Manual for the Beck Depression Inventory-II.
      ) and the neuroticism questions of the Eysenck Personality Questionnaire (
      • Eysenck H.J.
      • SBG E.
      Manual of the Eysenck Personality Questionnaire.
      ).

       Image Acquisition and Processing

      Magnetic resonance imaging data were acquired on a 3T Siemens (Erlangen, Germany) Trio scanner with an 8-channel head coil in the Research Imaging Institute, University of Texas Health Science Center San Antonio. Isotropic anatomic images (800 μm) were acquired for each subject using a retrospective motion-corrected protocol (
      • Kochunov P.
      • Lancaster J.
      • Glahn D.
      • Purdy D.
      • Laird A.
      • Gao F.
      • Fox P.
      Retrospective motion correction protocol for high-resolution anatomical MRI.
      ). This protocol included the acquisition of seven full-resolution volumes using a T1-weighted, three-dimensional TurboFlash sequence with the following scan parameters: echo time [TE]/repetition time [TR]/inversion time = 3.04/2100/785 milliseconds, flip angle = 13°. Surface-based image analyses were conducted with FreeSurfer (
      • Dale A.M.
      • Fischl B.
      • Sereno M.I.
      Cortical surface-based analysis I. Segmentation and surface reconstruction.
      ,
      • Fischl B.
      • Sereno M.I.
      • Dale A.M.
      Cortical surface-based analysis II: Inflation, flattening, and a surface-based coordinate system.
      ) following standardized protocols (
      • Winkler A.
      • Kochunov P.
      • Blangero J.
      • Almasy L.
      • Zilles K.
      • Fox P.
      • et al.
      Cortical thickness or grey matter volume? The importance of selecting the phenotype for imaging genetics studies.
      ). T1-weighted images were segmented into gray matter thickness measures for 53 cortical regions and 21 subcortical volumes (averaged across both hemispheres).
      T2-weighted imaging data were acquired using a 1-mm isotropic, turbo spin echo FLAIR sequence with the following parameters: TR/TE/inversion time/flip angle/echo train length = 5 seconds/353 milliseconds/1.8 seconds/180°/221. White-matter hyperintensities were manually delineated in three-dimensional space using in-house software by experienced neuroanatomists with high (r2 > .90) test-retest reproducibility (
      • Kochunov P.
      • Glahn D.
      • Winkler A.
      • Duggirala R.
      • Olvera R.
      • Cole S.
      • et al.
      Analysis of genetic variability and whole genome linkage of whole-brain, subcortical, and ependymal hyperintense white matter volume.
      ).
      Diffusion tensor imaging data acquisition used a single-shot single spin echo, echo planar imaging sequence with a spatial resolution of 1.7 × 1.7 × 3.0 mm (TR/TE = 8000/87 milliseconds, field of view = 200 mm, 55 directions, b = 0, and 800 seconds/mm2). Fractional anisotropy values were estimated for each subject on 13 tracts using Tract-Based Spatial Statistics software (
      • Smith S.M.
      • Jenkinson M.
      • Johansen-Berg H.
      • Rueckert D.
      • Nichols T.E.
      • Mackay C.E.
      • et al.
      Tract-based spatial statistics: Voxelwise analysis of multi-subject diffusion data.
      ). Diffusion tensor imaging images provided fractional anisotropy indices for 13 white-matter tracts (averaged across both hemispheres).

       Transcriptional Profiling

      Transcriptional profiling followed procedures described by Göring et al. (
      • Goring H.H.
      • Curran J.E.
      • Johnson M.P.
      • Dyer T.D.
      • Charlesworth J.
      • Cole S.A.
      • et al.
      Discovery of expression QTLs using large-scale transcriptional profiling in human lymphocytes.
      ). Total RNA was isolated from lymphocytes and hybridized to Illumina (San Diego, California) Sentrix Human Whole Genome (WG-6) Series 1 BeadChips. These BeadChips simultaneously probe ∼48,000 transcripts, representing more than 25,000 annotated human genes. Although we previously identified 20,413 quantitative transcripts in lymphocytes, we only examined those with heritabilities greater than or equal to .20 (n = 11,337) in the current analysis.

       Genotyping

      DNA extracted from lymphocytes was used in polymerase chain reactions (PCRs) for the amplification of individual DNA at 432 dinucleotide repeat microsatellite loci (short tandem repeats [STRs]), spaced approximately 10 cM intervals apart across the 22 autosomes, with fluorescently labeled primers from the MapPairs Human Screening Set, Versions 6 and 8 (Research Genetics, Huntsville, Alabama). Polymerase chain reactions were performed separately according to manufacturer specifications in Applied Biosystems 9700 thermocyclers (Applied Biosystems, Foster City, California). For each individual, the products of separate PCRs were pooled using the Robbins Hydra-96 Microdispenser (Robbins Scientific Corporation, Sunnyvale California), and a labeled size standard was added to each pool. The pooled PCR products were loaded into an ABI PRISM 377 or 3100 Genetic Analyzer (Applied Biosystems) for laser-based automated genotyping. The STRs and standards were detected and quantified, and genotypes were scored using the Genotyper software package (Applied Biosystems).

       Quantitative Genetic Analyses

      All analyses were conducted with SOLAR (
      • Almasy L.
      • Blangero J.
      Multipoint quantitative-trait linkage analysis in general pedigrees.
      ), which employs maximum likelihood variance decomposition methods to determine the relative importance of genetic and environmental influences by modeling the covariance among family members as a function of genetic proximity (see Supplement 1 for detail on variance components methods).
      The ERV represents the standardized genetic covariance between the endophenotype (denoted by the subscript, e) and illness (denoted by the subscript, i) and is defined as ERVie = |√hi2he2ρg|. Heritability (h2) represents the portion of the phenotypic variance accounted for by additive genetic variance (h2 = σ2g2p). Genetic correlation represents the common genetic covariance between two traits or pleiotropy (
      • Almasy L.
      • Dyer T.D.
      • Blangero J.
      Bivariate quantitative trait linkage analysis: Pleiotropy versus co-incident linkages.
      ). Bivariate quantitative genetic analysis was used to estimate the genetic (ρg) and environmental (ρe) correlations between each potential endophenotype and rMDD. The phenotypic correlation (ρp), which quantifies the overall relationship between the two traits, can be derived from the genetic and environmental correlations as ρp = ρg√(h2eh2i) + ρe√[(1-h2e)(1-h2i)]. These parameters are estimated by jointly utilizing all available pedigree information with a multivariate normal threshold model for combined dichotomous/continuous traits (
      • Williams J.T.
      • Van Eerdewegh P.
      • Almasy L.
      • Blangero J.
      Joint multipoint linkage analysis of multivariate qualitative and quantitative traits I. Likelihood formulation and simulation results.
      ,
      • Williams J.T.
      • Begleiter H.
      • Porjesz B.
      • Edenberg H.J.
      • Foroud T.
      • Reich T.
      • et al.
      Joint multipoint linkage analysis of multivariate qualitative and quantitative traits II. Alcoholism and event-related potentials.
      ). The significance of the ERV was tested by comparing the ln likelihood for the restricted null model (with ρg constrained to equal 0) against the ln likelihood for the alternative model in which the ρg parameter is estimated. The resultant likelihood ratio test is asymptotically distributed as a chi-square with a single degree of freedom. The corresponding p value is identical to that used for genetic correlation. Before analysis, endophenotypes were normalized using an inverse Gaussian transformation. Age, sex, age × sex, age2, and age2 × sex were included as covariates whose effects were simultaneously estimated in all analyses.

       Bivariate Linkage Analysis

      Bivariate linkage analysis exploits the genetic covariance between the endophenotype and the illness to improve the power to localize QTLs and to detect QTL-specific pleiotropic effects (
      • Williams J.T.
      • Van Eerdewegh P.
      • Almasy L.
      • Blangero J.
      Joint multipoint linkage analysis of multivariate qualitative and quantitative traits I. Likelihood formulation and simulation results.
      ). After addressing (by blanking, recalling, or retyping) mistyping errors identified using Simwalk II (Savannah Simulations AG, Herrliberg, Switzerland) (
      • Sobel E.
      • Papp J.C.
      • Lange K.
      Detection and integration of genotyping errors in statistical genetics.
      ), genotype data were used to compute maximum likelihood estimates of allele frequencies. Matrices of empirical estimates of identity-by-descent allele sharing at points throughout the genome for every relative pair were computed using the Loki package (
      • Heath S.C.
      Markov chain Monte Carlo segregation and linkage analysis for oligogenic models.
      ). We used high-resolution chromosomal maps provided by deCODE genetics (deCODE genetics, Reykjavik Iceland) (
      • Kong A.
      • Gudbjartsson D.F.
      • Sainz J.
      • Jonsdottir G.M.
      • Gudjonsson S.A.
      • Richardsson B.
      • et al.
      A high-resolution recombination map of the human genome.
      ). For the localization of QTLs, we performed both univariate and bivariate variance components linkage analyses by employing the models for combined analysis of quantitative and dichotomous phenotypes described by Williams et al. (
      • Williams J.T.
      • Van Eerdewegh P.
      • Almasy L.
      • Blangero J.
      Joint multipoint linkage analysis of multivariate qualitative and quantitative traits I. Likelihood formulation and simulation results.
      ,
      • Williams J.T.
      • Begleiter H.
      • Porjesz B.
      • Edenberg H.J.
      • Foroud T.
      • Reich T.
      • et al.
      Joint multipoint linkage analysis of multivariate qualitative and quantitative traits II. Alcoholism and event-related potentials.
      ). Once a genome-wide significant localization was made, formal single degree of freedom likelihood ratio tests for pleiotropy were performed to test the specific hypothesis that a QTL at that location influenced a given endophenotype/rMDD risk (
      • Almasy L.
      • Dyer T.D.
      • Blangero J.
      Bivariate quantitative trait linkage analysis: Pleiotropy versus co-incident linkages.
      ).

      Results

       Heritability of Recurrent Major Depression

      Two hundred fifteen individuals met criteria for lifetime rMDD (19% of the sample; 73% female subjects). Eighty-six individuals were clinically depressed at the time of the assessment. The estimated heritability for lifetime rMDD was h2 = .463 (standard error ± .12), p = 4.0 × 10−6. We previously demonstrated that this heritability estimate is not significantly influenced by common environmental factors as indexed by shared household (
      • Olvera R.L.
      • Bearden C.E.
      • Velligan D.I.
      • Almasy L.
      • Carless M.A.
      • Curran J.E.
      • et al.
      Common genetic influences on depression, alcohol, and substance use disorders in Mexican-American families.
      ). Additionally, there was no evidence for dominance (p = .14) or additive × additive epistasis (p = .18), suggesting that the heritability estimate reflects additive genetic factors.

       Potential Behavioral/Neurocognitive Endophenotypes

      The 10 top-ranked behavioral/cognitive endophenotypes are presented in Table 2. The BDI-II was the highest ranked endophenotype in this class. Although the BDI-II was developed as an index of mood state, the heritability of this measure was h2 = .254 (± .07), p = 5.6 × 10−5, demonstrating that 25% of the variability on this measure is due to additive genetic factors. The genetic correlation between the BDI-II and the neuroticism questions from the Eysenck Personality Questionnaire, the second best ranked endophenotype in this domain, was ρG = .870 (± .09), p = 3.3 × 10−4, suggesting significant pleiotropy and potential redundancy between these two measures. Top-ranked cognitive measures include indices of working and declarative memory, attention, and emotion recognition.
      Table 2Ten Top-Ranked Endophenotypes per Domain for Recurrent Major Depression
      EndophenotypesERVp ValueGenetic Correlation (ρg)Heritability (h2)
      Behavioral/Neurocognitive
       Beck Depression Inventory II.2631.9 × 10−5.825.253
       EPQ Neuroticism.2381.79 × 10−4.739.228
       Declarative Memory (CVLT Recognition).1365.49 × 10−2−.338.338
       Working Memory (Digit Span Forward).1425.69 × 10−2−.295.490
       Working Memory (Letter-Number Span).1356.39 × 10−2−.267.541
       Penn Facial Memory (Immediate).1276.99 × 10−2−.319.344
       Penn Facial Memory (Delayed).1348.19 × 10−2−.295.439
       Attention (CPT hits).1198.39 × 10−2−.387.202
       Attention (Trails A).1219.69 × 10−2.303.340
       Penn Emotion Recognition.1171.09 × 10−1−.288.347
      Neuroimaging
       Ventral diencephalon volume.2403.99 × 10−3−.425.694
       Parietal hyperintensity volume.2827.89 × 10−3.569.573
       Hippocampus volume.2041.29 × 10−2−.347.771
       Pallidum volume.2031.39 × 10−2−.396.562
       Cerebellar white matter volume.2181.39 × 10−2−.443.524
       Frontal hyperintensity volume.2551.39 × 10−2.483.635
       Corticospinal tract (FA).2082.19 × 10−2−.900.101
       Subcortical hyperintensity volume.2134.19 × 10−2.473.459
       Superior parietal gyrus thickness.1784.59 × 10−2.363.480
       Thalamus proper volume.1724.89 × 10−2−.294.739
      Transcriptional
      RNF123 (3p24).3235.29 × 10−6−.943.209
      PDXK (21q22).3311.19 × 10−5−.689.489
      ZFP64 (20q13).3522.09 × 10−5−.711.470
      RWDD2A (6q14).2602.39 × 10−5.666.337
      B4GALT7 (5q35).2763.69 × 10−5−.732.309
      MARK2 (11q12).1803.99 × 10−5−.399.412
      GADD45A (1p31).3444.09 × 10−5.729.432
      PIGN (18q21).2747.99 × 10−5.646.399
      HTT (4p16).2257.99 × 10−5−.546.358
      ABHD12 (20p11).2691.19 × 10−4.755.272
      Ten top-ranked endophenotypes for recurrent major depression in the categories of behavioral/cognitive, neuroimaging, and RNA transcripts (see Supplement 1 for the complete rankings). Genetic correlations are between the respective endophenotypes and lifetime affection status. Endophenotype heritability estimates were estimated as part of bivariate models.
      CPT, Continues Performance Test; CVLT, California Verbal Learning Test; EPQ, Eysenck Personality Questionnaire; ERV, endophenotype ranking value; FA, fractional anisotropy.

       Potential Neuroimaging Endophenotypes

      The top-ranked brain region was bilateral ventral diencephalon volume (Table 2), a region primarily comprised of the hypothalamus. As part of the hypothalamic-pituitary-adrenal axis, the hypothalamus mediates neuroendocrine and neurovegetative functions and has been consistently implicated in the neurobiology of depression (
      • Nestler E.
      • Barrot M.
      • DiLeone R.
      • Eisch A.
      • Gold S.
      • Monteggia L.
      Neurobiology of depression.
      ). Hypothalamic-pituitary-adrenal axis activity is regulated, in part, by the hippocampus and amygdala (
      • Nestler E.
      • Barrot M.
      • DiLeone R.
      • Eisch A.
      • Gold S.
      • Monteggia L.
      Neurobiology of depression.
      ), both regions with reasonably high ERV ranking (3rd and 13th ranked, respectively). Our results suggest that the genetic factors that influence the structure of these subcortical regions (Figure 1) also confer risk for rMDD. Additionally, white-matter hyperintensity measures, which are associated with aging, cerebrovascular dysfunction, smoking, and a host of other depression-related pathologies (
      • Fazekas F.
      • Kleinert R.
      • Offenbacher H.
      • Schmidt R.
      • Kleinert G.
      • Payer F.
      • et al.
      Pathologic correlates of incidental MRI white matter signal hyperintensities.
      ), were highly ranked endophenotypes for rMDD. This result is consistent with and extends the vascular depression hypothesis (
      • Alexopoulos G.
      • Meyers B.
      • Young R.
      • Campbell S.
      • Silbersweig D.
      • Charlson M.
      'Vascular depression' hypothesis.
      ), by suggesting common genetic factors increase risk for rMDD and white-matter hyperintensities.
      Figure thumbnail gr1
      Figure 1Endophenotype ranking value statistics for subcortical brain regions and recurrent major depression. Volume measurements of subcortical nuclei were found to share genetic variance with liability for recurrent major depression in extended pedigrees selected without regard to phenotype. Endophenotype ranking value statistics, which provide an unbiased and empirically derived method for choosing appropriate endophenotypes, were strongest for the ventral diencephalon volume, a region primarily comprised of the hypothalamus. For anatomical reference, in this image the cortex is shown as a semi-transparent structure. ERV, endophenotype ranking value; L, left; R, right.

       Potential Transcriptional Endophenotypes

      Endophenotype ranking value analyses on 11,337 transcripts are presented in Figure 2 and top-ranked transcriptional endophenotypes for rMDD are presented in Table 2. The top-ranking transcript, RNF123, is a member of the E3 ubiquitin-protein ligase family, which have diverse functions including protein degradation and modulation of protein assembly, structure, function, and localization (
      • Doolittle M.
      • Ben-Zeev O.
      • Bassilian S.
      • Whitelegge J.
      • Péterfy M.
      • Wong H.
      Hepatic lipase maturation: A partial proteome of interacting factors.
      ,
      • Deshaies R.
      • Joazeiro C.
      RING domain E3 ubiquitin ligases.
      ). Other members of the ubiquitin-proteosome system were previously implicated in anxiety, depression, and vulnerability to seizures (
      • Kim S.
      • Zhang S.
      • Choi K.
      • Reister R.
      • Do C.
      • Baykiz A.
      • Gershenfeld H.K.
      An E3 ubiquitin ligase, Really Interesting New Gene (RING) Finger 41, is a candidate gene for anxiety-like behavior and beta-carboline-induced seizures.
      ,
      • Nishioka G.
      • Yamada M.
      • Kudo K.
      • Takahashi K.
      • Kiuchi Y.
      • Higuchi T.
      • et al.
      Induction of kf-1 after repeated electroconvulsive treatment and chronic antidepressant treatment in rat frontal cortex and hippocampus.
      ). PDXK, an essential cofactor in the intermediate metabolism of amino acids and neurotransmitters, including serotonin and dopamine (
      • Gachon F.
      • Fonjallaz P.
      • Damiola F.
      • Gos P.
      • Kodama T.
      • Zakany J.
      • et al.
      The loss of circadian PAR bZip transcription factors results in epilepsy.
      ), confers risk for Parkinson disease (
      • Elstner M.
      • Morris C.
      • Heim K.
      • Lichtner P.
      • Bender A.
      • Mehta D.
      • et al.
      Single-cell expression profiling of dopaminergic neurons combined with association analysis identifies pyridoxal kinase as Parkinson's disease gene.
      ) and epilepsy (
      • Gachon F.
      • Fonjallaz P.
      • Damiola F.
      • Gos P.
      • Kodama T.
      • Zakany J.
      • et al.
      The loss of circadian PAR bZip transcription factors results in epilepsy.
      ). Additionally, MARK2 and ABHD12 have previously been implicated in neuronal migration (
      • Sapir T.
      • Sapoznik S.
      • Levy T.
      • Finkelshtein D.
      • Shmueli A.
      • Timm T.
      • et al.
      Accurate balance of the polarity kinase MARK2/Par-1 is required for proper cortical neuronal migration.
      ), degeneration (
      • Nishimura I.
      • Yang Y.
      • Lu B.
      PAR-1 kinase plays an initiator role in a temporally ordered phosphorylation process that confers tau toxicity in Drosophila.
      ), and regulation of endocannabinoid signaling pathways (
      • Blankman J.
      • Simon G.
      • Cravatt B.
      A comprehensive profile of brain enzymes that hydrolyze the endocannabinoid 2-arachidonoylglycerol.
      ), respectively. Although other identified transcripts are less obvious candidates for rMDD risk, they may represent novel genes whose functions are not fully understood and may extend to depression phenotypes.
      Figure thumbnail gr2
      Figure 2Manhattan plot depicting whole transcriptomic search for expression-based endophenotypes for recurrent major depression. Endophenotype ranking values were calculated for 11,337 detected lymphocyte-based transcripts and recurrent major depression. Dashed lines reflect cutoff points for false discovery rate < .10 (13 transcripts) and false discovery rate <.25 (29 transcripts).

       Genome-Wide Bivariate Linkage Analyses Using rMDD and Top-Ranked Endophenotypes

      We performed a genome-wide linkage-based search for pleiotropic quantitative trait loci influencing disease risk and the top-ranked endophenotype from each class: BDI-II, bilateral ventral diencephalon volume, and the RNF123 transcript. First, standard univariate linkage analyses were performed. Two traits exhibited genome-wide or near genome-wide significance QTLs. The best univariate score for rMDD was found on chromosome 4 at 47 cM (LOD = 2.98, nominal p = .00011). While not reaching traditional genome-wide significance, this result points to a potential disease-related QTL at chromosomal location 4p15. The bilateral ventral diencephalon exhibited an unequivocal genome-wide significant peak on chromosome 7 at 131 cM (LOD = 3.40, nominal p = 3.8 × 10−5). Neither BDI-II nor RNF123 expression levels showed strong evidence for causal QTLs in univariate analysis. Suggestive evidence for a QTL influencing BDI-II was found on chromosome 17 at 98 cM (LOD = 2.57, nominal p = .0003). We found little evidence for a QTL influencing quantitative RNF123 gene expression levels, with the single best univariate QTL location found on chromosome 6 at 53 cM (LOD = 1.81).
      Bivariate linkage analyses were performed to determine if QTL localization could be enhanced via simultaneous analysis with rMDD affection status. The most dramatic improvement in localization was seen for rMDD and RNF123 transcription levels. The bivariate analysis of this endophenotype/disease combination substantially improved the evidence for a QTL located at 4p15 seen in the univariate rMDD results. Figure 3 shows the QTL localization results for the bivariate analysis and the two related univariate analyses. The peak bivariate LOD (scaled to a standard single degree of freedom LOD) was 3.51 (nominal p = 3.8 × 10−5) at 45 cM, a marked improvement over that seen for rMDD alone. No other rMDD/endophenotype combination provided genome-wide evidence for QTLs.
      Figure thumbnail gr3
      Figure 3Detection of a quantitative trait locus influencing recurrent major depression and RNF123 expression levels on chromosome 4. Multipoint logarithm of odds (LOD) functions for chromosome 4 in 1122 individuals from large extended pedigrees from the Genetics of Brain Structure and Function Study. The black line represents the univariate linkage analysis for RNF123 expression levels alone. The blue line represents the univariate linkage analysis for recurrent major depression alone. The red line represents the bivariate linkage analysis for recurrent major depression and RNF123 and reaches genome-wide significance (LOD = 3.5) at 45 cM (chromosomal band 4p15). The vertical axis is in LOD score units, and the horizontal axis is in units of genetic distance (cM) from the p arm telomere.
      Table 3 shows the results of likelihood ratio statistic-based formal tests of pleiotropy at the chromosome 4:45 cM location obtained from the bivariate analysis of RNF123/rMDD. The marginal results are from univariate analysis (technically co-incident linkage [35]) and the strict test of pleiotropy that can be performed using the bivariate linkage model. The chromosome 4 locus significantly influences rMDD (p = 4.7 × 10−5), RNF123 (p = .0010), and diencephalon volume (p = .0290) and shows a trend for BDI-II (p = .1170). The fact that this QTL influences both risk of rMDD and two of our three best endophenotypes provides additional validation for endophenotype identification, with evidence for rMDD increasing by nearly an order of magnitude. These results strongly support a QTL influencing rMDD and related endophenotypes at chromosome 4p15.
      Table 3Tests of Pleiotropy at the Chromosome 4:45 cM Quantitative Trait Locus
      TraitPleiotropy p Value from Bivariate ModelCo-Incident Linkage p Value from Univariate Model
      Recurrent MDD4.7 × 10−51.1 × 10−4
      RNF123 Expression.0010.0219
      Ventral Diencephalon Volume.0290.0266
      BDI-II Score.1170.5000
      BDI-II, Beck Depression Inventory-II; MDD, major depressive disorder.
      Given evidence for a QTL influencing diencephalon volume on chromosome 7, we tested for pleiotropic effects. As expected, these tests revealed a major effect on diencephalon (pleiotropy p value = 1.6 × 10−5) and rMDD liability (pleiotropy p value = .0437). Both of these results are substantially improved over their univariate analogues and only with bivariate analysis do we detect a significant influence of this QTL on rMDD liability. The other two leading endophenotypes show no pleiotropic effects at this QTL.

      Discussion

      Our results demonstrate the utility of the ERV approach for formally identifying endophenotypes in high-dimensional data and provide a novel genome-wide significant QTL for recurrent major depression. Bivariate genetic analyses including a quantitative endophenotype and disease risk significantly improved QTL detection over that observed utilizing diagnosis alone. These results may reflect the improved statistical sensitivity of quantitative over qualitative traits or that endophenotypes index a somewhat less heterogeneous aspect of the pathophysiology associated with mental illnesses (
      • Blangero J.
      Localization and identification of human quantitative trait loci: King harvest has surely come.
      ). In either case, quantitative endophenotypes can significantly improve the potential to localize loci for complex disorders like rMDD, where multiple genes with varying effects and incomplete penetrance are thought to interact with environmental factors to determine illness susceptibility.
      The present experiment demonstrates the utility of gene expression measures in peripheral tissues for psychiatric phenotypes. Transcripts can be considered endophenotypes that, while removed from the phenomenology-based diagnosis, are close to gene action and in the case of primary cis-regulation, provide evidence for a gene's involvement in the illness. Although brain tissue is ideal for gene expression studies in psychiatry, difficulty obtaining this tissue in genetically informative samples necessitates the use of a surrogate marker and lymphocytes appear to be good surrogates for detection of mental disease-relevant genes (
      • Tsuang M.T.
      • Nossova N.
      • Yager T.
      • Tsuang M.M.
      • Guo S.C.
      • Shyu K.G.
      • et al.
      Assessing the validity of blood-based gene expression profiles for the classification of schizophrenia and bipolar disorder: A preliminary report.
      ,
      • Borovecki F.
      • Lovrecic L.
      • Zhou J.
      • Jeong H.
      • Then F.
      • Rosas H.D.
      • et al.
      Genome-wide expression profiling of human blood reveals biomarkers for Huntington's disease.
      ). The lymphocyte measures used in the present experiment were collected 12 to 15 years before the current assessments, minimizing the potential that these traits were influenced by acute variation in mood or medication usage (
      • Tsankova N.
      • Berton O.
      • Renthal W.
      • Kumar A.
      • Neve R.
      • Nestler E.
      Sustained hippocampal chromatin regulation in a mouse model of depression and antidepressant action.
      ). It is notable that the top-ranked transcriptional endophenotype for rMDD was ranked higher than any of the behavioral/cognitive or neuroimaging traits, including BDI-II, suggesting that transcripts may provide an important new set of markers for disease risk.
      Our single strongest ERV result was observed for quantitative messenger RNA levels of the RNF123 gene with risk for rMDD. This gene (also known as KPC1) encodes ring finger protein 123, which is likely involved in the regulation of neurite outgrowth via its modulation of the degradation of the cyclin-dependent kinase inhibitor p27(Kip1) (
      • Zheng Y.L.
      • Li B.S.
      • Rudrabhatla P.
      • Shukla V.
      • Amin N.D.
      • Maric D.
      • et al.
      Phosphorylation of p27Kip1 at Thr187 by cyclin-dependent kinase 5 modulates neural stem cell differentiation.
      ,
      • Zhao J.
      • Zhang S.
      • Wu X.
      • Huan W.
      • Liu Z.
      • Wei H.
      • et al.
      KPC1 expression and essential role after acute spinal cord injury in adult rat.
      ). Cyclin-dependent kinase inhibitor p27(Kip1) is involved in increased hippocampal neuronal differentiation via a glucocorticoid receptor function that is observed upon administration of the antidepressant sertraline (
      • Anacker C.
      • Zunszain P.A.
      • Cattaneo A.
      • Carvalho L.A.
      • Garabedian M.J.
      • Thuret S.
      • et al.
      Antidepressants increase human hippocampal neurogenesis by activating the glucocorticoid receptor.
      ). Thus, RNF123 appears to be a novel candidate involved in hippocampal neurogenesis of significant relevance to depression risk. We observed a significant negative genetic correlation between RNF123 expression level and disease risk consistent with evidence that RNF123 inhibits p27(Kip1) and depression amelioration. Thus, RNF123 represents a potential drug target for depression.
      The dominant paradigm in psychiatric genetic studies focuses on a specific disease itself. However, as with most disease states, this end point is relatively distant from the causal anatomic or physiological disruption. In contrast, we supplement disease status with quantitative endophenotypes, selected through an empirically derived process, to identify and characterize genes that influence rMDD. Since these endophenotypes vary within the normal population, it is possible to localize genes influencing them in samples ascertained without regard to a specific phenotype (illness). The endophenotype and normal variation strategy have been successfully applied to the study of other complex diseases such as heart disease (
      • Kathiresan S.
      • Willer C.
      • Peloso G.
      • Demissie S.
      • Musunuru K.
      • Schadt E.
      • et al.
      Common variants at 30 loci contribute to polygenic dyslipidemia.
      ,
      • Almasy L.
      • Hixson J.E.
      • Rainwater D.L.
      • Cole S.
      • Williams J.T.
      • Mahaney M.C.
      • et al.
      Human pedigree-based quantitative-trait-locus mapping: Localization of two genes influencing HDL-cholesterol metabolism.
      ,
      • Ayra R.
      • Blangero J.
      • Williams K.
      • Almasy L.
      • Dyer T.
      • Leach R.
      • et al.
      Factors of insulin resistance syndrome (IRS)-related phenotypes are linked to genetic locations on chromosomes 6 and 7 in nondiabetic Mexican Americans.
      ) obesity (
      • Comuzzie A.
      • Hixson J.
      • Almasy L.
      • Mitchell B.
      • Mahaney M.
      • Dyer T.
      • et al.
      A major quantitative trait locus determining serum leptin levels and fat mass is located on human chromosome 2.
      ,
      • Cai G.
      • Cole S.A.
      • Freeland-Graves J.H.
      • MacCluer J.W.
      • Blangero J.
      • Comuzzie A.G.
      Genome-wide scans reveal quantitative trait loci on 8p and 13q related to insulin action and glucose metabolism: The San Antonio Family Heart Study.
      ,
      • Willer C.
      • Speliotes E.
      • Loos R.
      • Li S.
      • Lindgren C.
      • Heid I.
      • et al.
      Six new loci associated with body mass index highlight a neuronal influence on body weight regulation.
      ), diabetes (
      • Cai G.
      • Cole S.A.
      • Bastarrachea-Sosa R.A.
      • Maccluer J.W.
      • Blangero J.
      • Comuzzie A.G.
      Quantitative trait locus determining dietary macronutrient intakes is located on human chromosome 2p22.
      ,
      • Mitchell B.D.
      • Cole S.A.
      • Hsueh W.C.
      • Comuzzie A.G.
      • Blangero J.
      • MacCluer J.W.
      • Hixson J.E.
      Linkage of serum insulin concentrations to chromosome 3p in Mexican Americans.
      ), hypertension (
      • Cho Y.
      • Go M.
      • Kim Y.
      • Heo J.
      • Oh J.
      • Ban H.
      • et al.
      A large-scale genome-wide association study of Asian populations uncovers genetic factors influencing eight quantitative traits.
      ,
      • Kammerer C.M.
      • Gouin N.
      • Samollow P.B.
      • VandeBerg J.F.
      • Hixson J.E.
      • Cole S.A.
      • et al.
      Two quantitative trait loci affect ACE activities in Mexican-Americans.
      ), and osteoporosis (
      • Kammerer C.M.
      • Schneider J.L.
      • Cole S.A.
      • Hixson J.E.
      • Samollow P.B.
      • O'Connell J.R.
      • et al.
      Quantitative trait loci on chromosomes 2p, 4p, and 13q influence bone mineral density of the forearm and hip in Mexican Americans.
      ,
      • Kiel D.
      • Demissie S.
      • Dupuis J.
      • Lunetta K.
      • Murabito J.
      • Karasik D.
      Genome-wide association with bone mass and geometry in the Framingham Heart Study.
      ). However, this strategy has not been effectively applied in the search for mental illness genes.
      There is debate regarding the definition of a good endophenotype or even if endophenotypes will benefit the search for mental illness genes. We propose that endophenotypes that are heritable and genetically correlated with disease liability can facilitate gene identification. Although both disease and endophenotype must be heritable for the ERV approach, there is no requirement that the endophenotype exhibit higher heritability than the disease itself. Higher heritability estimates do not imply a simpler genetic architecture or improve the potential to localize genes (
      • Glahn D.
      • Almasy L.
      • Barguil M.
      • Hare E.
      • Peralta J.
      • Kent J.J.
      • et al.
      Neurocognitive endophenotypes for bipolar disorder identified in multiplex multigenerational families.
      ). A quantitative endophenotype with a low but significant heritability estimate that is genetically correlated with disease still allows one to rank individuals along a continuous liability distribution (
      • Williams J.
      • Blangero J.
      Power of variance component linkage analysis-II Discrete traits.
      ), increasing power to identify genes. The ERV index includes no assumption about the genetic architecture of an endophenotype. While endophenotypes that are closer to gene action are desirable, this is not a requisite of an endophenotype, as information about the genetic simplicity of a particular endophenotype is generally not available or easily quantified. A putative endophenotype with a high ERV value will reflect the genetic component of disease liability better than one with a low ERV. Therefore, even quantitative endophenotypes with complex genetic architectures (involving many genes) can offer major advantages in genetic dissection of disease liability. Indeed, the gold standard endophenotype for heart disease, low-density lipoprotein cholesterol levels, is a complex quantitative trait that is not particularly close to gene action (given that it does not represent a single protein) that was successfully used to find cardiovascular disease risk genes (
      • Brown M.S.
      • Goldstein J.L.
      Familial hypercholesterolemia: A genetic defect in the low-density lipoprotein receptor.
      ,
      • Cohen J.
      • Pertsemlidis A.
      • Kotowski I.K.
      • Graham R.
      • Garcia C.K.
      • Hobbs H.H.
      Low LDL cholesterol in individuals of African descent resulting from frequent nonsense mutations in PCSK9.
      ).
      The present experiment establishes the value of randomly selected families in the search for common psychiatric illness genes. While we highlight the optimality of large families for the assessment of heritability, genetic correlations, and ERV calculations, we note that modern high-density typing now allows empirical assessment of deep kinship between unrelated individuals that could in principle be used to estimate these parameters (albeit very inefficiently due to the remoteness of relationships). Thus, very large previously collected data sets of unrelateds may be of some future value in ERV estimation.
      While we demonstrate the utlitiy of the ERV approach, the current experiment has several limitatons. For example, not all potential candidate ednophentypes for affective disorders were included (
      • Hasler G.
      • Drevets W.C.
      • Manji H.K.
      • Charney D.S.
      Discovering endophenotypes for major depression.
      ), as this is impractial in large-scale genetic studies. In addition, verification of endophenotypes in independent samples is warranted. However, when the goal of simultaneous evaluation of disease liability and endophenotype is focused on gene discovery, it may be folly to wait for such replication rather than immediately pursuing an independent discovery avenue like deep sequencing of a gene whose expression level is genetically correlated with disease liability. The formal testing and rigorous defining of endophenotypes for a given disease should speed the identification of risk genes and improve our understanding of the underlying pathobiological processes. Endophenotypes identified by emperical approaches like the ERV will likely outperform nonobjective expert-derived putative endophenotypes.
      The endophenotype strategy has the potential to significantly improve our understanding of the genetic architecture of psychiatric illnesses (
      • Gottesman I.I.
      • Gould T.D.
      The endophenotype concept in psychiatry: Etymology and strategic intentions.
      ). However, choosing optimal endophenotypes for brain-related illness is difficult when relying on theoretical factors alone. The ERV approach provides an unbiased method for selecting endophenotypes that is applicable to any heritable disease and should facilitate the use of endophentypes in the search for genes influencing brain-related illnesses. Objective formal identification of endophenotypes using the ERV procedure led to improved power to localize causal QTLs influencing risk of major depression and the identification of a novel potential player in depression risk focused on the RNF123 gene, its products, and its pathway.
      Financial support for this study was provided by the National Institute of Mental Health Grants MH0708143 (Principal Investigator [PI]: DCG), MH078111 (PI: JB), and MH083824 (PI: DCG). Theoretical development of the endophenotype ranking value and its implementation in SOLAR is supported by National Institute of Mental Health Grant MH59490 (PI: JI). This investigation was conducted, in part, in facilities constructed with support from Research Facilities Improvement Program Grant Numbers C06 RR13556 and C06 RR017515 from the National Center for Research Resources, National Institutes of Health .
      We thank the study participants, our research staffs, and Irving Gottesman for 50 years of championing endophenotypes in psychiatric genetics. Irving Gottesman is the true source of the endophenotype ranking value. We acknowledge the Azar and Shepperd families and ChemGenex Pharmaceuticals for supporting the transcriptional profiling, sequencing, genotyping, and statistical analysis. The supercomputing facilities used for this work at the AT&T Genomics Computing Center were supported, in part, by a gift from the SBC Foundation.
      All authors reported no biomedical financial interests or potential conflicts of interest.

      Supplementary data

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