Joachim Hallmayer

Genetic Heritability and Shared Environmental Factors Among Twin Pairs With Autism

CONTEXT Autism is considered the most heritable of neurodevelopmental disorders, mainly because of the large difference in concordance rates between monozygotic and dizygotic twins. OBJECTIVE To provide rigorous quantitative estimates of genetic heritability of autism and the effects of shared environment. DESIGN, SETTING, AND PARTICIPANTS Twin pairs with at least 1 twin with an autism spectrum disorder (ASD) born between 1987 and 2004 were identified through the California Department of Developmental Services. MAIN OUTCOME MEASURES Structured diagnostic assessments (Autism Diagnostic Interview-Revised and Autism Diagnostic Observation Schedule) were completed on 192 twin pairs. Concordance rates were calculated and parametric models were fitted for 2 definitions, 1 narrow (strict autism) and 1 broad (ASD). RESULTS For strict autism, probandwise concordance for male twins was 0.58 for 40 monozygotic pairs (95% confidence interval [CI], 0.42-0.74) and 0.21 for 31 dizygotic pairs (95% CI, 0.09-0.43); for female twins, the concordance was 0.60 for 7 monozygotic pairs (95% CI, 0.28-0.90) and 0.27 for 10 dizygotic pairs (95% CI, 0.09-0.69). For ASD, the probandwise concordance for male twins was 0.77 for 45 monozygotic pairs (95% CI, 0.65-0.86) and 0.31 for 45 dizygotic pairs (95% CI, 0.16-0.46); for female twins, the concordance was 0.50 for 9 monozygotic pairs (95% CI, 0.16-0.84) and 0.36 for 13 dizygotic pairs (95% CI, 0.11-0.60). A large proportion of the variance in liability can be explained by shared environmental factors (55%; 95% CI, 9%-81% for autism and 58%; 95% CI, 30%-80% for ASD) in addition to moderate genetic heritability (37%; 95% CI, 8%-84% for autism and 38%; 95% CI, 14%-67% for ASD). CONCLUSION Susceptibility to ASD has moderate genetic heritability and a substantial shared twin environmental component.

A Genomic Screen of Autism: Evidence for a Multilocus Etiology

We have conducted a genome screen of autism, by linkage analysis in an initial set of 90 multiplex sibships, with parents, containing 97 independent affected sib pairs (ASPs), with follow-up in 49 additional multiplex sibships, containing 50 ASPs. In total, 519 markers were genotyped, including 362 for the initial screen, and an additional 157 were genotyped in the follow-up. As a control, we also included in the analysis unaffected sibs, which provided 51 discordant sib pairs (DSPs) for the initial screen and 29 for the follow-up. In the initial phase of the work, we observed increased identity by descent (IBD) in the ASPs (sharing of 51.6%) compared with the DSPs (sharing of 50.8%). The excess sharing in the ASPs could not be attributed to the effect of a small number of loci but, rather, was due to the modest increase in the entire distribution of IBD. These results are most compatible with a model specifying a large number of loci (perhaps >/=15) and are less compatible with models specifying </=10 loci. The largest LOD score obtained in the initial scan was for a marker on chromosome 1p; this region also showed positive sharing in the replication family set, giving a maximum multipoint LOD score of 2.15 for both sets combined. Thus, there may exist a gene of moderate effect in this region. We had only modestly positive or negative linkage evidence in candidate regions identified in other studies. Our results suggest that positional cloning of susceptibility loci by linkage analysis may be a formidable task and that other approaches may be necessary.

Using induced pluripotent stem cells to investigate cardiac phenotypes in Timothy syndrome

Individuals with congenital or acquired prolongation of the QT interval, or long QT syndrome (LQTS), are at risk of life-threatening ventricular arrhythmia. LQTS is commonly genetic in origin but can also be caused or exacerbated by environmental factors. A missense mutation in the L-type calcium channel CaV1.2 leads to LQTS in patients with Timothy syndrome. To explore the effect of the Timothy syndrome mutation on the electrical activity and contraction of human cardiomyocytes, we reprogrammed human skin cells from Timothy syndrome patients to generate induced pluripotent stem cells, and differentiated these cells into cardiomyocytes. Electrophysiological recording and calcium (Ca2+) imaging studies of these cells revealed irregular contraction, excess Ca2+ influx, prolonged action potentials, irregular electrical activity and abnormal calcium transients in ventricular-like cells. We found that roscovitine, a compound that increases the voltage-dependent inactivation of CaV1.2 (refs 6–8), restored the electrical and Ca2+ signalling properties of cardiomyocytes from Timothy syndrome patients. This study provides new opportunities for studying the molecular and cellular mechanisms of cardiac arrhythmias in humans, and provides a robust assay for developing new drugs to treat these diseases.Individuals with congenital or acquired prolongation of the QT interval, or long QT syndrome (LQTS), are at risk of life threatening ventricular arrhythmia 1, 2. LQTS is commonly genetic in origin but can also be caused or exacerbated by environmental factors1, 3. A missense mutation in the L-type calcium channel CaV1.2 leads to LQTS in patients with Timothy syndrome (TS)4, 5. To explore the effect of the TS mutation on the electrical activity and contraction of human cardiomyocytes (CMs), we reprogrammed human skin cells from TS patients to generate induced pluripotent stem cells (iPSCs), and differentiated these cells into CMs. Electrophysiological recording and calcium (Ca2+) imaging studies of these cells revealed irregular contraction, excess Ca2+ influx, prolonged action potentials, irregular electrical activity and abnormal calcium transients in ventricular-like cells. We found that roscovitine (Ros), a compound that increases the voltage-dependent inactivation (VDI) of CaV1.26–8, restored the electrical and Ca2+ signaling properties of CMs from TS patients. This study opens new avenues for studying the molecular and cellular mechanisms of cardiac arrhythmias in humans, and provides a robust assay for developing new drugs to treat these diseases.

HPA-Axis Reactivity: A Mechanism Underlying the Associations Among 5-HTTLPR, Stress, and Depression

BACKGROUND Recent evidence indicates that individuals who are homozygous for the short (s) allele in the promoter region of the serotonin transporter gene have higher rates of depression and other psychiatric disorders as a function of exposure to increasing levels of stressful life events than do individuals who have one or two copies of the long (l) allele. Despite the reliability of this association, the mechanism by which this polymorphism confers risk for psychopathology in the presence of stress is not understood. This study was designed to examine the formulation that individuals who are homozygous for the s allele are characterized by a greater biological reactivity to stress than are their counterparts who have one or two copies of the l allele. METHODS Girls at high (n = 25) and low (n = 42) risk for depression by virtue of the presence or absence of a family history of this disorder were genotyped and exposed to a standardized laboratory stress task. Cortisol levels were assessed before the stressor, after the stressor, and during an extended recovery period. RESULTS Girls who were homozygous for the s allele produced higher and more prolonged levels of cortisol in response to the stressor than did girls with an l allele. CONCLUSIONS These findings indicate that the 5-HTTLPR polymorphism is associated with biological stress reactivity, which may increase susceptibility to depression in the face of stressful life events.

Support for Association of Schizophrenia with Genetic Variation in the 6p22.3 Gene, Dysbindin, in Sib-Pair Families with Linkage and in an Additional Sample of Triad Families

Genetic variants in a gene on 6p22.3, dysbindin, have been shown recently to be associated with schizophrenia (Straub et al. 2002a). There is no doubt that replication in other independent samples would enhance the significance of this finding considerably. Since the gene is located in the center of the linkage peak on chromosome 6p that we reported earlier, we decided to test six of the most positive DNA polymorphisms in a sib-pair sample and in an independently ascertained sample of triads comprising 203 families, including the families for which we detected linkage on chromosome 6p. Evidence for association was observed in the two samples separately as well as in the combined sample (P=.00068 for SNP rs760761). Multilocus haplotype analysis increased the significance further to .00002 for a two-locus haplotype and to .00001 for a three-locus haplotype. Estimation of frequencies for six-locus haplotypes revealed one common haplotype with a frequency of 73.4% in transmitted, and only 57.6% in nontransmitted, parental haplotypes. All other six-locus haplotypes occurring at a frequency of >1% were less often transmitted than nontransmitted. Our results represent a first successful replication of linkage disequilibrium in psychiatric genetics detected in a region with previous evidence of linkage and will encourage the search for causes of schizophrenia by the genetic approach.

Narcolepsy is strongly associated with the T-cell receptor alpha locus

Narcolepsy with cataplexy, characterized by sleepiness and rapid onset into REM sleep, affects 1 in 2,000 individuals. Narcolepsy was first shown to be tightly associated with HLA-DR2 (ref. 3) and later sublocalized to DQB1*0602 (ref. 4). Following studies in dogs and mice, a 95% loss of hypocretin-producing cells in postmortem hypothalami from narcoleptic individuals was reported. Using genome-wide association (GWA) in Caucasians with replication in three ethnic groups, we found association between narcolepsy and polymorphisms in the TRA@ (T-cell receptor alpha) locus, with highest significance at rs1154155 (average allelic odds ratio 1.69, genotypic odds ratios 1.94 and 2.55, P < 10−21, 1,830 cases, 2,164 controls). This is the first documented genetic involvement of the TRA@ locus, encoding the major receptor for HLA-peptide presentation, in any disease. It is still unclear how specific HLA alleles confer susceptibility to over 100 HLA-associated disorders; thus, narcolepsy will provide new insights on how HLA–TCR interactions contribute to organ-specific autoimmune targeting and may serve as a model for over 100 other HLA-associated disorders.

Using iPSC-derived neurons to uncover cellular phenotypes associated with Timothy syndrome

Monogenic neurodevelopmental disorders provide key insights into the pathogenesis of disease and help us understand how specific genes control the development of the human brain. Timothy syndrome is caused by a missense mutation in the L-type calcium channel Cav1.2 that is associated with developmental delay and autism. We generated cortical neuronal precursor cells and neurons from induced pluripotent stem cells derived from individuals with Timothy syndrome. Cells from these individuals have defects in calcium (Ca2+) signaling and activity-dependent gene expression. They also show abnormalities in differentiation, including decreased expression of genes that are expressed in lower cortical layers and in callosal projection neurons. In addition, neurons derived from individuals with Timothy syndrome show abnormal expression of tyrosine hydroxylase and increased production of norepinephrine and dopamine. This phenotype can be reversed by treatment with roscovitine, a cyclin-dependent kinase inhibitor and atypical L-type–channel blocker. These findings provide strong evidence that Cav1.2 regulates the differentiation of cortical neurons in humans and offer new insights into the causes of autism in individuals with Timothy syndrome.

SHANK3 and IGF1 restore synaptic deficits in neurons from 22q13 deletion syndrome patients

Phelan–McDermid syndrome (PMDS) is a complex neurodevelopmental disorder characterized by global developmental delay, severely impaired speech, intellectual disability, and an increased risk of autism spectrum disorders (ASDs). PMDS is caused by heterozygous deletions of chromosome 22q13.3. Among the genes in the deleted region is SHANK3, which encodes a protein in the postsynaptic density (PSD). Rare mutations in SHANK3 have been associated with idiopathic ASDs, non-syndromic intellectual disability, and schizophrenia. Although SHANK3 is considered to be the most likely candidate gene for the neurological abnormalities in PMDS patients, the cellular and molecular phenotypes associated with this syndrome in human neurons are unknown. We generated induced pluripotent stem (iPS) cells from individuals with PMDS and autism and used them to produce functional neurons. We show that PMDS neurons have reduced SHANK3 expression and major defects in excitatory, but not inhibitory, synaptic transmission. Excitatory synaptic transmission in PMDS neurons can be corrected by restoring SHANK3 expression or by treating neurons with insulin-like growth factor 1 (IGF1). IGF1 treatment promotes formation of mature excitatory synapses that lack SHANK3 but contain PSD95 and N-methyl-d-aspartate (NMDA) receptors with fast deactivation kinetics. Our findings provide direct evidence for a disruption in the ratio of cellular excitation and inhibition in PMDS neurons, and point to a molecular pathway that can be recruited to restore it.

A combined analysis of D22S278 marker alleles in affected sib-pairs: Support for a susceptibility locus for schizophrenia at chromosome 22q12

Several groups have reported weak evidence for linkage between schizophrenia and genetic markers located on chromosome 22q using the lod score method of analysis. However these findings involved different genetic markers and methods of analysis, and so were not directly comparable. To resolve this issue we have performed a combined analysis of genotypic data from the marker D22S278 in multiply affected schizophrenic families derived from 11 independent research groups worldwide. This marker was chosen because it showed maximum evidence for linkage in three independent datasets (Vallada et al., Am J Med Genet 60:139-146, 1995; Polymeropoulos et al., Neuropsychiatr Genet 54:93-99, 1994; Lasseter et al., Am J Med Genet, 60:172-173, 1995. Using the affected sib-pair method as implemented by the program ESPA, the combined dataset showed 252 alleles shared compared with 188 alleles not share (chi-square 9.31, 1df, P = 0.001) where parental genotype data was completely known. When sib-pairs for whom parental data was assigned according to probability were included the number of alleles shared was 514.1 compared with 437.8 not shared (chi-square 6.12, 1df, P = 0.006). Similar results were obtained when a likelihood ratio method for sib-pair analysis was used. These results indicate that may be a susceptibility locus for schizophrenia at 22q12.

Immunology : hepatitis A virus link to atopic disease

Atopic diseases, including asthma, allergic rhinitis and atopic dermatitis, are caused by both environmental and genetic factors. Here we show that infection by hepatitis A virus (HAV) may protect individuals from atopy if they carry a particular variant of the gene that encodes TIM-1 (also known as HAVcr-1) — the cell-surface receptor used by HAV to infect human cells. Exposure to HAV is associated with poor hygiene, large family size and attendance at day-care centres, all factors that are also inversely associated with atopy. Our discovery indicates that interaction between HAV and TIM-1 genotype may contribute to the aetiology of atopic diseases, and provides a mechanism to account for the hygiene hypothesis.