assignment - review of vissers et al. (2010), "a de novo paradigm for mental retardation."

A de novo paradigm for mental retardation

Presented by Kelly Clemenza. Wednesday, April 10th 2013.

Vissers L. E. (2010). Nature Genetics. 42: 1109-1113

Stangor C. (2011). Introduction to Psychology, v. 1.0.

“Mental retardation (MR) ” - A basic definition

But it’s really not that simple…

Diagnoses for Mental Retardation as defined in both the DSM-IV-TR and ICD-10!DSM = Diagnostics and Statistics Manual for Mental Illness (United States) ICD = International Classifcation of Disease (International)

Class ! !IQ ‘ Borderline intellectual functioning: 70–84 Mild mental retardation: 50–69 Moderate mental retardation: 35–49 Severe mental retardation: 20–34 Profound mental retardation: Below 20

A varied global understanding

World Health Organization. (2007). Atlas: global resources for persons with intellectual disabilities. Verney S. P., et al. (2005) . Culture-fair cognitive ability assessment: information processing and psychophysiological approaches. Assessment. 12: 303-319

•  Understanding mental retardation on a global scale is incredibly difficult considering there is not one universally accepted term or definition for what it means to be ‘cognitively impaired.’ !

•  When dealing with psychiatric disorders it may be important to understand that a person may find the demands of one culture perfectly manageable but may find the demands of another culture completely overwhelming.

•  To this day many scientists and psychologists argue that the metrics used for measuring IQ are largely biased toward western cultural constructs, sensibilities, and values. Can IQ really provide a global understanding of mental retardation?

Toward a new definition: Intellectual Developmental Disorder

  IDD generally involves difficulties with verbal comprehension, perceptual reasoning, working memory and processing speed.

  This cognitive impairment will lead to difficulties in different domains of learning, including academic and practical knowledge.

  IDD also involves difficulties in adaptive behavior; that is, meeting the demands of daily life expected for one’s age peers, cultural, and community environment.

  Persons with IDD often have difficulties in managing their behavior, emotions, and interpersonal relationships, and maintaining motivation in the learning process.

  IDD is a life span condition requiring consideration of developmental stages and life transitions.!

Salavador-Carulla L., et al. (2011). Toward a new name, de!nition and framework for “mental retardation/intellectual disability” in ICD-11. World Psychiatry. 10: 174-180

According the the ICD-11 Beta Draft: “Intellectual developmental disorder (IDD) is characterized by a marked impairment of core cognitive functions necessary for the development of knowledge, reasoning, and symbolic representation of the level expected of one’s age peers, cultural and community environment.”

•  The ICD, being an international guide, is better suited to provide a globally-culturally-conscious definition of what it means to be ‘mentally retarded.’ ICD-11 is in the beta stages, so it is refered to with the caveat that the definitions are not final and are changing every day. However, updated definitions are assumed to be the best informed, drawing from the most up-to-date research.

•  When using any guide to diagnose mental retardation/intellectual developmental disorder, it is extremely important to keep in mind that all degrees and varieties of mental retardation will present with their own unique cognitive impairments - and that one case of mental retardation is not representative of all cases.

Mental retardation is a symptom Mental retardation is not in itself a disorder, but is a symptom of any number of pathologies. Some genetic causes of mental retardation are well characterized, while others remain a total mystery:

Down’s Syndrome (chromosomal duplication - trisomy 21)

Fragile X Syndrome (chromosomal structural abnormality) …And maybe hundreds of other x-linked disorders

DiGeorge syndrome (chromosomal deletion – 22q11.2)

De novo copy number variation (CNVs) De novo point mutations (this will lead us to our study!) Calles J. L. (2011). Cognitive-adaptive disabilities. Pediatr Clin North Am. 58: 189-203

•  Mental retardation is a symptom of an overwhelming number of pathologies.

•  Non-genetic causes of mental retardation include malnutrition, lead and other toxin poisonings, pre-natal and post-natal infections, fetal alcohol syndrome, head injury, among other factors; all of which are essentially preventable.

**It is interesting to note that these causes of mental retardation are much more prevalent in low-income countries, where nutrition, education and medical care are less available. However, GENETIC causes of MR are roughly equally represented across all nations, regardless of income.

•  Per-generation mutation rate for humans is between 7.6 ! 10"9 and 2.2 ! 10"8

•  This results in 50 to 100 new mutations in each offspring’s genome.

•  Those nucleotide mutations will yield an average of 0.86 new amino-acid–altering mutations.

Resolving the paradox of common,harmful, heritable mental disorders:Which evolutionary genetic modelswork best?

Matthew C. KellerVirginia Institute for Psychiatric and Behavioral Genetics, Virginia

Commonwealth University, Richmond, VA 23219.

[email protected] www.matthewckeller.com

Geoffrey MillerDepartment of Psychology, University of New Mexico, Albuquerque,

NM 87131-1161.

[email protected] www.unm.edu/!psych/faculty/gmiller.html

Abstract: Given that natural selection is so powerful at optimizing complex adaptations, why does it seem unable to eliminate genes(susceptibility alleles) that predispose to common, harmful, heritable mental disorders, such as schizophrenia or bipolar disorder? Weassess three leading explanations for this apparent paradox from evolutionary genetic theory: (1) ancestral neutrality (susceptibilityalleles were not harmful among ancestors), (2) balancing selection (susceptibility alleles sometimes increased fitness), and(3) polygenic mutation-selection balance (mental disorders reflect the inevitable mutational load on the thousands of genesunderlying human behavior). The first two explanations are commonly assumed in psychiatric genetics and Darwinian psychiatry,while mutation-selection has often been discounted. All three models can explain persistent genetic variance in some traits undersome conditions, but the first two have serious problems in explaining human mental disorders. Ancestral neutrality fails to explainlow mental disorder frequencies and requires implausibly small selection coefficients against mental disorders given the data on thereproductive costs and impairment of mental disorders. Balancing selection (including spatio-temporal variation in selection,heterozygote advantage, antagonistic pleiotropy, and frequency-dependent selection) tends to favor environmentally contingentadaptations (which would show no heritability) or high-frequency alleles (which psychiatric genetics would have already found).Only polygenic mutation-selection balance seems consistent with the data on mental disorder prevalence rates, fitness costs, thelikely rarity of susceptibility alleles, and the increased risks of mental disorders with brain trauma, inbreeding, and paternal age.This evolutionary genetic framework for mental disorders has wide-ranging implications for psychology, psychiatry, behaviorgenetics, molecular genetics, and evolutionary approaches to studying human behavior.

Keywords: adaptation; behavior genetics; Darwinian psychiatry; evolution; evolutionary genetics; evolutionary psychology; mentaldisorders; mutation-selection balance; psychiatric genetics; quantitative trait loci (QTL)

1. Introduction

Mental disorders such as schizophrenia, depression,phobias, obsessive-compulsive disorder, and mentalretardation are surprisingly prevalent and disabling. Inindustrialized countries such as the United States, an esti-mated 4% of people have a severe mental disorder(National Institute of Mental Health 1998), and almosthalf of people will meet the criteria for some type of lesssevere mental disorder at some point in their lives(Kessler et al. 2005). The annual economic costs in treat-ment and lost productivity are in the hundreds of billionsof dollars (Rice et al. 1992). The less quantifiable personalcosts of mental disorders to sufferers, families, and friendsare even more distressing. For example, schizophreniaaffects about 1% of people worldwide (Jablensky et al.1992), typically beginning in early adulthood and oftenfollowing a chronic lifelong course. People with

schizophrenia often imagine hostile, confusing voices;they have trouble thinking clearly, feeling normalemotions, or communicating effectively; and they tend tolose jobs, friendships, and sexual partners. In response,many people with schizophrenia kill themselves, and amuch larger proportion dies childless.

This is an evolutionary puzzle, because differences inthe risk of developing schizophrenia and other common,debilitating mental disorders are due, in large part, todifferences in people’s genes. Given that natural selectionhas built the most exquisitely complex machinery known tohumankind – millions of species of organic life-forms –why do so many people suffer from such debilitating andheritable mental disorders? If these mental disorders areas disabling as they appear, natural selection should haveeliminated the genetic variants (susceptibility alleles)that predispose to them long ago. Does the prevalenceof heritable mental disorders therefore imply that mental

BEHAVIORAL AND BRAIN SCIENCES (2006) 29, 385–452Printed in the United States of America

# 2006 Cambridge University Press 0140-525X/06 $12.50 385

MR is present in ~3% of the population, why?

The paradox of common, harmful, heritable mental disorders: !Why does mental illness persist in the population at an almost steady rate, despite it’s obviously deleterious affects to health and reproduction?

Each mental disorder may have it’s own evolutionary genetic model; however, it seems mental retardation may share some evolutionary mechanisms with schizophrenia, bipolar disorder and autism.

Adapted from Huang K. (2011). De novo paradigm: the ultimate answer to the paradox in mental retardation? Clin Genet. 79: 427-428

Stages where de novo mutations may occur

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� This study set out not only to prove this hypothesis but also to develop a rigorous pipeline for de novo point mutation discovery as a way to test this hypothesis.

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•  10 case-parent trios, eight male patients and two female patients

•  Patients all had moderate to severe mental retardation (IQ •  Negative family history •  Clinical evaluation eliminated possibility of syndromic to etiological diagnoses •  Repeat expansion analysis eliminated possibility of Fragile X Syndrome •  Cytogenic analysis showed no chromosomal abnormalities

Clinical evaluations were given to eliminate any non-de novo genetic causes of mental retardation. Requirements included:!•  Subjects result from uncomplicated pregnancies •  Parents be non-consanguinous (not related through any recent ancestors) •  Absence of known post-natal causes of mental retardation, including malnutrition, infections, head injury, etc. •  Any abnormal health history must be either unrelated to MR or caused by MR, but cannot be suspected to be the cause of MR.

Subject Sample •  10 case-parent trios, eight male patients and two female patients

•  Patients all had moderate to severe mental retardation •  Negative family history •  Clinical evaluation eliminated possibility of syndromic to etiological diagnoses •  Repeat expansion analysis eliminated possibility of Fragile X Syndrome •  Cytogenic analysis showed no chromosomal abnormalities •  Array-based genomic profiling discounted any known CNV’s associated with MR

edented resolution. The value of their use for routinediagnostic applications is less obvious and is fraught withdifficulties that will be discussed below.

A more defined and targeted array is one designed fora specific region(s) of the genome for the purpose ofevaluating that targeted segment. It may be designed tostudy a specific chromosome 10,11 or chromosomal seg-

ment12–16 or to identify and evaluate specific DNA dos-age abnormalities in individuals with suspected microde-letion syndromes3 or subtelomeric rearrangements.17

The crucial goal of a targeted microarray in medicalpractice is to provide clinically useful results for diagno-sis, genetic counseling, prognosis, and clinical manage-ment of unbalanced cytogenetic abnormalities. Thus, a

Figure 1. Schematic representation of CGH microarray technology. Whole genomic DNA from a control or reference (left) and genomic DNA from a test or patient(right) are differentially labeled with two different fluorophores. The two genomic DNA samples are competitively cohybridized with large-insert clone DNAtargets that have been robotically printed onto the microarray (middle). Computer imaging programs assess the relative fluorescence levels of each DNA for eachtarget on the array (lower left). The ratio between control and test DNA for each clone can be linearly plotted using data analysis software to visualize dosagevariations (lower right), indicated by a deviation from the normal log2 ratio of zero.

Targeted Array CGH for Diagnostics 529JMD November 2006, Vol. 8, No. 5

edented resolution. The value of their use for routinediagnostic applications is less obvious and is fraught withdifficulties that will be discussed below.

A more defined and targeted array is one designed fora specific region(s) of the genome for the purpose ofevaluating that targeted segment. It may be designed tostudy a specific chromosome 10,11 or chromosomal seg-

ment12–16 or to identify and evaluate specific DNA dos-age abnormalities in individuals with suspected microde-letion syndromes3 or subtelomeric rearrangements.17

The crucial goal of a targeted microarray in medicalpractice is to provide clinically useful results for diagno-sis, genetic counseling, prognosis, and clinical manage-ment of unbalanced cytogenetic abnormalities. Thus, a

Figure 1. Schematic representation of CGH microarray technology. Whole genomic DNA from a control or reference (left) and genomic DNA from a test or patient(right) are differentially labeled with two different fluorophores. The two genomic DNA samples are competitively cohybridized with large-insert clone DNAtargets that have been robotically printed onto the microarray (middle). Computer imaging programs assess the relative fluorescence levels of each DNA for eachtarget on the array (lower left). The ratio between control and test DNA for each clone can be linearly plotted using data analysis software to visualize dosagevariations (lower right), indicated by a deviation from the normal log2 ratio of zero.

Targeted Array CGH for Diagnostics 529JMD November 2006, Vol. 8, No. 5

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Array-based genomic profiling is only useful when you already know the genes/variants you are looking for. Probes are synthesized to match to genomic areas of interest. (In this study we don’t know our areas of interest!)

Bejjani B. A. and Scha!er L. G. (2006). Application of Array-Based Comparative Genomic Hybridization to Clinical Diagnostics. J Mol Diagn. 8: 528-533

1110 VOLUME 42 | NUMBER 12 | DECEMBER 2010 NATURE GENETICS

L E T T E R S

(covered by a median of 17 variant reads). Parental analysis validated the de novo occurrence for 9 of these 13 mutations, detected in seven different individuals (Table 2 and Supplementary Figs. 3 and 4). We did not identify these mutations in a total of 1,664 control chro-mosomes, nor did we see other likely pathogenic mutations identi-fied in the affected genes in these control chromosomes, indicating that the population frequency of these types of de novo mutations in these genes will be lower than 0.22% (power = 0.95, = 0.05). Eight of the de novo mutations were present in a heterozygous state on the autosomes and one was present in a hemizygous state on the X chromosome. All de novo mutations occurred in different genes, including two genes recently implicated in mental retardation (Table 2). In addition to using a dominant disease model, we also analyzed the data for recessive forms of mental retardation. In the affected male of trio 10, we identified a maternally inherited non-synonymous variant in JARID1C (Table 2), which is a well-described X-linked mental retardation gene12. Subsequent analysis of this vari-ant in DNA obtained from the affected individual’s grandparents indi-cated that the mutation had occurred de novo in the mother of this proband. No conclusive evidence for autosomal recessive inheritance, either homozygous or compound heterozygous, was obtained for the other affected individuals.

Next, we evaluated the function of each mutated gene in relation to the disorder (Table 2). Three genes do not seem to play a role in biological pathways linked to mental retardation. BPIL3 is involved in the innate immune response13, whereas PGA5 is involved in pro-tease activity in the stomach14. The function of ZNF599 is currently unknown. For the six other genes affected by de novo mutations, func-tional evidence suggests a role in mental retardation. Two mutations occurred in genes (RAB39B and SYNGAP1) that, when disrupted, are known to cause mental retardation (Table 2)15,16. For the remaining four mutated genes, evidence for a causal link with mental retardation is provided by model organisms and protein-protein interaction stud-ies. DYNC1H1 encodes a cytoplasmic dynein that acts as a motor for intracellular retrograde axonal transport. Heterozygous Dync1h1+/! mutant mice exhibit sensory neuropathy17, and studies in zebrafish have shown the importance of dync1h1 in correct nuclear position-ing. Mislocalization of nuclei in the vertebrate central nervous system is likely to result in profound patterning defects and severely com-promised function18. Notably, DYNC1H1 interacts with PAFAH1B1, the gene associated with type I lissencephaly, which involves gross disorganization of the neurons within the cerebral cortex19. YY1 encodes the ubiquitously expressed transcription factor yin-yang 1 and directs histone deacetylases and histone acetyltransferases, impli-cating chromatin remodeling as its main function. Complete abla-tion of Yy1 in mice results in early embryonic lethality, whereas Yy1 heterozygous mice display growth retardation, neurulation defects and brain abnormalities20. Recent studies show that YY1 inter-acts directly with MECP2; MECP2 is mutated in Rett syndrome21. DEAF1 encodes a transcription factor that regulates the 5-HT1A receptor in the human brain. Mutations in the Drosophila DEAF1 ortholog result in early embryonic arrest, suggesting an essential role

for the gene in early development22. Additional evidence is provided by Deaf1-deficient mice, which show neural tube defects including exencephaly23. Finally, CIC is a member of the HMG-box transcrip-tion factor superfamily, which is associated with neuronal and glial development of the nervous system. CIC is predominantly and tran-siently expressed in immature granule cells of the cerebellum, hippo-campus and neocortex, suggesting a critical role in central nervous system development24.

We next examined the evolutionary conservation of affected nucleo-tides (using the phyloP score), as well as the potential of the de novo mutations to affect the structure or function of the resulting proteins (using the Grantham score; Table 2). All de novo missense mutations and the inherited X-linked mutation were included in this analysis; no Grantham scores were available for the additional nonsense and frameshift mutations. Of note, de novo mutations in genes with a functional link to mental retardation showed a higher phyloP (mean, 4.7) and Grantham score (mean, 135) than mutations in genes with-out such a functional indication (mean phyloP score, !0.5 and mean Grantham score, 38). We also compared these scores to those for all non-synonymous variants in the dbSNP database as well as those in the Human Gene Mutation Database (HGMD). The distribution of phyloP scores and Grantham scores differed markedly between dbSNP and the HGMD (Online Methods and Supplementary Fig. 5). The four mutations in genes functionally linked to mental retardation all showed higher probability values for being observed in HGMD

Table 1 Overview of all variants detected per proband and impact of the prioritization steps for selecting candidate non-synonymous de novo mutationsTrio 1 2 3 4 5 6 7 8 9 10 Average

High-confidence variant calls 20,810 21,658 21,338 22,647 17,694 22,333 21,369 22,658 24,085 22,962 21,755After exclusion of nongenic, intronic and synonymous variants

5,556 5,665 5,691 5,991 4,607 5,567 5,716 5,628 5,985 5,994 5,640

After exclusion of known variants 165 159 157 155 120 136 120 149 96 171 143After exclusion of inherited variants 4 7 3 7 7 2 2 6 6 7 5

Exome data of 10 mental retardation casessequenced on SOLiD 3 Plus System

Read mapping andvariant calling

Default mapping settingsHigh-stringency variant callingExclude low quality

Exclude nongenic, intronic and synonymousExclude known SNPs and in-house databaseExclude inherited

Exclude non-validatedExclude inheritedTest occurrence in control cohort

Mutation impactGene function

Variant analysis

Validation

Interpretation

Figure 1 Experimental work flow for detecting and prioritizing sequence variants. For all ten mental retardation trios, prioritization of variants observed in the probands was based on selection for non-synonymous changes of high quality only and exclusion of all variants previously observed in healthy individuals, together with those variants that were inherited from an unaffected parent. Interpretation of de novo variants was based on gene function and the impact of the mutation.

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Analysis pipeline for detecting & prioritizing variants

Why only the exome? •  Time, money and computational contraints.

•  Only about 2% of the human genome is comprised of protein-coding DNA.

•  For this reason, the exome is better understood, while the non-coding genome is still largly a mystery!

•  The exome can be wildly informative in detecting mendelian disorders.

•  Exome sequencing has already discovered CNV’s relating to MR.

A disorder of mendelian inherentence is a single-mutation disorder. !Short of total gene activation/inactivation, most non-coding changes won’t have the potential for such profound phenotypic affects as seen with mental retardation.

Bamshad M. J., et al. (2011). Exome sequencing as a tool for Mendelian disease gene discovery. Nat Rev Genet. 11: 745-755

Exome Sequencing 1.  DNA was isolated from peripheral blood of probands and parents using QIAamp DNA Mini Kit

2.  Enriched exome libraries were created using the AB SOLiD Optimized SureSelect Human Exome Kit Contains the primers for the exonic sequences of ~18,000 genes and covering a total of ~37 Mb of genomic sequence, specific for the human genome.

3.  Exome libraries were used for emulsion PCRs – SOLiD Sequencing


L E T T E R S







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Exome Sequencing – SOLiD Sequencing

Metzker M. L. (2010) Sequencing technologies — the next generation. Nature. 11: 31-46

Nature Reviews | Genetics

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Figure 3 | Next-generation sequencing technologies that use emulsion PCR. a | A four-colour sequencing by ligation method using Life/APG’s support oligonucleotide ligation detection (SOLiD) platform is shown. Upon the annealing of a universal primer, a library of 1,2-probes is added. Unlike polymerization, the ligation of a probe to the primer can be performed bi-directionally from either its 5 -PO

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group. The SOLiD cycle is repeated nine more times. The extended primer is then stripped and four more ligation rounds are performed, each with ten ligation cycles. The 1,2-probes are designed to interrogate the first (x) and second (y) positions adjacent to the hybridized primer, such that the 16 dinucleotides are encoded by four dyes (coloured stars). The probes also contain inosine bases (z) to reduce the complexity of the 1,2-probe library and a phosphorothiolate linkage between the fifth and six nucleotides of the probe sequence, which is cleaved with silver ions106. Other cleavable probe designs include RNA nucleotides107,108 and internucleosidic

phosphoramidates107, which are cleaved by ribonucleases and acid, respectively. b | A two-base encoding scheme in which four dinucleotide sequences are associated with one colour (for example, AA, CC, GG and TT are coded with a blue dye). Each template base is interrogated twice and compiled into a string of colour-space data bits. The colour-space reads are aligned to a colour-space reference sequence to decode the DNA sequence. c | Pyrosequencing using Roche/454’s Titanium platform. Following loading of the DNA-amplified beads into individual PicoTiterPlate (PTP) wells, additional beads, coupled with sulphurylase and luciferase, are added. In this example, a single type of 2 -deoxyribonucleoside triphosphate (dNTP) — cytosine — is shown flowing across the PTP wells. The fibre-optic slide is mounted in a flow chamber, enabling the delivery of sequencing reagents to the bead-packed wells. The underneath of the fibre-optic slide is directly attached to a high-resolution charge-coupled device (CCD) camera, which allows detection of the light generated from each PTP well undergoing the pyrosequencing reaction. d | The light generated by the enzymatic cascade is recorded as a series of peaks called a flowgram. PP

i, inorganic pyrophosphate.

REVIEWS

38 | JANUARY 2010 | VOLUME 11 www.nature.com/reviews/genetics

Nature Reviews | Genetics

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Roche/454 — PyrosequencingLife/APG — Sequencing by ligationca

Figure 3 | Next-generation sequencing technologies that use emulsion PCR. a | A four-colour sequencing by ligation method using Life/APG’s support oligonucleotide ligation detection (SOLiD) platform is shown. Upon the annealing of a universal primer, a library of 1,2-probes is added. Unlike polymerization, the ligation of a probe to the primer can be performed bi-directionally from either its 5 -PO

4 or 3 -OH end. Appropriate

conditions enable the selective hybridization and ligation of probes to complementary positions. Following four-colour imaging, the ligated 1,2-probes are chemically cleaved with silver ions to generate a 5 -PO

4

group. The SOLiD cycle is repeated nine more times. The extended primer is then stripped and four more ligation rounds are performed, each with ten ligation cycles. The 1,2-probes are designed to interrogate the first (x) and second (y) positions adjacent to the hybridized primer, such that the 16 dinucleotides are encoded by four dyes (coloured stars). The probes also contain inosine bases (z) to reduce the complexity of the 1,2-probe library and a phosphorothiolate linkage between the fifth and six nucleotides of the probe sequence, which is cleaved with silver ions106. Other cleavable probe designs include RNA nucleotides107,108 and internucleosidic

phosphoramidates107, which are cleaved by ribonucleases and acid, respectively. b | A two-base encoding scheme in which four dinucleotide sequences are associated with one colour (for example, AA, CC, GG and TT are coded with a blue dye). Each template base is interrogated twice and compiled into a string of colour-space data bits. The colour-space reads are aligned to a colour-space reference sequence to decode the DNA sequence. c | Pyrosequencing using Roche/454’s Titanium platform. Following loading of the DNA-amplified beads into individual PicoTiterPlate (PTP) wells, additional beads, coupled with sulphurylase and luciferase, are added. In this example, a single type of 2 -deoxyribonucleoside triphosphate (dNTP) — cytosine — is shown flowing across the PTP wells. The fibre-optic slide is mounted in a flow chamber, enabling the delivery of sequencing reagents to the bead-packed wells. The underneath of the fibre-optic slide is directly attached to a high-resolution charge-coupled device (CCD) camera, which allows detection of the light generated from each PTP well undergoing the pyrosequencing reaction. d | The light generated by the enzymatic cascade is recorded as a series of peaks called a flowgram. PP

i, inorganic pyrophosphate.

REVIEWS

38 | JANUARY 2010 | VOLUME 11 www.nature.com/reviews/genetics

SOLiD Sequencing

Source: Wikipedia, because look there’s too many citations in the table to include them all!

Read Mapping and Variant Calling 1.  Obtained 3.1 Gb of mappable sequence data

per individual

2.  After mapping to reference genome: • An average of 79.6% of bases originated from the targeted exome • 90% of the targeted exons were covered at least 10x • Median exon coverage was 42-fold!

3.  Identified an average of 21,755 genetic variants per individual of high confidence


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Supplementary Figures

Supplementary Figure 1: Coverage plots of all 30 individuals

Figure legend

Coverage for all exons targeted by enrichment was evaluated. The median coverage for all 30 individuals was 42-fold, with on average 90% of all targets covered at least 10-fold. The numbers in the figure legends refer to the corresponding MR trios; M: Mother; F: Father; C: Child

Nature Genetics: doi: 10.1038/ng.712

The exon coverage was excellent and indicated that the majority of variants present in each exome could be robustly detected using the researcher’s bioinformatics pipeline.

A coverage plot shows the variation between fold coverage and percent of targets per each individual’s exome, as representation by each colored line (each line is labeled #_A where # is the trio number and A is either M, F, or C for mother, father and child. This chart shows that almost every single target was covered at least once, with an average coverage of 42-fold for all 30 individuals.

Variant Analysis

1.  Exlcluding all nongenic, intronic, and synonymous variants other than those occuring at canonical splice sites reduce the # of candidates to an average of 5,640."

2.  This number was further reduced to 143 by excluding known, likely benign, variants by referencing data from dbSNP database v130 and an in-house variant database.

3.  Comparing patient’s data to parent’s exome reduce the results to an average of 5 candidate de novo mutations, with 51 total candidate mutations."


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The observed ratio of non-synonymous-to-synonymous de novo mutations is far greater than would be expected for protein-coding genes under purifying selection. This indicates that many of these mutations will result in a reproductive disadvantage.

**Purifying selection is the evolutionary process by which the selective pressure to maintain a certain variant is so strong that all other variants are rapidly removed as soon as they are introduced into a population.

Sanger validation of de novo mutations

Patient!

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Supplementary Figure 4: Sanger validation of de novo mutations

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Supplementary Figure 4: Sanger validation of de novo mutations

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Sequence Chromatograms


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Only 13 variants could be validated, 9 of these variants confirmed as de novo mutations

•  DNA sequences are generated using algorithms that can read thisgraphical display and convert the visual output into text – A,C,G,T. •  Verification by eye is important here because the algorithms aren’t perfect and may call the wrong base if there is overlap of peaks (but overlap is the only visual indicator of heterozygosity!)

•  M is the IUPAC symbol for heterozygous A and C. R is the IUPAC symbol for heterozygous G and A. •  Notice how the mother and father are homozygous A, a de novo mutation from A to C has occurred on one of the chromosomes.

•  The goal of this step is to validate the mutations observed in the probands and (validate the absence of the mutations in the parental DNA. Thirty-eight candidates could not be validated.

Study subjects vs. control cohort •  None of the 9 variants could be found in 1,664 control chromosomes.

•  No likely pathogenic mutations were identified in the genes containing the 9 variants. This indicates the population frequency of de novo mutations in these genes will be lower than 22%.

•  All 9 variants occurred In different genes, including two that have been previously implicated in MR.

•  8 mutations were found in a heterozygous state, and 1 was present in a hemizygous state on the X chromosome.

•  The hemizygous variant was actually inhereted, it had occurred occurred de novo in the patient’s mother. This was a non-synonymous variant in JARID1C, which is an X-linked mental retardation gene.


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(mean, 0.83) than for being observed in dbSNP (mean, 0.17). The three mutations in genes without a functional link to mental retar-dation showed an average probability of 0.94 for being observed in dbSNP and an average probability of 0.06 for being observed in HGMD (Table 2). Additionally, the inherited JARID1C mutation showed a probability of 1.00 for being in HGMD versus 2.09 ! 10"6 for being in dbSNP.

This analysis of the mutated nucleotides and their impact on gene function strongly supports pathogenicity for six of the nine de novo mutations. Importantly, these six mutations occurred in genes with a functional link to mental retardation, two of which are known mental retardation genes. In contrast, three de novo variants in genes without a functional link did not appear to significantly affect protein func-tion. Moreover, we identified a maternally inherited mutation in a known X-linked mental retardation gene that arose de novo in the proband’s mother. Although we have not provided individual func-tional tests to prove causality, these data collectively provide strong evidence for a major role of de novo mutations in mental retardation. The identification of recurrent mutations in these genes in unrelated cases would provide additional proof for disease causality, but this may require the evaluation of thousands of affected individuals. The identification of subtle CNVs encompassing (part of) these genes may also provide additional proof for disease causality, as was shown recently for mutations in X-linked mental retardation genes25. As of yet, no such CNVs have been reported, nor have we found such CNVs in our diagnostic cohort of ~4,500 individuals with mental retarda-tion (data not shown).

The discovery of nine de novo non-synonymous mutations in this cohort of ten affected individuals is concordant with the recently

estimated background mutation rate of 0.86 amino-acid–altering mutations per newborn in controls2, but it will be important to com-pare this result to similar data from healthy control trios when avail-able. Notably, after applying the same systematic filtering approach and Sanger sequencing, we could only validate a single de novo syn-onymous mutation, which occurred in GRIN1 (c.351C>T, seen in trio 10). This base pair is not conserved through evolution (phyloP score = "3.2) and does not seem to alter splicing, suggesting that this mutation is an unlikely candidate for causing mental retarda-tion. Of note, the individual carrying this mutation also carries the JARID1C mutation. The observed ratio of non-synonymous to syn-onymous de novo mutations is far greater than would be expected for protein-coding genes under purifying selection and indicates that many of these mutations will result in a reproductive dis-advantage. In contrast, the average non-synonymous to synonymous ratio reported in dbSNP for the six genes with predicted pathogenic mutations is significantly lower than that of the three genes with mutations reflecting the background mutation rate (Fisher’s Exact test, P = 0.0016), which is to be expected for disease genes in the normal population.

In summary, our results suggest that de novo mutations are a major cause of unexplained mental retardation. These muta-tions can readily be identified using a family based exome sequencing approach and require only limited follow-up by Sanger sequencing. Our findings have implications for preven-tive and diagnostic strategies in mental retardation. Systematic genome-wide resequencing in parent-child trios may uncover further examples of this de novo paradigm for other human neurodevelopmental disorders.

Table 2 Overview of all de novo variants identified by exome sequencing in ten individuals with unexplained mental retardation

Gene Trio Sexa NM numbercDNA level

changeProtein level

changePhyloP score

Grantham score

Probability of being observed in

dbSNPb

Probability of being observed

in HGMDb Gene function

De novo mutationsDYNC1H1 1 M NM_001376 c.11465A>C p.His3822Pro 5.5 77 0.20 0.80 Retrograde axonal transporter;

interacts with PAFAH1B1 (mutation of which causes lissencephaly, a neurodevel-opmental disorder)

ZNF599 1 M NM_001007248 c.532C>T p.Leu187Phe –1.5 22 1.00 2.65 ! 10"4 UnknownRAB39B 2 M NM_171998 c.557G>A p.Trp186X 4.8 – – – Known X-linked mental

retardation geneYY1 3 M NM_003403 c.1138G>T p.Asp380Tyr 6.9 160 2.27 ! 10"6 1.00 Ubiquitously expressed

transcription factor; mouse knockdown results in growth retardation, neurulation defects and brain abnormalities; interacts with MECP2, a known mental retardation gene

BPIL3 3 M NM_174897 c.887G>A p.Arg269His 0.5 29 0.97 0.03 Innate immune responsePGA5 4 F NM_014224 c.1058T>C p.Val353Ala 0.7 64 0.84 0.16 Precursor of pepsinDEAF1 5 M NM_021008 c.683T>G p.Ile228Ser 4.9 142 0.01 0.99 Transcription factor; regulator

of 5-HT1A receptor in the brain; mouse knockout causes neural tube defects

CIC 6 M NM_015125 c.1474C>T p.Arg492Trp 2.6 101 0.46 0.54 Granule cell development in central nervous system

SYNGAP1 8 F NM_006772 c.998_999del p.Val333AlafsX 3.3 – – – Known autosomal dominant mental retardation gene

X-linked inherited mutationsJARID1C 10 M NM_001146702 c.1919G>A p.Cys640Tyr 5.1 194 2.09 ! 10"6 1.00 Known X-linked mental

retardation geneaSex of proband, with M for male and F for female. bVisual representation of probabilities are provided in Supplementary Figure 5. Grantham scores for nonsense (in RAB39B) and frameshift mutations (in SYNGAP1) could not be calculated.

•  ZNF599, BPIL3, and PGA5 are not known to have a functional link mental retardation.

Genes of interest include: •  DYNC1H1, which is implicated in lissencephaly, which is a condition where the brain gyri and sulci are much less pronounced, leading to a ‘smoothi’ appearance of the brain and drastically

reducing the surface area for neuronal connections. •  DEAF1, which is a regulator of the 5-HT1A receptor, a receptor for the neurotransmitter serotonin. Serotonin is ubiquitously expresed throughout the brain and is very important for pre-natal

neuronal migrations and maturation.!

Impact of variants on function


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changeProtein level

changePhyloP score

Grantham score


dbSNPb














PhyloP score described the evolutionary conservation of affected nucleotides.

Grantham score describes the potential of the de novo mutations to affect the structure or function of the resulting proteins

Most genes tend to vary linearly when plotting their PhyloP scores against their Grantham scores. !A higher PhylopP score means the gene has been subject to greater purifying selection, any changes to that gene will usually not last in the population. This is frequently because

any genetic changes will causes too great a change to the protein structure - the protein will be unable to carry out the very function that has been under such selective pressure. This kind of gene would have also a higher Grantham score.


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changeProtein level

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Supplementary Figure 5: Distribution of PhyloP and Grantham scores for dbSNP, HGMD and the de novo mutations identified in this study

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Supplementary Figure 5: Distribution of PhyloP and Grantham scores for dbSNP, HGMD and the de novo mutations identified in this study

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Comparing with other human variants

dbSNP = Single Nucleotide Polymorphism Database HGMD = Human Gene Mutation Database

dbSNP is a database of variation across all domains of life. It of course includes the human species and all known human genetic variation. The HGMD is a database cataloguing specifically deleterious, or disease causing variations found in the human population. !•  The four de novo mutations in genes functionally linked to mental retardation all showed a higher probability for being observed in HGMD (mean probability value of 0.83), than for being observed in

dbSNP (mean probability value of 0.17.) The inherited JARID1C mutation showed a probability of 1.00 for being in HGMD versus 2.09 ! 10"6 for being in dbSNP.

•  The three mutations in genes without a functional link to MR showed an average probability of 0.94 for being observed in dbSNP and an average probability of 0.06 for being observed in HGMD.

•  So this is good news! It looks like the newly discovered de novo mutations with a functional link to mental retardation may be the ACTUAL causes of each patient’s MR. !

.

(2013)

Future Directions

This study has set a precedent for de novo point mutation discoveriy in psychiatric disorders. Recent studies have begun to extened the ‘de novo paradigm’ to autism and schizophrenia, disorders which have previously shown to involve a high number of de novo CNV’s just as in MR.

Further research has implicated a mostly paternal influence on de novo CNV mutations in mental illness as well, counteracting the mostly maternal influence of chromosomal arrangement disorders like Down’s Syndrome. {Hehir-Kwa J. Y. Et al (2011)}

In addition, understanding the genetic causes of any pathology will always hold the promise of developing new screenings and therapies to treat that pathology, and this certainly applies to MR. Maybe most important, a better scientific understanding will foster more respect and outreach toward those individuals and families dealing with cognitive deficiences.

Conclusions •  I really like the paper! I thought it was exceedingly well organized…

•  …which was made more important considering this paper set out to design a specific set of methods for variant discovery that would hopefully be used in the future by others.

•  The experimental design was simple yet fairly effective. •  Those variants found in genes functionally implicated in MR were most likely the ones that caused MR.

•  Comparing PhyloP with Granthma scores, and referencing their relationship across dbSNP and HGMD was a simple, yet intuitive method for verifying de novo mutations.

•  Were the variants in the genes ‘not functionally implicated’ in MR somehow, maybe indirectly, still the cause of MR?

•  If not, this study failed to detect the cause of MR in those three probands."

•  There are surely many variations in non-coding DNA, as well as epi-genetic factors, that can cause MR - and this method won’t find them.

But this was a great start!

References 1.  Stangor C. (2011). Introduction to Psychology, v. 1.0.

2.  World Health Organization. (2007). Atlas: global resources for persons with intellectual disabilities.

3.  Verney S. P., et al. (2005) . Culture-fair cognitive ability assessment: information processing and psychophysiological approaches. Assessment. 12: 303-319

4.  Salavador-Carulla L., et al. (2011). Intellectual developmental disorders: towards a new name, definition and framework for 'mental retardation/intellectual disability' in ICD-11. World Psychiatry. 10: 174-180

5.  Calles J. L. (2011). Cognitive-adaptive disabilities. Pediatr Clin North Am. 58: 189-203

6.  Keller M. C. and Miller G. (2006) Resolving the paradox of common, harmful, heritable mental disorders: Which evolutionary genetic models work best? Behav Brain Sci. 29: 385-452

7.  Bejjani B. A. and Schaffer L. G. (2006). Application of Array-Based Comparative Genomic Hybridization to Clinical Diagnostics. J Mol Diagn. 8: 528-533

8.  Bamshad M. J., et al. (2011). Exome sequencing as a tool for Mendelian disease gene discovery. Nat Rev Genet. 11: 745-755

9.  Robinson P. N. (2010). Whole-exome sequencing for finding de novo mutations in sporadic mental retardation. Genome Biol. 11:144

10.  Metzker M. L. (2010) Sequencing technologies — the next generation. Nature. 11: 31-46

11.  Hehir-Kwa J. Y. (2011). De novo copy number variants associated with intellectual disability have a paternal origin and age bias. J Med Genet. 11: 776-778.

12.  Ellison J. W., Rosenfeld J. A. and Shaffer L. G. (2013) Genetic Basis of Intellectual Disability. Annu Rev Med. 64: 441-450

13.  O'Roak B. J., et al. (2011). Exome sequencing in sporadic autism spectrum disorders identifies severe de novo mutations. Nat Genet. 44: 471.

14.  Xu B., et al. (2011). Exome sequencing supports a de novo mutational paradigm for schizophrenia. Nat Genet. 43: 864-868.

Thank you!

assignment - review of vissers et al. (2010), "a de novo paradigm for mental retardation."

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