Genetics of Autism and Related Neurodevelopmental Disabilities: An Update David B. Everman, MD Greenwood Genetic Center

Objectives • Illustrate the clinical utility of identifying genetic factors that underlie ASDs and related neurodevelopmental disabilities • Review how alterations in chromosomes and genes can cause or contribute to ASDs and other disabilities • Understand various genetic testing methods and strategies for the interpretation of genetic variants • Learn about current genetic models of autism and how to apply genetic testing in a practical way • Understand ways in which genetic discoveries are being translated into treatment efforts

Recent Developments in Genetics • Success of the Human Genome Project – Initiatives to understand how genes, chromosomes, and biological pathways contribute to development and disease – Importance of genetic variation

• Rapid pace of new discoveries in genetics and biology • Emergence of new genetic approaches and technologies – Array comparative genomic hybridization (microarray) – Next generation sequencing – Non-invasive prenatal screening (cell-free fetal DNA analysis)

• Treatment initiatives

Nature 470:204-213, 2011.

Neurodevelopmental Disabilities • Developmental delay • Intellectual disability • Autism spectrum disorders (ASDs) – With/without co-existing intellectual disability

• Developmental regression

Benefits of Identifying a Genetic Cause • DIAGNOSIS – getting better “answers” for the patient/family • Recurrence risk assessment and prenatal counseling • Education and support • Identification of associated problems • Prevention of condition or its complications • Management and treatment

Medical Implications of a Microarray Finding

Deletion of this gene causes renal cysts and diabetes

Isolated vs. Syndromic ASDs • Isolated – ASD is the primary condition

• Syndromic/Complex – ASD is one component of a larger pattern of findings that may also include: • Atypical physical features (“dysmorphism”) • Growth concerns • Major medical issues

Syndromic/Complex ASDs

Genet Med 2013, 15:399-404.

Syndromic/Complex ASDs Smith-Magenis syndrome

PTEN-related disorder

http://jmg.bmj.com/

J Med Genet 42:318-321, 2005.

Associated Findings That May Provide Clues to a Specific Genetic Cause • • • • • • • • • •

Atypical physical features or congenital anomalies Family history Growth problems (reduced or excessive) Microcephaly or macrocephaly Cranial MRI abnormalities Hypotonia or hypertonia Seizures Feeding, behavior, or sleep difficulties Vision or hearing problems Other medical problems

Evidence for Genetic Factors in Autism • Increased recurrence risk in families with one or more affected children – 5-10% recurrence risk in sibs when one child is affected – 25-35% (or higher) recurrence risk in sibs when two or more children are affected

• Higher concordance rate between monozygotic versus dizygotic twins (70% vs. 3%) • 3-4 times more common in males • Overall heritability is approximately 70-80%

Genetic Basis of ASDs • Most cases (85-94%) have no identifiable cause through traditional testing approaches • Heterogeneous group of disorders with multifactorial inheritance – Small effects from multiple genes and non-genetic factors add up to produce a particular phenotype

Each chromosome is one continuous DNA sequence numbered from the tip of the p arm to the tip of the q arm

Exons Enhancer 5’

3’ Promoter

Gene

Unspliced mRNA Transcript Start

Stop

Spliced mRNA Transcript

Genetic Signaling Pathways

A complex network of signals . . .

Types of Genetic Alterations X SINGLE GENE MUTATION

Enhancer

Gene 1

Gene 2

NORMAL

X

DELETION

DUPLICATION

CHROMOSOME REARRANGEMENT

Genetic Variation

Enhancer

Gene 1

Gene 2

NORMAL

* * SINGLE NUCLEOTIDE POLYMORPHISM (SNP) COPY NUMBER VARIANT (CNV)

Genetic Testing Methods

Chromosome analysis

Microarray

DNA sequencing

Looks for large missing or extra segments and balanced rearrangements

Looks for small missing or extra segments

Looks for misspellings

The “Book” level

The “Page” level

The “Word” level

Testing for Chromosome Abnormalities • High resolution chromosome analysis – Looks for balanced or unbalanced chromosome rearrangements that are microscopically visible

• Microarray analysis – Also called array comparative genomic hybridization (CGH) – Looks for submicroscopic chromosome deletions/duplications – Has largely replaced FISH analysis unless targeted testing of a specific region is needed – Has not completely replaced chromosome analysis because it does not detect balanced rearrangements or translocations

Testing for Single Gene Alterations • General techniques – Sequence analysis to look for “misspellings” • Sanger sequencing (older method) • Next-Generation sequencing (newer method) – faster and cheaper

– Deletion/duplication (dosage) analysis to look for missing or extra copy an entire gene or a small missing or extra segment within a gene (such as a single exon deletion or duplication) • MLPA analysis • Custom microarray analysis

• Different approaches for different clinical situations – Targeted analysis of a specific gene (Tuberous sclerosis) – Gene panel (Autism, intellectual disability) – Whole exome sequencing (Unexplained complex phenotype)

Whole Exome Sequencing (WES) • Initially used as a research modality for gene discovery • Now a clinical test that can be routinely ordered for patients and billed to insurance • Idea is to sequence essentially all coding exons in search of a cause of a patient’s unexplained finding(s)

Epigenetics • Modulation of gene expression as a result of chemical modifications of the DNA • In other words, changes are made “on top of” the primary DNA sequence • Genomic imprinting – Epigenetic modifications depend on parent from which a chromosome/gene is inherited

DNA Methylation

Current Landscape of Genetic Testing • Targeted testing – Many individual genes can be sequenced and tested for deletions/duplications

• General testing – analysis of larger portions of the genome in a single test – Array CGH (Microarray) – Gene panels – Whole exome sequencing

Effects of Genetic Alterations • Benign (polymorphisms or SNPs) • Detrimental (Pathogenic variants or “mutations”) – Loss of function – gene/protein no longer do what they are supposed to do – Gain of function – gene/protein do too much of what they are supposed to do, or something entirely new

• If detrimental, an alteration can be the entire causative or only partially responsible for a phenotype (i.e. risk/contributing factor) • Variant of uncertain significance (VUS or VOUS) • Keep in mind that for each gene, the impact of an alteration depends on whether it affects one or both copies or whether it is on the X chromosome

Variability and Penetrance • Penetrance – if you have a condition, you show some sign of it • Variability – variation in signs of a condition shown by different affected people

Non-penetrant individual

Rare vs. Common Variants

Dialogues Clin Neurosci 17:69-78, 2015.

Interpreting Variants – General Concepts • Location and specific gene(s) involved • Type of alteration and its potential effect on gene function • Number of chromosome/gene copies affected • Disorders/phenotypes associated with genes and their mechanisms of inheritance – Autosomal vs. X-linked; dominant vs. recessive

• Parental/family studies to determine segregration pattern of variant in family relative to phenotype (de novo vs. inherited) • Incomplete penetrance and variable expression

Genetic Testing – Issues to Consider • Potential complexity of results – Difficulties with interpretation • • • •

Normal variants Pathogenic alterations Variants of uncertain significance Incomplete penetrance or variable expression of phenotype

– Unanticipated outcomes - finding things that you were not looking for • Disease/disability susceptibility • Consanguinity • Mistaken parentage

• Importance of pre-test counseling • Cost and patient access

Etiology of ASDs – Current Concepts • Chromosomal copy number variants (CNVs) are important identifiable causes or predisposing factors with incomplete penetrance and variable clinical effects • Alterations in single genes cause a small percentage of cases, but no major single gene has been identified – FMR1, MECP2, PTEN, SHANK3, NLGN3, NLGN4, etc.

• Major functions of ASD-related genes – Synapses, protein synthesis, transcriptional regulation, chromatin remodeling – 400 -1000 genes may be involved in ASD susceptibility!

• Other potentially important factors – Epigenetic, metabolic, inflammatory, immune

N Engl J Med 366:733, 2012.

Sahin M, Sur M. Science 350:926, 2015

Am J Hum Genet 98:149-164, 2016.

How do we utilize all of this information in a practical way for persons with ASDs and related neurodevelopmental disabilities?

Am J Hum Genet, 86, 749-764, May 14, 2010.

Genet Med 2013, 15:399-404.

Genet Med 2013, 15:399-404.

Diagnostic Yields • Chromosome analysis – 3% • Microarray – 10% overall; ~30% in syndromic/complex cases • Single gene disorders – Fragile X syndrome (males and some females) – 1-5% – MECP2-related disorder (females) – 4% – PTEN -related disorder (OFC > +2.5 SD) – 5% – Other – ?10% (~ 1% for some specific genes) – Autism gene panel – low for isolated;