Saudi Human Genome Program Building a Foundation for Personalized Medicine

Brian Meyer, PhD What is Personalized Medicine?

• The ability to determine an individual's unique molecular characteristics – to use those genetic distinctions to diagnose more finely an individual's disease – select treatments that increase the chances of a successful outcome – reduce possible adverse reactions. • Personalized medicine also is the ability to predict an individual's susceptibility to diseases and thus to try to shape steps that may help avoid or reduce the extent to which an individual will experience a disease. What does it require?

• Essential features for personalized medicine to be a fully functioning reality at the clinical level include: – an electronic medical record – personalized genomic data – physician access to electronic decision support tools – personalized treatments – personal clinical information available for research use Why Personalized Medicine?

• The past decade has seen a revolution in human genetics – it is having a very significant impact on virtually all specialties of medicine • Several scientific advances are responsible for this revolution – First is the recognition and validation that the genetic composition of individuals play a significant role in that individual's health and predisposition to common diseases such as heart disease and cancer. – Second is the availability of the human genome sequence and the many high throughput technologies developed and advanced during and since the Human Genome Project. – Third is the imminent ability to completely sequence and analyze the human genome in real time at relatively low cost.

OMIM 2012/2013

9 Characteristics of the Saudi Population

─Homogeneity Iraq

Iran ─Tribal organization (multiple independent Jordan RUWALLA Kuwait

homogeneous units with limited founders) ENAZA SHAMMAR Awazim Arabian ─8 tribes MUTAIR Gulf

Ajman ─Tribal affiliation maintained Juhainah Qatar Gulf of HARB SUHUL OTAIBAH Oman

Jahadila SUBAI Manasir U.A.E. Egypt Oman ─Consanguinity Quraish Buqum Ghamid MURRA ─57% Consanguinity rate Red Sea Zahran QAHTAN Dawasir ASIR ─Up to 98% in specialty clinics Sudan Yam Arabian ─Mostly first cousin marriages Yemen Sea ─Large family size ─Nuclear family size of 8-10 is common ─High incidence of rare recessive Mendelian traits and common diseases such as type 2 diabetes The Saudi Human Genome (SGP) is a national program which aims to sequence 100,000 samples (normal and disease) from the Saudi population in the next 5 years. Create multiple satellite centers across the Kingdom WORKFLOWS There are three (3) main work flows:

Gene panels: 13 panels specifically designed for inherited diseases. These panels cover all genes in OMIM; unique to SHGP

Exome Sequencing: Predominantly Index cases from families with Autosomal recessive diseases for gene discovery

Genome Sequencing: For cataloging population variants in health and disease

GENOME vs. EXOME vs. MENDELIOME / GENE PANELS

“Actionable” Monogenic disorders are encountered by every discipline of medicine

• Investigation of underlying causes • Whole Genome Sequencing • Most comprehensive but expensive, inefficient and has ethical questions that need to be answered

• Growing number of large Whole Exome Studies • Expensive, ethical questions (Clinical Exome $2,500; long turnaround) • Significant technical commitment & interpretation required • ~25% yield

• Mendeliome/Gene Panels • ~3,200+ known Mendelian genes • Requires less technical expertise and interpretation, cheaper, faster • Specifically applied • Jaundice  GI panel • Recurrent infection  Primary Immune Deficiency (PID) panel • Unusual facial appearance  Dysmorphology Mendeliome - Gene Panel Design

 Two Medical Geneticists developed the gene content of the “SHGP/Mendeliome” panels

 Defining the Mendeliome: - monogenic disorders [autosomal, X-linked, mtDNA] - symptom/sign based design: (i.e. neuro, ophth, derm, renal, heme, etc.)

~ 4,300 monogenic disorders - OMIM; PubMed; Genetic Testing Registry (GTR); & Gene Tests (https://www.genetests.org/)

Number of Entries in OMIM (Updated 9/15/2015, http://omim.org/statistics/entry

Prefix Autosomal X Linked Y -Linked Mito-

* Gene description 14,234 700 48 35

+ Gene & phenotype, combined 82 2 0 2

# Phenotype description, molecular basis known 4,214 297 4 29

% Phenotype description/locus, ? molecular basis 1,511 129 5 0

Phenotypes with suspected mendelian basis 1,706 112 2 0

Totals 21,747 1,240 59 66 Gene Panel Design • Develop multiplexed gene panels using Ion AmpliSeq Designer software

• Primer design was based upon generating amplicons with an average length of 200 bp providing 90% minimum coverage of the CDS, and on average 10 bp flanking regions of associated exons.

• Following in silico design coverage was assessed for compliance with design criteria and manual processes applied on a gene by gene basis to ensure adequate coverage and resolve factors such as 3’- SNPs that could impact primer efficiency. MENDELIOME GENE PANELS Gene Panel Signs & Symptoms # Genes IEM AA, OA, FAO, Mito-ETC, Peroxisomal, LSDs, GSD, SLCs, Steroids, Neurotransmitters 570 CARDIO-VASCULAR Cardiomyopathies (HCM/DCM/Restrictive), Arrhythmias, CHD, CAD/MI 426 DEAFNESS Sensorineural hearing loss 124 Alopecia/Hypotrichosis/Hypertrichosis/abnormal sweating; Bullous lesions, Cafe au lait spots/lentigines, DERM Diffuse pigmentation disorders, Hyperkeratosis, Icthyosis, Lax/tight skin, Peeling Skin deficiency/pits, 214 Unexplained facial papules/rash, Unexplained itch, Urticaria/angioedema, White mouth, Xanthomas DYSM/DYSPLASIA Dysmorphism, Skeletal dysplasia 422 DM, Growth Failure-Short Stature, Hyperlipidemias, HTN, Hypoglycemia-Hyperinsulinemia, ENDOCRINE Hypogonadism, Infertility/Ovarian failure/Ambig.Genitalia, Obesity, Disorders of Pit., Para/Thyroid 311 Aganglionosis, Bowel rupture/ diverticulosis, Cholelithiasis, Chronic diarrhea/malabsorption, Chronic vomiting, Esophageal leio myomatosis/ strictures/perforation, GI bleeding, Hepatomegaly/cirrhosis/liver GASTRO-INTESTINAL failure, Heterotaxy, Imperforate anus, Jaundice, Omphalocele, Pancreatic insufficiency/agenesis/ 189 inflammation, Pyloric stenosis/atresia, Unexplained abdominal pain HEME Anemias hemolytic, Aplastic Anemias, Excessive Bleeding-Clotting 405

Ataxia/Dystonia/Spasticity/Myotonia; Brain malformation, Brain malformation/ Microcephaly/ 758 NEUROGENETICS Macrocephaly, Epilepsy, Hypotonia/Weakness/hypoventilation, ID/speech delay/autism, Lactic acidosis, Narcolepsy, Neurodegeneration, Neuropathy Abn. Lymphocytes, Agamma, Allergic Rhinitis, Candidiasis, Complement def., Eczema-Urticaria, PID Hemophagocytosis/FHL, Auto-Inflammatory syndromes, chronic Meningitis, ID + Herpes simplex, 265 Osteomyelitis, Phagocyte abn., Pneumonia/Sinusitis, SCIDS, Autoimmunity synd, recurrent Viral infxns PULMONARY Surfactant, Apnea/Narcolepsy, Cilia/Bronchiectasis/Emphysema 120 CKD, Cystic KD, Hematuria, Nephrolithiasis/ Nephrocalcinosis, Nephrotic syndrome, Renal agenesis/ RENAL hypoplasia/dysplasia, Renal tubular diseases, VUR 96 Abnormal cornea, Apraxia, Cataract, Ectopia lentis, Glaucoma, Microphthalmia/Coloboma, Ocular VISION albinism, Ophthalmoplegia/apraxia, Optic atrophy, Retinal dystrophy/ achromatopsia 334 - 15% redundancy among different panels

SHGP Neuro - Panel

= 24 samples 24 x 758 genes (18093 amplicons)

~162 x coverage

Absent in Total Variants: QS>100: Coding or Missense/ dbSNP,1000G, Homozygous: 1106 717 splicing: 643 Nonsense: 263 1000 Arabs, 1000 1 Saudi: 21 Variant Analysis Pipeline - All Panels

Panel Input Functional Public Pop. SGP Quality Zygosity sites DBs Pop. DB Cardiovascular 746 338 76 26 9 2 Deafness 828 257 63 50 17 6 Dermatology 1113 271 71 41 17 5 Dysmorphology/Dysplasia 1529 369 80 43 15 2 Endocrinology 1129 326 61 42 19 5 Gastroenterology 362 190 60 20 6 1 Hematology 1474 324 79 39 18 3 IEM 1955 571 94 54 24 4 Neurology 2885 718 158 87 29 4 PID 633 309 111 22 6 1 Pulmonology 723 230 74 39 21 3 Renal 507 132 35 21 7 1 Vision 906 341 75 51 17 3 Total (averages) 1138 337 80 41 16 3 The table entries are the average number of variants per panel after applying the respective filter in the column title. The column titled ‘Input’ is the input without applying any filter. The column titled ‘Functional sites’ is for the step of filtering intronic, UTR, synonymous variants. The column titled ‘Public Pop. DBs’ is for the step of filtering variants based on the 1000 Genomes Project database. The column titled ‘SGP Pop. DBs’ is for the step of filtering variants based on the in-house database. The column titled ‘Quality’ is for the step of filtering based on the quality criteria. The column titled ‘Zygosity’ is for the step of excluding non-homozygous variants

Genome Biol. 2015 Jun 26;16:134. “MENDELIOME ASSAY” CLINICAL SENSITIVTY Total patients Overall clinical Selected subgroup clinical Gene Panel type run sensitivity sensitivity Cardiovascular 243 28% Cardiomyopathy 32% Congenital heart disease 10% Arrhythmias 31% Aneurysms 29% Deafness 147 54% Hearing loss - Dermatology 68 62% Various derm. features - Dysmorphology/Dysplasia 354 38% Skeletal dysplasia 45% Dysmorphism 32% Endocrinology 36 61% Pituitary and thyroid disorders - Gastroenterology 73 29% Persistent jaundice - Hematology 33 24% Aplastic anemia - IEM 122 59% Metabolic disorders - Neurology 524 40% Syndromic DD/ID (recognizable 47% syndromes) Syndromic DD/ID NYD (not yet 25% determined, unrecognizable syndrome) Structural brain (cerebral/cerebellar/brain 34% stem) and spinal malformations/anomaliesa Non-syndromic DD/ID NYD (not yet 11% determined, Unrecognizable) Neurodegenerative disorders 42% Coordination/movement disorders 69% Peripheral neuropathy 33% Myopathies/joint abnormalities 56% Genome Biol. 2015 Jun 26;16:134. “MENDELIOME ASSAY” CLINICAL SENSITIVTY

Total Overall clinical Selected subgroup clinical Gene Panel type patients run sensitivity sensitivity PID 196 37% Primary immunodeficiency disorders - Pulmonology 36 36% Chronic lung infection suspected - Cystic Fibrosis Renal 107 57% Glomerular/tubular disorders 41% Cystic kidney disease 63% Kidney malformation 69% Vision 418 52% Retinal dystrophy (syndromic, non- 65% syndromic, RP, CRD, macular dystrophy, FEVR, GFS) - Sanger confirmation of a Cataractlikely (syndromic variant and non - 34% syndromic) - OVERALL: 43% Aniridia 33% Micro/anophthalmia (+/- coloboma) 30% Corneal dystrophy (CHED & all other 40% subtypes) Others 23% aPrimary microcephaly cases are included in this group. bNon-syndromic cases of autism/mental disorder and epilepsy are included under this group cAtaxia cases secondary to cerebellar hypoplasia are included under the structural brain abnormalities group. dCases with arthrogryposis multiplex syndromes are included under the myopathies group. CHED corneal hereditary endothelial dystrophy, CRD cone-rod dystrophy, DD developmental delay, FEVR familial exudative vitreoretinopathy, GFS Goldmann-Favre. syndrome, ID intellectual disability, NYD not yet determined, PID primary immunodeficiency, RP retinitis pigmentosa

Genome Biol. 2015 Jun 26;16:134. MENDELIOME ASSAY: Improved Annotation of the Human Variome

• Confirmed Candidate (OMIM) genes: e.g. ARL14EP, ZNF526, WDR45B, and WDR81 • 433 novel (total = 788) disease alleles – largest number from a single study; • A very large number of polymorphic variants attributed to individuals of Arab ethnicity • Our study identified 342 HGMD variants including 133 with MAF >5% • 137 variants with MAF >1 % in our study have MAF < 1% in the 1000 Genomes database • Several inactivating mutations of disease causing genes, in the homozygous state at high frequency or in patients with no phenotype Saudi Mendeliome Group (alphabetical order): Abdulwahab, Firdous; Abouelhoda, Mohamed; Abouthuraya, Rula; Abumansour, Iman; Ahmed, Syed O.; Al Rubeaan, Khalid; Al Tassan, Nada; AlAbdulaziz, Basma; AlAbdulrahman, Khalid; Alamer, FH; Alazami, Anas; Al-Baik, Lina A.; Aldahmesh, Mohammed; Al-Dhekri, Hasan; AlDusery, Haya; Algazlan, Sulaiman; Al-Ghonaium, Abdulaziz; Alhamed, Mohammed; Alhashem, Amal; Alhissi, Safa Ahmed; AlIssa, Abdulelah; Aljurf, Mahmoud D.; Alkuraya, Fowzan S; Alkuraya, Hisham; Allam, Rabab; Almasharawi, Iman J; Almoisheer, Agaadir; AlMostafa, Abeer; Al-Mousa, Hamoud; Al-Muhsen, Saleh; Almutairy, Eid A; Alnader, Noukha; AlNaqeb, Dhekra; ALOtaibi, AB; Alotibi, Afaf; Al-Qattan, Sarah; Al-Saud, Bandar; Al-Saud, Haya; Alshammari, M; Alsharif, Hadeel; Alsheikh, Abdulmoneem H; Al-Sulaiman, Ayman; Altamimi, AS; Al-Tayeb, Hamsa; Alwadaee, SM; Al-Younes, B; Alzahrani, Fatima; Anazi, Shamsa; Arnaout, Rand; Bashiri, Fahad; Binamer, Yousef; Binhumaid, FS; Boholega, Saeed; Broering, Dieter; Burdelski, Martin; Dasouki, Majed Jamil; Dzimiri, Nduna Francis; Elamin, Tanziel; El-Kalioby, Mohamed; Elsiesy, Hussien; Faqeih, Eissa; Faquih, Tariq; Hagos, Samya; Hagr, Abdulrahman A; Hashem, Mais; Hawwari, Abbas; Hazzaa, Selwa; Ibrahim, Niema; Imtiaz, Faiqa; Jabr, Amal; Kattan, Rana; Kaya, Namik; Kentab, Amal; Khalil, Dania; Khan, Arif O; Khier, Omnia; Meyer, Brian F; Mohamed, Jawahir; Monies, Dorota; Muiya, Paul N.; Murad, Hatem; Naim, EA; Owaidah, Tarek; Patel, Nisha; Ramzan, Khushnooda; Salih, Mustafa A; Shagrani, Mohammad; Shaheen, Ranad; Shamseldin, Hanan; Sogaty, Sameera; Subhani, Shazia; Taibah, Khalid; Wakil, Salma M.

Genome Biol. 2015 Jun 26;16:134.

Right elbow contracture and wrist joint deformity

Gene underlying JIA

Syndromic Dystonia with Myoclonus and Tremor • Asymmetric, generalized Tremor: – involving head, neck, trunk and extremities – 4-5Hz frequency at rest, waxing and waning in amplitude, and worsening with posture and action – side-to-side (“no” type) movement of neck – pronation-supination and abduction- adduction movements in arms and wrists, respectively – dysarthric and tremulous speech • Focal or segmental Dystonia – retrocollis shoulder elevation – hyperextension of fingers • Variable degrees of Spasticity – affecting legs more than arms – some patients require walking aids

WES – Novel Gene: CAMTA2A Trio Analysis

De novo Recessive

Child Filtered Child Filtered data data Mother Father Mother Father Filtered data Filtered data Filtered data Filtered data

HET variant only HOM variant present in child, present in child, HTZ variant in both absent in parents parents Comp. HTZ

Child Filtered data Mother Father Filtered data Filtered data 2 HET variants in same gene, present in child, one from each parent Sporadic Mutation – Trio Analysis

Family Clinical Description Sporadic Mutation Family 1 Female with severe intellectual disability, dysmorphic MECP2:NM_001110792:exon3:c.C433T:p.R145C features (big dysplastic ears, deep set eyes), epilepsy. Family 2 Male with intellectual disability, microcephaly, CTNNB1:NM_001098209:exon3:c.C232T:p.Q78X hyperekplexia (startle disease) , impaired upward gaze . Family 3 Male with delayed psychomotor development, brain COL4A1:NM_001845:exon39:c.3326G>A:p.G1109E malformations (migration disorder), cataract, urethral posterior valve. Family 4 Male with severe intellectual disability, epilepsy, hip OCRL:NM_000276:exon12:splicing:AGdel dysplasia, hypotonia, renal insufficiency, massive fibromatosis on feet and hands Family 5 Female with intellectual disability, dysmorphic features (high forehead, long and curly eyelashes, syndactyly of second and third toes, hypotonia of trunk muscles, psychomotor delay, epilepsy.

SUMMARY

High (43%) yield of the Mendeliome assay vs. WES (25%)

Highest yields were Pediatric .Opthalmology (52%), Neurological Disorders (39%), D/D (37%), PID (35%)

Specificity of presentation impacts on assay sensitivity: * D/D panel: Skeletal dysplasia (45%) vs. Dysmorphia (30%) * Neuro panel: muscular dystrophy/myopathy (55%), neurodegenerative disorders (44%) non-syndromic Dev Delay/ID (10%) *Vision panel: retinal dystrophies (64%) vs. whole vision (52%)

WES studies often identify mutations in known genes

Improved Annotation of the Human Variome

Unique allelic distribution of pharmacogenetically relevant genes

The Saudi Human Genome Project aims to address the issue of inherited disease throughout the entire “Circle of Life”

Pre-Marital Screening Pre-implantation & Counseling Genetic Diagnosis (PGD)

Genetic and Biochemical Testing Pre - Natal Testing Molecular Amniocentesis CVS Diagnostics Cell-free fetal DNA

Newborn Screening Thank You For Your Attention