Use of Whole-Exome Sequencing to Define Genetic Causes of Primary Ciliary Dyskinesia Primary Ciliary Diskinesia (PCD)

2 Defining PCD-Causing Genes

Predominantly autosomal recessive Few reports of autosomal dominant and X-linked traits

Many genes participate ~250 different polypeptide identified in the ciliary axoneme

Locus heterogeneity

Allelic heterogeneity

3 Known PCD Genes

• DNAI1 and DNAH5 mutations in ~ 1/3rd of all PCD

• DNAH11 mutations in ~22% of normal dynein arms (DA) (Normal DA is small fraction of all PCD)

• Mutations in other genes in a small fraction of all PCD (KTU, RSPH9, RSPH4A, TXNDC3, DNAI2, CCDC39, CCDC40)

• Over 50% PCD patients still do not have defined mutations

4 Exome Sequencing: PCD Clinical Manifestations Type of Defect Outer Dynein Arm (ODA) 7 Inner Dynein Arm (IDA) 2 ODA + IDA 2 IDA + Central pair (CP) 3 Possible IDA + CP 1 CP / Radial Spoke (RS) 3 Normal 5 Acilia / Oligocilia 1

Total 24 5 Exome Sequencing

• Barcoded shotgun libraries generated • Nimblegen v1 capture (CCDS 2008) • Multiplexed paired-end sequencing (PE76) • BWA, SAMTOOLS used for variant calling • Average mean fold coverage (all 24 samples): 87X • 98% target bases covered at least 1X • 93% target bases covered at 8X

6 Average Results for 24 PCD Exomes

Total genes with variation 8000 Total SNPs 17000 Coding synonymous 8000 Missense 6400 Nonsense 50 Splice site 18

7 Exome Results Summary

Cilial Genes # of Known Genes Exome Hits Defect targeted? Exomes DNAI1, DNAI2, DNAH5 (n = 2) ODA Yes 7 DNAH5, TXNDC3, DNAI1 (n = 3) KTU, LRRC50, ODA+IDA DNAI1, DNAI2, Yes 2 0 DNAH5, TXNDC3, IDA None -- 2 0 IDA+CP CCDC39, CCDC40 No 4 0 RS/CP RSPH9, RSPH4A Yes 3 0 Yes (RSPH9) Normal DNAH11, RSPH9 5 RSPH9 (n = 1) No (DNAH11) Acilia None -- 1 0

8 Result 1: Novel founder DNAI2 nonsense mutation

Clinical manifestation Known Genes ODA defects DNAI1, DNAH5, DNAH11? (n = 7) DNAI2, TXNDC3, KTU? LRRC50?

• 3 unrelated cases had identical homozygous nonsense mutation in DNAI2

• All families were of Ashkenazi Jewish ethnicity

• Haplotype analysis showed ~158kb shared haplotype suggesting founder mutation

9 Result 2: Compound het DNAH5 mutations

Clinical manifestation Known Genes

ODA defects DNAI1, DNAH5, DNAH11? (n = 7) DNAI2, TXNDC3, KTU? LRRC50?

2 unrelated families

- 1st family: Compound mutation in DNAH5 (one missense/splice & one insT)

- DNAH5 insT mutation: Initial false negative in Sanger validation

- 2nd family: Compound missense mutations 10 Result 3: Novel RSPH9 deletion

Clinical manifestation Known Genes Normal DA DNAH11 (n = 5) RSPH9

• One patient had homozygous 1bp deletion in RSPH9

• Caucasian female, parental consanguinity

• Clinical manifestation: Ciliary ultrastructure normal, low nNO

11 Summary Exome Results

• PCD mutations identified in 6 of 24 patients: RSPH9, DNAI2, DNAH5 (previously known PCD genes)

• Whole-exome sequencing can successfully define disease-causing gene/mutations in genetically heterogeneous disorder such as PCD

• We still need to define possible genetic causes in the remaining 18 families

12 Primary Ciliary Dyskinesia Research Teams PCD Group: UNC-CH PCD Clinical testing group: UNC-CH RDCRN - GDMCC Consortium Michael Knowles, MD Karen Weck, MD W. Univ, St. Louis, MO Maimoona Zariwala, PhD Jim Evans, MD Thomas Ferkol, MD Margaret Leigh, MD Jonathan Berg, MD Jeff Atkinson, MD Staphanie Davis, MD Kay Chao, PhD Jane Quante, RN Peadar Noone, MD Other Collaborators Child. Hospital, Denver, CO Johnny Carson, PhD Heymut Omran, MD, Germany Scott Sagel, MD Milan Hazucha, PhD Shelley Mann, RN, Larry Ostrowski, PhD UW Genome Sciences, Seattle, WA Child. Hospital, Seattle, WA Adam Shapiro, MD Jay Shendure, MD, PhD Margaret Rosenfeld, MD Jessica Pittman, MD Debbie Nickersson, PhD Sharon McNamara, RN Deepika Polineni, MD Michael Bamshad, MD Stanford Univ., CA Leigh Ann Daniels, MD Carlos Milla, MD Susan Minnix, RN Grant Support: Jacquelyn Zirbes, RN Lu Huang, MS ORDR, NHLBI, NIH NIAID, NIH, MD Brock Barker, BS NCRR, NIH Ken Olivier, MD Caroline LaFave, BS NHLBI/NHGRI Exome project Reginald Claypool, CAPT, USPHS Kim Burns, BS RR00046, Heather Root Kristin Thompson, BS UNC/URC Hosp. Sick Kids, Toronto, Canada Rhonda Pace, BS NIH/ORD CETT Sharon Dell, MD Elizabeth Godwin, BS RS&G, U Wash. NHLBI Melody Miki, RN Cindy Sell, BS MRG/NC Biotech Center DMCC, Tempa, FL Jeff Krischer, PhD Jennifer Pilger, MA We thank Michele Manion and the PCD Foundation 13 AND All the PCD Patients and Families Known PCD–Causing Genes in 2011 Genes Class DNAH5 Outer Dynein Arms heavy chain DNAI1 Outer Dynein Arms intermediate chain DNAH11 Outer Dynein Arms heavy chain DNAI2 Outer Dynein Arms intermediate chain KTU / PF13 Cytoplasmic, pre-assembly of dynein arm LRRC50 Cytoplasmic, pre-assembly of dynein arm RSPH9 Radial spoke head RSPH4A Radial spoke head TXNDC3 Thioredoxin family CCDC39 Dynein Regulatory complex CCDC40 Dynein Regulatory complex PCD co-segregated with Other syndromes RPGR: Several families with PCD & X-linked Retinits Pigmentosa 14 OFD1: 1 family with PCD & X-linked Mental Retardation Diagnosis of PCD is Challenging

Clinical phenotype: Variable & overlaps with other airways disease

Ultrastructural analysis: Variable (ODA+/-IDA defects, RS or CP defects or normal DA & few cases with no cilia on multiple bx). Technically cumbersome and requires special training

Immunofluorescence Analysis: Requires development of cilia specific antibodies. One center

Beat frequency measurements: Useful only if highly reduced (but problematic due to the temp dependent variations). Few centers

Nasal NO measurement: Useful if CF is ruled out and in individuals >5 yo. Levels low (~20-30 nl/min compared to normal ~300 nl/min) 15 Result 1: RSPH9 deletion

Ultrastructure Known PCD- PCD-Causing Genes PCD-Causing Causing Genes Tested Prior to Genes NOT Tested Capture Prior to Capture n=5 normal DA DNAH11, RSPH9 DNAH11 RSPH9

Patient: Patient had delT +/- Caucasian homozygous Female PCD: Clinical Manifestation 1bp deletion in Ciliary Ultrastructure Normal RSPH9 Low nNO Parental Consanguinity delT/delT

16 Work continues……..

• Still validating some promising variants • Reanalyzing data (keeping rare variants, looking carefully at species conservation) • Annotating splice site variants better • Wherever possible, do exome capture for the affected sibling with the latest chip • For IDA+CP defects patients, Sanger sequencing CCDC40 and CCDC39 (Heymut)

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