De Novo Variants: Can these Inform Clinical Phenotypes?€¦ · De Novo Variants: Can these Inform Clinical Phenotypes? Christine Seidman, MD Thomas W. Smith Professor of Medicine

De Novo Variants: Can these Inform Clinical Phenotypes?

Christine Seidman, MD Thomas W. Smith Professor of Medicine & Genetics Investigator, Howard Hughes Medical Institutes

Tetralogy of Fallot Hypoplastic Left Heart Syndrome

Transposition of Great Arteries

Schematics Courtesy of the Center for Disease Control

National Center Birth Defects/Disabilities

Prevalence:1.45/1000 children 0.4 in 1000 adults

Mean Age (2000): 17 years

11% Congenital anomaly 9-50% Neurodevelopmental deficits

(NDD)

Pediatric Cardiac Genomics Consortium

Hypothesis: De Novo Gene Mutations Cause Critical CHD Mutations are Damaging

Loss of Function (LoF): Premature Stop, Frameshift

Deleterious Missense Resides

Brigham & Women’s Hospital Children’s Hospital Boston Columbia Medical Center Children’s Hospital Philadelphia Children’s Hospital Los Angeles

Mount Sinai Medical Center Rochester Medical Center UCLA Yale School of Medicine NHLBI

Collaborating Sites: PCGC

Whole Exome Sequencing of Critical CHD Trios

1220 critical CHD Trios Exclude Syndromic Cases Unaffected parents Family history negative

vs. Observed in 900 Control Trios Unaffected parents Unaffected sibling of Autistic Child Simons Simplex Cohort Collaborators:

Matthew State, Michael Wigler, Eric Eichler

PCGC

vs. Expected Number per Exome Based on the probability of mutation at

each base, controlling for a) Local sequence context

b) Depth of coverage c) Divergences score based on human-macaque differences d) Exclude in-frame insertions/deletions e) Meta-SVM for Missense variants

(Samocha et al., Nature Genetics, 2014)

7DEOH �

7DEOH �� GH QRYR (QULFKPHQW LQ &DVHV YV� &RQWUROV

&DVHV� 1 �� &RQWUROV� 1 ��2EVHUYHG ([SHFWHG

(QULFKPHQW S2EVHUYHG ([SHFWHG

(QULFKPHQW SQ 5DWH Q 5DWH Q 5DWH Q 5DWH

$OO JHQHV7RWDO �� 6\QRQ\PRXV �� 0LVVHQVH �� '�0LV �� [�� /R) �� 'DPDJLQJ �� [��

++( JHQHV7RWDO �� [�� 6\QRQ\PRXV �� 0LVVHQVH �� '�0LV �� [�� /R) �� [�� 'DPDJLQJ �� [��

/+( JHQHV7RWDO �� 6\QRQ\PRXV �� 0LVVHQVH �� '�0LV �� /R) �� 'DPDJLQJ ��

Q� 1XPEHU RI GH QRYR PXWDWLRQV� 5DWH� 1XPEHU RI GH QRYR PXWDWLRQV GLYLGHG E\ QXPEHU RI LQGLYLGXDOV LQ FRKRUW �1�� ++(� +LJK +HDUW ([SUHVVHGJHQHV �WRS TXDUWLOH RI H[SUHVVLRQ�� /+(� /RZHU +HDUW ([SUHVVHG JHQHV �ERWWRP WKUHH TXDUWLOHV RI H[SUHVVLRQ�� '�0LV� 'DPDJLQJ 0LVVHQVH SUHGLFWHGE\ 0HWD�690� 'DPDJLQJ� '�0LV � /R)� %ROG� (QULFKPHQW ! � RU S � ��

CHD Cases, N = 1220 Controls, N = 900

Homsy et al, Science 2015

Genes Highly Expressed in Developing Heart

Observed

Damaging De Novo Variants: Enriched in CHD Cases vs Expected or Observed

Observed Expected Expected Enrichment Enrichment p p

Evidence for Pathogenicity: 21 Genes with Recurrent Damaging De Novo Variants

De Novo Copy Number Variants Detected by WES Glessner*, Bick,* et al, Circ Res 2014

Z-sc

ore

of N

orm

aliz

ed R

ead

Dep

th

-10

-

5

0

5 11 Targets, 105 bases (chr 22: 36013197-36156089)

O 1-00425

RBFOX2

De Novo RBFOX2 LoF Variants 3 SNVs (frameshifts/stop) 1 CNV(del) 4 De Novo vs expected: p=1.60E-8 All with HLHS

De Novo Copy Number Variants Detected by WES Glessner*, Bick,* et al, Circ Res 2014

RBFOX2

RBFOX2 : Binds mRNA splicing enhancer element (UGCAUG) Contributes to tissue-specific exon splicing in pre-mRNAs Highly expressed in heart (>250 reads/million) Targets ~7% Human ES cell Genes (Yeo et al; Nat Struct Molec Bio, 2009)

Genotype:Phenotype Assessments: De Novo, Damaging Variants Enriched in CHD Subsets

Extra: Non-cardiac Congenital Anomaly NDD: Neurodevelopmental Delay/Deficits


Genes Harboring De Novo Variants: Common to Both CHD and NDD Cohorts


Known NDD Genes HHE & NDD Genes

Enric

hmen

t

Functional Annotation of Genes with De Novo, Damaging Variants

2. Cardiovascular Developmental Processes (p=8.6x10-8) 3. Anatomic Structure Morphogenesis Modification (p=1.1 x 10-14)

1. Neurologic Development (p=1.8 x10-5)

4. Chromatin Modification (p=7.5x10-20)


Evidence/Support that De Novo Variants are not VUS

Ø  Unlikely Chance Co-Occurrence of De Novo, Damaging Mutations in “Constrained” Residues and Critical CHD

Ø  Genes with De Novo Variants: Highly Expressed in Affected Tissues Ø  Additional (Unappreciated) Clinical Phenotypes are “Explained” by

De Novo Variants Ø  Recurrent De Novo, Damaging Gene Variants among Probands with

“Like” phenotypes Ø  Proteins encoded by Genes with De Novo Variants, like Familial

Mutations Function in Related Biologic Pathways

Familial CHD: Cardiac TxF

Evidence/Support that Damaging De Novo Variants do Not Fully Cause Clinical Phenotypes

Ø  69 Genes with De Novo Mutations (n=85) were shared in CHD and

NDD cohorts. Highly UNLIKELY that NDD-ascertained patients with these variants had severe CHD.

Ø  Not all CHD patients with De Novo Mutations in Genes identified in NDD-ascertained patients have NDD

Ø  Mutations = Definitive Causes of Disease: Asymmetric Limb Phenotypes in CHD patients

Ø  Can Clinical Identification of De Novo Mutations be Diagnostic in Single Patients?

Strategies to Functionally Annotate CHD Mutations: Define Cardiac Developmental Transcription in Single Cells

Delaughter et al, in Review

A1

A2

KCNN3 WNT2 TNNI3

Atrial Cardiomyocytes Ventricular Cardiomyocytes

1

2

3

Acadl Mlf1 Cav1 Gja1 Casq2 Kcnk3 Dhrs7 Ckm Cox8b Mt1 Nfia Slc24a5 Sdpr Mgp Mgst1 Ckb Ddt Hmga2 Wnt2 Tagln Acta2 Gm100 Tnni3 Ndufa5 Pam Cox6a2 Smpx Fxyd1 Nppa Sln

Strategies to Functionally Annotate CHD Mutations: Define Cardiac Developmental Transcription in Single Cells


Modeling Human Mutations in Isogenic iPSC-derived Cardiomyocytes

iPSC-derived Cardiomyocytes

Adult Human Myocyte

iPSC-CM

KDM5B (1341)

KDM5A^ (452)

CHD7 (471)

115

200

125 171

136

20

834

KDM5B (2244)

KDM5A^ (858)

CHD7 (1051)

296

508

124

56

170

191

1270

Transcriptional Differences between Isogenic WT and CHD-mutant Cardiomyocytes

Increased Decreased

^KDM5A+/R1508W & KDM5A+/- >90%Congruent

hES-CM (1 yr)

Developmental age of Cardiomyocytes Differentiated from Mouse/Human Embryonic Stem Cells


E9.5 E14.5

-0.10 0.0

20

30

40

P0

10

20

30

40

10

20

30

40

10

P21

E14.5

20

30

40

10

0.05 -0.05

P0

P21 %

Cel

ls

A2

0%

50%

100%

E14.5 P0 P7 P21

*** **

WT Nkx2-5+/-

*** **

0.10

------- WT Nkx2-5

Developmental Delay in Cardiomyocytes from Nkx2-5 Mice


CHD Genetics Jason Homsy James Ware David McKean Michael Parvenof Steve DePalma Alireza Haghighi Alex Bick Akl Fahed Karou Ito

iPS-Cardiomyocytes Travis Hinson Calvin Sheng Tarsha Ward Navid Nafissi Warren Tai David Conner

RNAseq Studies Dan Delaughter Alex Bick Arin Kim

Pediatric CV Genetics Consortia Boston Children’s Hospital: Jane Newburger, Amy Roberts Children’s Hospital of Pennsylvania: Elizabeth Goldmuntz Columbia: Wendy Chung Icahn School of Medicine at Mt Sinai: Bruce Gelb Yale: Martina Brueckner & Richard Lifton NIH: Jonathan Kaltman and Charlene Shramm

Documents

De Novo Variants: Can these Inform Clinical Phenotypes?€¦ · De Novo Variants: Can these Inform Clinical Phenotypes? Christine Seidman, MD Thomas W. Smith Professor of Medicine