1. Genética en la Clínica Diaria
Carlos E. Prada, MD
Clinical & Biochemical Geneticist
Division of Human Genetics
Cincinnati Children’s Hospital Medical Center
Director, Centro de Medicina Genómica y Metabolismo
Fundación Cardiovascular de Colombia

2. • Genetic testing uses
laboratory methods to
look at your genes.
• Identify increased
risks of health
problems
• Choose treatments
• Response to
therapies
What Can I learn from genetic
testing?

3. Types of Genetic Testing
• Diagnostic testing
• Predictive and pre-symptomatic genetic tests
• Carrier testing
• Prenatal testing
• Newborn screening
• Pharmacogenomic testing
• Research genetic testing

4. Benefits and Drawbacks
• Rule in or rule out a disease
• Eliminating uncertainty
• Treatment recommendations
• Decision making process - Screening
• Emotional burden (Guilt, angry, depression)
• Financial difficulties
• Discrimination – GINA law 2008
• Limitations of techonology

5. How do I decide about tests?
• Doctors recommendations based on personal
and family history
• Testing is voluntary
• Talk about genetic test benefits and
limitations
• Emotional support

6. What is Whole Exome Sequencing?
• The genome is the entirety of an individual’s
DNA
• The exome is the coding region of the genome
(1.5%)
– Location of majority of mutations responsible for disease

7. Whole Exome Sequencing
(WES)
• Characteristics:
– Examines the coding regions of most genes at one
time.
– One of the most comprehensive genetic tests
available
• Mutations found in ~25%
• Results may be used to:
– Guide a patient’s treatment or management
– Genetic counseling
– End diagnostic odyssey for some cases

8. History of Next Generation Sequencing

9. Differences between
NGS and traditional sequencing

10. Next generation sequencing:
• is a short read shotgun approach
• has no precise control over the sequence initiation point in the
template
• is dependent on the existence of a reference sequence and
requires bioinformatic processing for alignment
Differences between
NGS and traditional sequencing

11. Next Generation Sequencing

12. Aligned reads
Data Processing
Image analysis
Sequencing by synthesis
Cluster generation
Library preparation
Template
NGS Process
Illumina HiSeq2000

13. – Break down template
• 200-500bp fragments
• Random breakpoints
• Sonication, nebulization,
enzymatic cleavage
– Ligate Y-adapter at ends
• Anchor point for
sequencing primer
• Produce asymmetric ends
when denatured
Aligned reads
Data Processing
Image analysis
Sequencing by synthesis
Cluster generation
Library preparation
Template
A
AT
T
NGS Process

14. • Cluster generation
– Isolate individual library
molecules (ssDNA) on
the sequencing slide
• Equivalent to plating step
– Replicate the molecule
• Boost signal
Aligned reads
Data Processing
Image analysis
Sequencing by synthesis
Cluster generation
Library preparation
Template
NGS Process

15. Cluster Generation

16. Aligned reads
Data Processing
Image analysis
Sequencing by synthesis
Cluster generation
Library preparation
Template 1. Bind sequencing primer
2. Elongate with fluorescent
nucleotides
– 3’ blocked (single
incorporation)
– 4 color system, one dye per
base
3. Record the color
incorporated
– Laser excitation of dyes
4. Remove 3’ block
– Reversible terminator
chemistry
5. Repeat cycle
NGS Process

17. NGS Process
• Image analysis
– Transform image at each
cycle into strings of base
calls
• Data processing
– Align reads to a reference
genome/transcriptome
Aligned reads
Data Processing
Image analysis
Sequencing by synthesis
Cluster generation
Library preparation
Template
NGS Process

18. mRNA-
seq
Exome
Genomic
Enrich.
ChIP-seqmiR-seq
Genome
Meta
genomics
NGS Applications

19. Technical Limitations of WES
• Capturing the exome
– 85-99% examined
• Certain types of mutations not
detected
– Large del/dups, rearrangements,
trinucleotide repeat expansions,
mtDNA
• Bioinformatics pipeline
– Assumptions made based on clinical
info
– Accurate medical and family histories
are crucial
• Current knowledge of the exome

20. Future Developments in NGS
Life Technologies
Ion Proton sequencer, chip
Nanopore Technologies
GridION and MinION
sequencing nodes
$1,000 genome in a day Single molecule sequencing
Native DNA

21. Newborn Screen by NGS

23. Methods for Exome Sequencing
• Singleton
• Only proband
• Trio
• Affected + two parents
• Trio + unaffected sibling
• Affected + two parents + unaffected sibling

24. Exome Data
• Variant Filtering
– Quality
– Read depth
– False positives removed
– Common variants removed
– Variant calls: Non-pathogenic, non-exon
removed
– Inheritance pattern: AR (homozygous,
compound heterozygous), AD, XL, De novo

25. Data Analysis
• Heuristic filtering to
identify novel genes
for Mendelian
disorders
Stitziel et al, Genome Biol 2011

26. Pipelines for Filtering Data
Inheritance models
to trio-D, AR, XLD, XLR, digenic
Prioritize phenotype filter, ACMG – 57 genes,
common mutation (>1% in HGMD)
Annotate all model files
Visual inspect variants to identify and remove
false-positives
Apply in-house control script , keep freq <5%
Gene function, functional domain, pseudogene
OMIM/HGMD genes
AD AR XLD XLR Digenic
Variant
MAF< 1% , dbSNP, 1000K G, ESP5400,
OMIM, HGMD
SIFT, Conservation phyloP, Gratham score
Organize by AD, AR, XLD, XLR, digenic
Mutation type – nonsense, frameshift,
splicing, missense, in-frame, reported
synonomous
segregation
Pathway analysis
Gene expression
Gene phenotype association – human, animal
models
Literature support
Candidate gene ranking
NextGENe Golden Helix

27. Dominant
De novo
Germline mosaicism
XL
Male
Sex-limited
Recessive
Homozygous
Compound Het
Filtering by Inheritance Model

28. Compound heterozygotes
Rare /
Common
De novo /
De novo
Rare /
Rare
Rare /
De novo
Filtering by Inheritance Model

29. Glaucoma with and without aniridia
FOXC1 (c.313_314insA;p.Tyr105*)

30. Indications for Clinical WES
• The patient’s symptoms or family history
suggest a genetic condition but
– there is an atypical clinical presentation
– negative previous genetic testing
• The suspected condition is genetically
heterogeneous and multi-gene panels
are unavailable/impractical
• Requires a relation with managing physician

31. – Probable disease-causing mutation(s) related to
the patient’s phenotype with supporting evidence
– Additional gene variants of unknown clinical
significance related to patient’s phenotype
What Is Included in the Report?

32. Genetic Counseling Considerations in WES
• Patient perception of WES as definitive test
• Looking for diagnosis
• Genetic discrimination questions
• Decision making regarding incidental findings
• Coping with uncertainty
• Parental guilt

33. Patient 1
9 month old male with:
• Immunodeficiency: T-,B+, NK+
SCID with dermatitis and hair loss
• Congenital anomalies: cervical and
lumbar kyphosis, basilar skull
anomaly, short stature
• Dysmorphic features: bilateral
microtia, malar prominence, narrow
alae nasi, cupid bow lip, retrognathia

34. Cervical vertebral body hypoplasia, --“wedged”, “beaked”
Basilar skull anomaly—narrow foramen magnum
Lumbar vertebral anomalies—”wedged”, “beaked”
Skeletal changes not consistent with storage disorders

35. Patient 1
• Previous genetic testing:
– SCID panel
– DOCK8
– VCFS FISH
– CHD7
– FOXP3
• Microarray revealed pericentric region on chromosome 20 with LOH:
– 20p11.23p11.1(18,236,237-26,293,985)x2 hmz,
– 20q11.21q12(29,522,520-40,987,446)x2 hmz
• Exome Sequencing Revealed:
– Homozygous c.463_465del (p.Asn155del) mutation in PAX1:
Genetic diagnosis of Otofaciocervical syndrome
• SCID phenotype not explained
– Mouse model have showed T cell developmental defects.

36. Patient 2. Familial Dominant Parkinson
45 yo
Onset of symptoms at age 30 years
Improves with alcohol per patient
report (DYT15?)
No cognitive decline
Normal Brain MRI
Dystonia and SCA panel negative

37. Patient 3. Familial Dominant Parkinson
38 yo – Sister
Onset of symptoms at age 28 years
No cognitive decline
Dystonia on exam

38. Patient 4. Familial Dominant Parkinson
34 yo
Onset of symptoms at age 25
years
No cognitive decline

39. Patient 5. Familial Dominant Parkinson
8 year old
Difficulty writing and tremors
Normal development

40. Curr Genomics. Dec 2013; 14(8): 560–567.
Genetic Causes

41. 126,752
103,431
28,875
14,312
2,943
1,801
769
14 heterozygous variants
No known disease genes – 4 with brain expression
Quality Control
Exome
Focus on parts that make protein
Focus on important protein changes
Healthy Population 1
Dominant analysis
Healthy Population 2
Healthy Population 3
Filtered out,
do not review

42. Candidate Genes
Gene Symbol Alignment Chromosome
AKAP5 Real 14
ATXN2 Real 12
SH2D2A Real 1
ADORA3 Real 1
LENG8 Real 19
ERAP2 Real 5
CHRM2 Real 7

43. AKAP5
Important in depolarization of
neurons.

45. UCSC Genome Browser - conservation

48. Patient 6. Polyneuropathy and
Parkinsonism
38 yo previously healthy
Tremor and difficulty walking.
Chronic pain.
Normal brain MRI. No cognitive
changes.
Neurophysiological studies:
polyneuropathy

49. Patient 6. Polyneuropathy and
Parkinsonism

50. Pathway analysis

51. PLP1 network

52. Patient 7. Grandson of patient 6.
Spasticity
Nystagmus
Unable to walk
Brain MRI -
hypomyelination

53. Patient 8
• 17 year old female with leukoencephalopathy,
global developmental delay, hypotonia,
cryptogenic partial complex epilepsy, and
dysphagia.
• Seizures and developmental delay began in
infancy and progressively worsened with age
• Epilepsy Panel NGS:
– de novo c.1217G>A(p.H406R) in STXBP1

54. • STXBP1 (MUNC18-1)
encodes syntaxin
binding protein 1
• An evolutionarily
conserved protein
expressed in the
brains of humans and
rodents
• Involved in release of
neurotransmitters
through regulation of
syntaxin

55. Previously reported de novo STXBP1 mutations in patients with EIEE

56. Patient 9
6 yo with multiple joint subluxations
and dislocations (larsen-like
phenotype). Epileptic encephalopathy.
Previous tessting: FLNB sequencing
negative. Normal chromosomes.
Development: holds head, smiles, tracks
lights and noises. Not ambulatory. No
language development.

57. • Family history: Parents are first cousins. Healthy brother. No
other affected members.
• Exam: hypotonia, sterotypies, hypermobility, rotoscoliosis,
clinodactyly, and brachydactily.
• Brain MRI: frontotemporal atrophy. No leucodystrophy.
• No prenatal complications.
• Epilepsy panel study detected a homozygous mutation in
PGAP1.
Patient 9

58. Patient 10
• 3 y.o. male
• Immunological phenotype: Hypogamma-
globulinemia, recurrent infections, fevers
• Other features: fine motor and speech delay, feeding
problems/FTT, gait abnormality, hypotonia,
macrocephaly, deep set eyes, prominent forehead,
thin upper lip, long fingers and toes, persistent fetal
fingerpads.

59. Previous testing
• Normal microarray
• Normal 22q deletion testing
• MRI: “prominence of subarachnoid space
over both cerebral convexities”

60. De novo mutation in FBN1 - Marfan
syndrome
• Missense mutation:
c.5873G>A(p.1958C>Y) affecting cysteine
residue
• Previously reported as pathogenic in
literature, (Ogawa et al.)
• De novo mutation
• Sanger confirmed

61. PREGUNTAS
Contacto:
carlosprada@fcv.org

62. OFTALMOGENETICA
Carlos E. Prada, MD
Clinical & Biochemical Geneticist
Division of Human Genetics
Cincinnati Children’s Hospital Medical Center
Director, Centro de Medicina Genómica y Metabolismo
Fundación Cardiovascular de Colombia

63. Visual perception and loss

64. WUSTL
Hofer et al, 2005
Color vision in the vertebrate retina

65. • Retinitis Pigmentosa
• Leber Congenital Amaurosis
• Congenital Stationary Night Blindness
• Cone-Rod Dystrophy
• Cone Dystrophy
• Achromatopsia (complete vs incomplete)
• Enhanced S-cone syndrome
• Stargardt Disease
• Syndromic Retinopathies (PNPLA6)
Retinal Dystrophies and Degeneration

66. Retinitis Pigmentosa
• Affects 1:2,500-3,000
• Rods initially affected, later cones
• Peripheral – Central progression
• Vision loss late 1st-2nd decades
• >60 associated genes
or loci
• AD, AR, and XL forms
Rpfightingblindness.uk
Molecular Vision

67. Leber Congenital Amaurosis
• Affects 1:80,000-100,000
• Cone and Rod involvement
• Early vision loss (often <1yo)
• Myopia, nystagmus
• Progressive, ~Severe RP
• AD and AR (XL reported)
• Foveal hypoplasia
• Associated finding in ciliopathies
(Joubert, Meckel-Gruber, Senor-
Loken, Bardet-Biedl)
• 15+ associated genes or loci

68. Allelic and Locus Heterogeneity in LCA

69. LCA and Foveal Hypoplasia
A B

70. Incomplete Achromatopsia
• = Blue cone monochromacy (BCM)
• Affects 1:100,000
• Cone function defect, no loss?
• Early, static vision loss
• Incomplete (Red+Green) – XL
• (OPN1MW + OPN1LW)
• Typically normal fovea exam, can have
mild foveal hypoplasia
• Good quality of life, usually cannot
drive

71. Stargardt Disease
• Affects 1:8,000-10,000
• Photoreceptor-RPE disease
• Early macular degeneration
(2nd-3rd decade)
• RPE atrophy  PR death
• Areolar choroidal dystrophy
• Progressive, similar to AMD
• ABCA4, ELOVL4

72. OFTALMOGENETICA
Profound neuropathy target esterase
impairment results in Oliver-
McFarlane Syndrome

73. Oliver-McFarlane Syndrome
• Hypopituitarism-chorioretinopathy-trichomegaly
• 14 patients reported since 1965 (2 sib pairs)
• Congenital anterior hypopituitarism (short stature, ID)
• Progressive chorioretinal degeneration in childhood
• Ataxia, neuropathy, spastic paraplegia (2nd-4th decade)
• Our patients: 3rd sibship, 7yo+9yo
• No family history
• Normal SNP microarray
• No genetic cause

74. A
D E F

75. 99,710
Quality >20, Depth >10
Whole exome analysis
Recessive inheritance
74,187
Remove if synonomous, noncoding,
predicted likely benign/tolerated
11,363
108
Homozygous
AR
Compound Heterozygous
59
Remove if frequency >1%
Visual inspection
Remove false positives
0
>1%
3
1PNPLA6
p.Arg1099Gln;p.Gly1176Ser

76. Pt3. c.1390-27_2287+1200[2];p.Val1215Ala
Pt4. c.1973+2T>G;p.Val1215Ala

77. Pt5. Patton et al. Am J Ophthalmol (1986). p.Arg1031Glnfs*38;p.Gly1129Arg
Pt6. p.Arg1031Glnfs*38;p.Gly1129Arg

78. Pt5. p.Arg1031Glnfs*38;p.Gly1129Arg
H&E SMI31
GFAP IBA1
Human Cerebellar Degeneration

79. Oliver and McFarlane, 1965 Pt2
Pt2 Pt1
Pt4
1965 Pt6

80. Phenotype
Oliver-McFarlane
1965
Laurence-Moon
1866
Bardet-Biedl
1920-1922
Trichomegaly, Alopecia + – –
Intellectual disability + + +
Retinal degeneration + + +
Choroidal atrophy + + –
Anterior hypopituitarism + + –
Short stature + + –
Hypogonadism + + +/–
Ataxia, SP, PN + +++ –
Obesity – – +
Polydactyly – – +
Oliver-McFarlane and Laurence-Moon Syndromes

81. Laurence-Moon Syndrome
Poster 2929S: H. Dollfus, M. Prasad, C. Stoetzel
Pt7-10. Chalvon-Demersay et al. Archives de Pédiatrie (1993).
p.Gly726Arg;p.Arg1031Glnfs*38

82. PNPLA6 (Neuropathy Target Esterase)
Poster 2976T: G. Arno, S. Hull, V. Plagnol, T. Moore Hou et al, 2009

83. Spastic Paraplegia 39 (SPG39)
Adult onset
Ataxia, +/- Cerebellar atrophy
Spastic Paraplegia
Peripheral Neuropathy
Gordon-Holmes Syndrome
Boucher-Neuhauser Syndrome
Late childhood/Adolescent onset
Ataxia, SP, PN, Cerebellar atrophy
Hypogonadotropic Hypogonadism
Chorioretinal Degeneration (BNHS)
Laurence-Moon Syndrome
Oliver-McFarlane Syndrome
Congenital/Childhood onset
Ataxia, SP, PN, Cerebellar atrophy
Anterior Hypopituitarism, atrophy
Chorioretinal Atrophy
Hair Anomalies (OMS)
Synofzik et al, 2013
Topaloglu et al, 2014
Rainier et al, 2008
PNPLA6 spectrum of disorders

84. PNPLA6 spectrum of disorders
Hypothesis: spectrum of congenital  adult
PNPLA6 diseases corresponds to the severity of
NTE loss-of-function: SPG39↓ OMS↓↓↓
1) Validate OMS/LMS mutation pathogenicity in vivo
2) Examine PNPLA6 expression during embryogenesis
3) Compare NTE enzymatic activity across disease states

85. Normal Mild Intermediate Severe
Zebrafish Morpholino – Rescue
OMSSPG39WT
Morpholino: Song et al, 2013
vs Wt RNA
* p<0.05
** p<0.01
***p<0.001

86. Human Embryonic PNPLA6 Expression
CS23 (GA 8 weeks)

87. Human NTE Activity Assay
Skin biopsy
Fibroblast culture
Phenol
Valerate
Phenol
NTE
NTE Cellular Assay
Wild type – 2 controls
SPG39 – carrier
SPG39 – homozygote
OMS – parent carriers
OMS – affected patientsParaoxon
Assay: Hein et al, 2010

88. OMSSPG39WT
Human NTE Activity
vs Wt/Wt
* p<0.05
** p<0.01
***p<0.001

89. PNPLA6-opathy Disease Model
Healthy
NTE Activity
Phenotype
SPG39
Laurence-Moon
Oliver-McFarlane
GHS BNHS

90. Summary
1) Oliver-McFarlane and Laurence-Moon syndromes are
caused by PNPLA6 mutations and NTE loss-of-function
2) Human expression and pathology studies support
a spectrum of tricho-oculo-neurologic PNPLA6-opathies
3) Phenotype is dose-dependent – patients with OMS have
three-fold loss of NTE activity compared to SPG39

91. Acknowledgements
The families
Robert.Hufnagel@cchmc.org
Carlos.Prada@cchmc.org
carlosprada@fcv.org