Paediatric Neurogenetics

Prof Martin Delatycki

Clinical Genetics Austin Health

Bruce Lefroy Centre for Genetic Health Research, MCRI

What will be covered

• Dynamic mutations

• NF

• Tuberous sclerosis

• Prenatal diagnosis and preimplantation diagnosis

• Genetic disease searches

Genes- Nuclear and Mitochondrial

• Nuclear- most genes

(~23,000)

• Mitochondrial- 37 genes- 13

encode proteins

– No introns

– 93% of mitochondrial

genome is coding DNA

(nuclear 2%)

– Mitochondrial DNA

maternally inherited

Dynamic mutations

DYNAMIC MUTATIONS

• = a mutation which changes upon transmission

• Trinucleotide repeat disorders are the best example

• = three nucleotides which are present in increased number

TRINUCLEOTIDE REPEATS

• Normal

• Disease causing when expanded beyond a certain

threshold

• Below that threshold they are stable both in mitosis and

meiosis

• Beyond a certain number the repeat can be unstable in

meiosis ± mitosis

DYNAMIC MUTATIONSREPEAT DISEASES INHERITANCE PARENTAL

GENDER BIAS

CGG/GCC FRAX A, E XL MATERNAL

GAA FRIEDREICH ATAXIA AR MATERNAL

CTG MYOTONIC

DYSTROPHY, SCA8?

AD MATERNAL

CAG HD, DRPLA, SCA

1,2,3,6,7, 17 (TBP),

SBMA,

AD, XL PATERNAL

12 MER PROGRESSIVE

MYOCLONUS

EPILEPSY

AD

5 MER SCA 10 AD

CCTG DM2 AD

SITE OF TRINUCLEOTIDE REPEAT

5’

ATG

TAA

3’

CCG GAA CAG CTG

FRAXA

CAG

SCA 12

FRDA HD

SBMA

SCA1,2,3,6,7

DM

?SCA8

DYNAMIC MUTATIONS

• INTERGENERATIONAL INSTABILITY

• ANTICIPATION

• PREMUTATIONS

INTERGENERATIONAL INSTABILITY

• REPEAT CHANGES IN SIZE FROM PARENT TO

OFFSPRING

• SEX OF TRANSMITTING PARENT IMPORTANT

• SOME MORE UNSTABLE FROM FATHER, OTHERS

FROM MOTHER

ANTICIPATION

• MORE SEVERE PHENOTYPE WITH SUCCESSIVE

GENERATIONS

• BEST EXAMPLE IS MYOTONIC DYSTROPHY

MYOTONIC DYSTROPHY

• Worse with succeeding generations= anticipation

• Most severe= congenital myotonic dystrophy

• Very floppy, may need ventilator

• If survives, intellectual disability

ANTICIPATION

Myotonic dystrophy

MYOTONIC DYSTROPHY

• CTG repeat

• <37- no problem

• >50- disease

• 50-100- generally mild

• Congenital form often >1000

• Congenital form almost always maternally inherited

PREMUTATIONS

• REPEAT SIZE WHICH IS UNSTABLE BUT DOES NOT

RESULT IN A PHENOTYPE

• BEST EXAMPLE IS FRAGILE X SYNDROME

Fragile X syndrome

Fragile X syndrome

FRAGILE X SYNDROME

• CGG repeat

• <55- normal and no risk for offspring (45-54 grey zone)

• 55-200= premutation- normal intellect but risk to offspring of

females

• >200- males have intellectual disability but intellect in

females is variably affected

FRAGILE X PREMUTATION

• Not truly a premutation because female premutation

carriers have a higher rate of

– premature ovarian failure

– “shy” personality

• Males and females with a premutation are at higher risk of

late onset cerebellar tremor/ ataxia syndrome (FXTAS)

FRAXA Cerebellar Tremor/ Ataxia Syndrome

• Progressive action tremor, ataxia, cognitive decline

• 45% males, 17% females >50yrs

GENOTYPE- PHENOTYPE

• For most dynamic mutation disorders, the larger the repeat,

the earlier the onset

• CANNOT use the repeat size to predict phenotype with

accuracy

• eg: myotonic dystrophy prenatal

• Huntington disease predictive test

• NOT true for all dynamic mutations- eg: CCTG repeat in

DM2

Friedreich Ataxia

Friedreich Ataxia

Autosomal Recessive

Commonest Hereditary Ataxia

Prevalence 1:30 000 based on molecular data

Carrier Frequency 1:85

Friedreich Ataxia

• Clinical Features

» progressive ataxia of limbs

» absent lower limb reflexes

» reduction in vibration and proprioception

» extensor plantar responses

» scoliosis

» foot deformity

» cardiomyopathy

» diabetes mellitus

Friedreich Ataxia

9q13 - q21.1

GAA triplet repeat in intron 1 of FXN gene

98% of affected alleles have an expansion of this trinucleotiderepeat

Normal 7-36

Affected 67-1300

Point mutations and deletions in FXN described

12345a

Intron 1

GAAn

Friedreich Ataxia

• FXN produces frataxin

• Nuclear encoded, mitochondrial localisation

GAA Expansion

0

5

10

15

20

25

30

35

100 300 500 700 900 1100 1300 More

GAA repeat number

Num

ber

of a

llele

s in

ran

ge

0

510

15

2025

30

0 500 1000 1500

GAA repeat size of smaller allele

Ag

e o

f o

nse

t

Age of onset v GAA size smaller allele (R2=0.39)

P- value: complication v small allele size

FEATURE Delatycki Dürr Filla Montermini

Wheelchair 0.02 - <0.01 <0.0001

Cardiomyopathy 0.11 <0.05 <0.05 NS

Diabetes 0.08 NS <0.001 NS

Scoliosis 0.0001 <0.005 - -

Iron in FRDA

• Knockout of the yeast frataxin homologue leads to

mitochondrial iron accumulation

• Iron deposits found in the myocardium of FRDA patients

Iron levels in whole cells- FRDA v control (p=0.55)

0

0.5

1

1.5

2

2.5

3

3.5

FRDA Control

ng

Fe

/ ce

ll x

10000

Mitochondrial iron/protein (p=0.01)

0

1

2

3

4

5

6

FRDA Control

ug

Fe

/ u

g p

rote

in x

10000

Mitochondrial copper/protein (p=0.77)

0

0.2

0.4

0.6

0.8

1

1.2

FRDA Control

ug

Cu

/ u

g p

rote

in x

10000

Pathogenesis

• Frataxin involved in iron sulphur cluster synthesis

• Fe-S crucial in various protein including some respiratory chain

proteins

• Deficiency leads to increased susceptibility to oxidative stress

• Increased Fe results in oxidative damage 2 to Fenton chemistry

FRDA Treatment - Iron chelation

Must preferentially remove mitochondrial rather than cytosolic iron

Desferioxamine not successful (Kaplan unpublished data)

Deferiprone (L1) has shown promise (Boddaert, Cabantchik Blood, 2007)

Reduced dentate nucleus Fe with six months treatment with 20-30 mg/kg/day deferiprone

? Improved neurological parameters- open label

Des Richardson- mitochondrial specific chelators

Deferiprone trial

• Placebo controlled, 3 doses- 20, 40, 60 mg/kg

• International multi-centre- Europe, Australia, Canada

• Recruited 10 subjects here

• High dose group permanent stop

• 20 and 40mg/kg – improved cardiac parameters, 40mg/kg

worse neurology scales

FRDA Treatment - Anti-oxidants

• Treats down-stream effects

• Idebenone, mitoquinone, CoQ10, vitamin E

Idebenone

Late 90’s shown to reduce cardiac hypertrophy in some

Buyse et al Neurology 2003- improved cardiac strain and strain rate

Di Prospero et at Lancet Neurology 2007

Phase II placebo controlled multi dose

Subjects < 18 years

12 subjects per arm- placebo, 5mg, 15mg, 45mg/kg/day

Trend to dose dependent response as measured by ICARS

Two phase III trials

USA- 6/12 trial no benefit

Europe- 12/12 trial- no benefit but close and 12% of placebo subjects took idebenone!

Idebenone trial

Upregulation of FXN

In FRDA normal frataxin produced but at low levels

An increase in frataxin production should ameliorate

disease

A few-fold increase in frataxin may stop disease

progression

Upregulation of FXN

Erythropoietin

HDAC inhibitors

Resveratrol

Dynamic Mutations-Practical Implications

• Diagnosis

• Predictive testing

• Therapeutic implications

PREDICTIVE TESTING

• Testing people at increased risk for a disease prior to the

clinical onset of that disease

• Huntington disease= greatest experience

• Over 5000 predictive tests for HD done worldwide

PREDICTIVE TESTING

• Process

– 1) discussion of genetics

– 2) neurological examination

– 3) counselling regarding what a positive and negative result

may mean for that person

– 4) result given

– 5) post-result counselling as required

PREDICTIVE TESTING- UPTAKE

• Prior to availability- 75%

• Reality- less than 20%

Predictive testing in minors

• Guidelines recommend against this

• Evidence is scarce

Neurofibromatosis Type 1

Neurofibromatosis Type 1- facts

• Incidence about 1:3000

• Affects all ethnic groups

Neurofibromatosis Type 1- diagnostic criteria

• Require two or more of the following:

– 6 café au lait patches (>5mm prepubertal; >1.5cm

postpubertal)

– 2 neurofibromas or 1 plexiform neurofibroma

– Axillary and/or inguinal freckling

– Optic glioma

– 2 Lisch nodules

Neurofibromatosis Type 1- diagnostic criteria

(cont)

– Distinctive osseous

lesion inc: sphenoid

wing dysplasia,

thinning of long bone

cortex ±

pseudoarthrosis

– A 1st degree relative

with NF1 by the

above criteria

Plexiform neurofibromas

• 30%

• Severe lesion of head or neck- 2%

• Majority of disfiguring plexiform neurofibromas evident by 2

years

• Variable natural history

• Can undergo malignant transformation

Plexiform Neurofibromas- Rx

• Surgery of limited value- regrowth

• Medical treatment-

– 2interferon

– cis-retinoic acid

– thalidomide

– farnesyl protein transferase inhibitor

– etoposide

• Problem of assessing efficacy

Neurofibromatosis Type 1- complications

• Learning difficulties- 50%. Mean full scale IQ- 88.5 (51-

129):

– severe (IQ< 70)- 6%

– about 45% require special educational assistance

– UBOs (T2 weighted MRI signal)- ? association with learning

difficulties

• Seizures- 4.4%

Neurofibromatosis Type 1- complications

• Symptomatic CNS tumours including optic nerve gliomas-

2% Subclinical optic nerve gliomas- 15%

• Peripheral nerve malignancy- 10%- often in a pre-existing

plexiform

• Scoliosis:

– requiring surgery- 4.4%

– mild- 5.2%

Optic Nerve Gliomas

• Asymptomatic 15%

• Symptomatic 2%

• F:M= 2:1

• Often regress

• Association with eyelid plexiform

• Treatment with carboplatin successful in some with

symptomatic lesions

UBOs

• Hyperintense regions on T2 MRI images

• Present in about 2/3- cerebellum> brainstem> internal

capsule

• Increase in size till about 10yrs then often disappear

• Benign

• ? Should be added to diagnostic criteria

CNS Tumours

• 5x more common in NF1 than in general population but

after 10 years 100x

• Apart from optic nerve gliomas CNS tumours are not very

common (but increased compared to people without NF1)

• Include

– non-optic nerve gliomas

– meningiomas

– astrocytomas

CNS tumours

• Childhood astrocytomas tend to be low grade

• Adult astrocytomas often high grade

Spinal Cord

• Spinal neurofibromas

• Malignant peripheral

nerve sheath tumour

infiltrating the spinal

canal

• Rare

• Children> Adults

Aqueduct Stenosis

• Increased incidence in NF1

– tumour

– idiopathic

• Childhood or adult presentation

• Responds well to shunting

Malignant Peripheral Nerve Sheath Tumours

(MPNST)

• Neurofibrosarcoma

• Lifetime risk about 10%

• Most arise in deep plexiform neurofibromas and fewer in

superficial cutaneous plexiform neurofibromas

• Present with pain and rapid growth

• PET scanning useful for diagnosis

• Risk increased by radiotherapy

• Treatment- surgical

Neurofibromatosis Type 1- complications

• Hypertension- 6%

– Renal artery stenosis- 1.5%

– Phaeochromocytoma- 0.7%

• Disturbances of puberty- rare and when present usually

associated with a CNS lesion

• Mortality- mean age at death 54 (males), 59 (females) cf:

gen pop 70, 74

Timeline for Complications

• 0-2: Café au lait, plexiform neurofibromas

• 1-6: Symptomatic optic nerve glioma, skinfold freckling

• Preschool onwards: learning difficulties

• > 2: hypertension

• >6: neurofibromas, neurofibrosarcomas

• 6-16: scoliosis

Neurofibromatosis Type 1- genetics

• Autosomal dominant

• 30-50% are new mutations

• Due to mutations in a gene called NF1

• Product= neurofibromin= tumour suppressor gene

• Huge gene!

• Numerous mutations

• Virtually 100% penetrant by 5 years

Neurofibromatosis Type 1- diagnosis

• Clinical

• Gene too big with too many mutations to be able to use

mutation detection as a clinical tool

Neurofibromatosis Type 1- clinical evaluation

• The vast majority of children with multiple café au lait

patches will go on to fulfil the diagnostic criteria for NF1

although a few families with dominant café au lait patches

exist

• Disease severity CANNOT be predicted in a child. There is

great intrafamilial variability including identical twins.

Neurofibromatosis Type 1- clinical evaluation

• Children-

– 12/12 review including:

» learning evaluation formal psychometric assessment as

necessary

» neurological assessment

» BP

» scoliosis

– 12/12 ophthalmological review

• Routine neuroimaging not recommended (controversial)

Neurofibromatosis Type 1- genetic counselling

• Must examine parents

– to confirm diagnosis in child

– to provide risk assessments in future pregnancies

– to look for complications in them

• Prenatal diagnosis/PGD rarely requested

Prenatal Advice

• If child inherits NF1 mutation:

– 40% medical problems

– 50% learning problems, ID- 6%

– 4.5% problem in childhood resulting in lifelong morbidity

– Malignant CNS tumour 2.3%

Malignancies in NF1

Non nervous system

GIST 6%

Somatostatinoma ?

Phaeochromocytoma 1%

Breast cancer 8.4% < 50 years (4x background)

Rhabdomyosarcoma 0.5% (20x background)

Nervous system

Astrocytoma ?

MPNST 8-13%

Neuroblastoma ?

Tuberous Sclerosis

Tuberous Sclerosis

• = Bourneville disease

• 1:5000 of those < 5 years

• 1:25 000 of all ages

Diagnostic Criteria

• Major Features

– Facial angiomas of forehead plaques

– Nontraumatic ungual or periungual fibroma

– >3 hypomelanotic macules

– Shagreen patch

– Multiple retinal nodular hamartomas

– Cortical Tuber

– Subependymal nodule

Diagnostic Criteria

• Major features (cont)

– Subependymal giant cell astrocytoma

– Cardiac rhabdomyoma 1

– Lymphangiomyomatosis

– Renal angiomyolipoma

Diagnostic Criteria

• Minor features

– Multiple pits in dental enamel

– Hamartomatous rectal polyps

– Bone cysts

– Cerebral white matter migration lines

– Gingival fibromas

– Nonrenal hamartomas

– Retinal achromic patch

Diagnostic Criteria

• Minor features (cont)

– “Confetti” skin lesions

– Multiple renal cysts

Diagnostic Criteria

• Definite TS

– 2 major OR 1 major + 2 minor

• Probable TS

– 1 major + 1 minor

• Possible TS

– 1 major OR 2 minor

TS- Adenoma

Sebaceum-

severe

TS- Adenoma

Sebaceum- mild

TS-

Hypomelanotic

Macule

TS- Periungual Fibroma

TS- Shagreen Patch

TS- Forehead plaque

TS- MRI showing a

tuber and

subependymal

nodules

TS-

Hyperpigmented

macule= café au

lait

TS- Enamel

pits

CNS

• 85% CNS complication

– Seizures

– Intellectual disability

– Behavioural problems

– Tumours

Seizures

• 75%

• Infantile spasms (20% will have TS)

• Myoclonic, partial, GTCS

• Presence in 1st 2yrs- association with MR- particularly

infantile spasms

• Can be refractory to treatment

Development and Behaviour

• About 50% have intellectual disability- mild- severe

• 30% profound ID

• Behaviour- ADHD, autistic like, sleep problems

Tumours

• >75% have a brain abnormality inc:

– tubers

– subependymal glial nodules

– white matter radial migration

• Giant cell astrocytomas- highest risk is late childhood/

adolescence but can affect adults

• Usually cause hydrocephalus

Skin

• Depigmented patches may only be visible using a Woods

lamp- UV light- present in nearly all

• Adenoma sebaceum= angiofibromata- butterfly distribution-

present in >75%

• Forehead plaque- 25%

• Shagreen patch- “leathery”- 60%

Ophthalmology

• Retinal phakoma- rarely affect vision

• “mulberry” lesions

CVS

• Cardiac

rhabdomyomata

• Mostly asymptomatic

• Rarely to arrhythmia

or CCF

• Tend to regress

spontaneously

Renal complications

• Commonest cause of TS related death

• Angiomyolipomata

– Generally bilateral and multiple (~80% of adults with TS)

– Usually asymptomatic

– Increase in frequency and severity with age

– Can bleed

Renal complications

• Renal cysts (contiguous gene deletion TSC2 and PKD1)

• Renal cell carcinoma ~3%

Respiratory

• Lymphangiomyomatosis

• F>>M

• Poor prognosis

Genetics

• Autosomal dominant

• 2/3 sporadic, 1/3 inherited

• 2 genes

• TSC1- 9q34

• TSC2- 16p13.3

• Tumour suppressor genes

Gene

% of Probands

with Definite TSC

and an

Identifiable

Mutation in This

Gene

Mutation Detection Frequency by

Gene, Family History, and Test

Method

Familial Cases Simplex Cases

TSC1 ~31% ~30% ~15%

TSC2 ~69% 51% ~60%-70%

mTOR inhibitors in TSC

• mTOR = mammalian target of rapamycin

• mTOR activated by mutations in TSC1 and 2

• Sirolimus, mTOR inhibitor, used for 12 months, resulted in

a reduction in size of angiomyolipomas and improved PFTs

in some with lymphangiomyomatosis

• Everolimus treatment reduced the size of subependymal

giant cell astrocytomas and reduced seizure frequency

• Topical rapamycin shown to reduce size of facial

angiofibromas

Genetics

• TSC1:TSC2= 1:1 in families

• TSC2:TSC1= 5:1 in sporadic cases

• ?TSC2 more severe than TSC1

• TSC1- hamartin

• TSC2- tuberin

• No homology between the two

• Appear to function in the same pathway

Genetics

• Mutations tend to cluster but the majority are family specific

• Not widely available

• US$2000

• Mutation found in about 80%

Genetic Counselling

• Parent with TS- 50% risk to offspring

• If affected about 50% chance MR- therefore 25% risk MR

• 75% risk epilepsy

Genetic Counselling

• Apparent sporadic case

• MUST assess parents

– Skin examination including Woods lamp

– CT ± MRI brain

– Renal ultrasound

– Ophthalmology

• If normal, recurrence risk 2% (gonadal mosaicism)

Prenatal diagnosis

Preimplantation diagnosis

Prenatal Options

• Traditional prenatal diagnosis

– Chorionic villus sampling

– Amniocentesis

• Preimplantation genetic diagnosis

Chorion Villous Sampling (CVS)

Amniocentesis

•CVS from 11 weeks

•Amnio from 15 weeks

•Diagnosis

– Chromosome abnorm.

– DNA Studies

– Biochemical studies

•CVS 1:100 miscarriage

•Amnio 1:200 miscarriage

CVS Amnio

Preimplantation Genetic Diagnosis- PGD

• In the context of IVF

• Testing by embryo biopsy

• Chromosomal abnormalities

• Selected single gene disorders

• Most common reason an objection to TOP or previous TOP

following PND.

• No apparent increase in birth defects

PGD

+

PGD

- DNA extracted and mutation

detection testing done

Method of embryo biopsy

The hole in the zona allows

entry of a micro pipette to

aspirate 1 or 2 cells from the

embryo.

The cell can then be fixed to

a slide, or placed in solution

to allow genetic analysis.

PGD for single gene disorders

• Requested by couples wishing to avoid TOP

• 97% diagnostic accuracy

• 20% pregnancy rate per cycle

• Cystic fibrosis most common indication

• Counselling by both Genetics and IVF team

Next generation sequencing

Next Generation Sequencing (NGS)

• = Massively parallel sequencing (MPS)

• Whole exome sequencing = sequencing of all ~200,000

exons (~50x106 bp)

• Whole genome sequencing = sequencing of 3x109 bp of

genome

Whole exome sequencing

• Most disease causing mutations are in the protein coding

part of the genome (ie: exons)

• Cost ~ $1,000 but ~$3000 with interpretation

• ~40,000 variants per exome!

AATCCGATTTCGGCTAAGCCTAAGGCTGTTACGTACCCG

AATCCGATTTTGGCTAAGCCTAAGGCTGTTACGTACCCG

AATCCGATTTCGGCTAAGCCTAAGGGTGTTACGTACCCG

AATCCGATTTCGGCTAAGCCTAAGGCTGTTACGTACCCG

AATCCGATTTCGGCTAAGCCTAAGGCTGTTACGTACCCG

AATCCAATTTCGGCTAAGCCTAAGGCTGTTACGTACCCG

AATCCGATTTCGGCTAAGCCTAAGGCTGTTACGTACCCG

AATCCGATTTCGGCTAAGCCTCAGGCTGTTACGTACCCG

AATCCGATTTCGGCTAAGCCTAAGGCTGTTACGTACCCG

AATCCGATTTCGGCTAAGCCTAAGGCTGTTACGTACCCG

NGS- uses

• Research- gene discovery

– Now possible to identify genes for rare diseases with few

families or in one family with relatively few affected

individuals

– New genes (and old genes causing new phenotypes) being

identified daily

Clinical uses

• It is much cheaper to do whole exome or targeted gene

sequencing and look at genes of interest than to sequence

two or more genes

• Here gene panels developed- technology focusses on the

genes of interest- targeted capture to get very high

coverage of genes of interest

– eg: Charcot Marie Tooth, Leukodystrophy, HSP, muscular

dystrophy

• ~$1000 cf TS-$2000 for 2 genes!

Typical whole exome dataset

Statistics summary ASD1 37509-1

Total reads 27,617,622 52,616,952

Total yield (bp) 3,037,938,420 5,787,864,720

Average read length (bp) 110 110

Mappable reads (=reads mapped to human genome) 22,786,188 47,217,845

Mappable yield (bp) 2,362,112,810 4,936,651,971

% Mappable reads (out of total reads) 82.50% 89.70%

On-target reads (=reads mapped to target regions) 18,178,648 35,664,336

On-target yield (bp) 1,536,584,594 3,004,107,432

% On-target reads (out of mappable reads) 79.80% 75.50%

% On-target reads (out of total reads) 65.80% 67.80%

Target regions (bp) 62,085,286 62,085,286

% Coverage of target regions (more than 1X) 88.20% 93.20%

% Coverage of target regions (more than 10X) 66.70% 82.10%

Median read depth of target regions 20.0X 44.0X

Whole genome sequence

• 1st human genome sequence- 13 years, $2.7 billion

• Now ~$5K (aim is for $1K)- this is for the test- more $$$ for interpretation

• 98% more data than whole exome sequence

• Much more variation

NGS- challenges

• Generates huge volumes of data- whole exome ~40,000 variants identified per study- “separate wheat from chaff”

– Bioinformaticians are the most important people in genetics!

• Not as accurate as Sanger sequencing so generally need to confirm findings but this is changing

• Not all exons captured with current technology- very important issue- can make this technology inappropriate for some genes/conditions

Variants of unknown significance

• = VOUS

• = unclassified variants

• Some alterations clearly pathogenic

• Some alteration clearly benign polymorphisms

• Often can’t tell which is the case

• Already a major issue in genetic testing

• With NGS being used, this will increase exponentially

Variants of unknown significance

• Generally missense mutations (changes one amino acid for

another)

• Various programs can assist with defining pathogenicity

• Based on conservation of the amino acid across species

• Is the amino acid changed from one type to a very different

or similar type?

– Eg: large basic to small acidic v large basic to large basic

• Common in population = likely to be polymorphism

• Functional studies- can take years!

Problems of VOUS

• 8 year old child

• Parents concerned with various symptoms

• Possibility of connective tissue disorder mentioned

• Parents went to internet

• At parent’s request, paediatrician ordered FBN1 and

COL3A1 mutation detection

Reports

• FBN1- “probable pathogenic mutation” identified

• BUT no signs Marfan syndrome! Probable polymorphism-

test parents (who have no signs Marfan syndrome)- if one

has it, can be confident it is not pathogenic

• COL3A1- silent mutation (base substitution but no change

in amino acid sequence)- extremely unlikely to be

pathogenic (child has no signs of EDS III)- parents still

concerned- want to have testing to be sure it is inherited

Extrapolate to WES and WGS

• 1000s of alterations

• We will quickly become better at knowing what is

pathogenic and what is not but there will always be

unknowns

• This case has taken 4 hours of clinical geneticist time

already

• Who will counsel individuals/families?

• Charge per alterations discussed??!!

NEJM October 3 2012

• 100 people with ID where cause not known (microarray,

fraX), IQ<50, healthy parents

• Whole exome sequencing

• 765 others with ID used to assess new genes

• 79 de novo alterations in 53 patients

• 10 autosomal mutations and 3 X-linked inherited mutations

in males previously described as pathogenic

• Potentially causative de novo mutations in 22 others

• These genes sequenced in 765 others with ID

• Mutations in 3 “new ID genes” identified in others with

similar phenotype

• No autosomal recessive mutations found

• Overall, 16% received a diagnosis

• 19 unclassified variants

• Shows that most undiagnosed genetic ID is new dominant mutations

• Surprising how little is recessive

• This will enter clinical practice

• Will potentially diagnose as many causes of ID as microarray

• Whole genome sequencing will likely eventually replace whole exome AND microarray

Whole genome sequencing

• Mercy Children’s Hospital Kansas- whole genome

sequencing and preliminary report in 50 hours from receipt

of sample for neonates (Stat-seq)- $13,500

• Not in clinical use yet in Australia but will be soon no doubt

Non-invasive prenatal diagnosis

• Detects foetal DNA in maternal blood

• Utilises NGS

• Currently fairly limited in how many things can be tested

(trisomy 13, 18,21, sex chromosome disorders) but will

increase rapidly

• Likely will be able to do whole exome/genome in the

relatively near future

• Will CVS/amnio become obsolete?

Ethical issues

• NGS can reveal things that were

not being sought

– Incest

– Mutations in other genes- eg:

BRCA

• What to do when such information

is identified?

Ethical guidelines

• Generally say if the variant leads to preventable disease risk (eg: BRCA), the person should be told

• If the variant leads to disease that is not treatable/preventable and the disease is unrelated to the purpose of the test (eg: Huntington disease mutation in a person being tested for cause of ID) then they should not be told

• BUT what if preventions become available?

• What if families would want to use the information for avoiding future children having those mutations?

Ethical issues

• Can people consent for the level of information they want?

– Just severe disorders (eg: always causes ID)

– Risk findings (eg: this finding gives a 15% risk ASD)

– Everything (eg: your son’s likely IQ will be 93, brown hair,

175cm, poor sporting prowess)

Online genetics resources

• OMIM

• Genereviews

Online Mendelian Inheritance in Man

• OMIM

• Lists practically every Mendelian condition and every gene

associated with a phenotype

• Lists some mutations

• Cross referenced to Pubmed and gene databases for those

interested in in-depth information about genes and

mutations underlying the various conditions

Autosomal X-

Linked

Y-

Linked

Mitochondrial Total

* Gene with known sequence 13980 683 48 35 14746

+ Gene with known sequence

and phenotype

87 2 0 2 91

# Phenotype description,

molecular basis known

3962 286 4 28 4280

% Mendelian phenotype or locus

,

molecular basis unknown

1541 134 5 0 1680

Other, mainly phenotypes with

suspected mendelian basis

1732 113 2 0 1847

Total 21302 1218 59 65 22644

Thank you!