Ban the Bullet!!

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Maria Jackson 31/05/2015

• Technological advances are lending pressure to expand NBS.

• It is possible now to screen newborns easily and cheaply for >50 rare disorders .

• Increased understanding of the natural history and treatment of inherited disorder .

NBS Expansion Reasons:

Reasons for expanding NBS

Technological advances generate pressure to

expand NBS

It’s now possible to screen cheaply & easily for >50 rare disorders

Increased understanding of natural history and treatment of inherited disorders

ROLE OF SR PROTEINS

HPV gene expression- controlled by alternative splicing (AS) (RNA processing event).

One of the main regulators of AS- serine-arginine (SR)

proteins, also called serine-arginine splicing factors (SRSFs) which include SRSFs1-9.

Their role- gene expression from transcriptional elongation-

mRNA splicing, RNA export to translation, thus involving these proteins in cell survival and proliferation.

organizing gene networks and maintenance of genome

stability and progression of cell cycle, thus defining their role in development of cancer.

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Role of SR proteins

HPV genome HPV proteins

Alternative splicing of RNA

SR proteins Serine-arginine proteins

Serine-arginine splicing factors (SRSFs)

SRSFs 1-9 Roles in: • transcription elongation • mRNA splicing • RNA export

Key for: • Cell survival & proliferation • Organising gene networks • Maintenance of genome stability • Cell cycle progression

Role in cancer development

Intellectual disability [id]

• Causes

– Environmental factor

– Genetic problem

• De novo

• X-linked pattern

– account 5-10% of male ID patients (Tzschach et al., 2015)

• Autosomal recessive

Causes of intellectual disability (ID)

ID

Environmental factors

Genetic factors

De novo

X-linked (15% male ID*)

Autosomal recessive

Inherited

*Tzschach et al., 2015

Prenatal diagnosis

Prenatal diagnosis uses different diagnostic techniques to collect fetus or embryo’s genetic information

Different abnormalities can be detected: • Chromosomal abnormalities

• Aneuploydies • Translocations • Inversions • Deletions

• Monogenic diseases • Polygenic diseases Two types of prenatal diagnosis 1. Invasive 2. Non-invasive screening

Prenatal diagnosis

INVASIVE versus NON-INVASIVE techniques for assessing:

Risk factors

• Age

• Family history

• Menstrual history

• Age at first pregnancy

• Personal history of benign breast disease

• Radiation

• Lifestyle

• Hormone replacement therapy

Breast cancer

risk

Age

Lifestyle Radiation

Family history

Menstrual history

Age at first pregnancy

Hormone replacement

therapy

Personal history of benign breast

disease

Candidate Genes

• Five array- CGH studies have identified submicroscopic regions of genomic imbalances.

• Recurrent, de novo regions: 17q12, 22q11, 16p11.2 and 1q21.1

• A new candidate gene LIM homeobox 1 (LHX1) was discovered

– Sequence analysis identified LHX1 as a monogenic cause for MRKH.

Candidate regions / genes

aCGH analysis recurrent de novo imbalances

17q12 22q11 16p11.2 1q21.1

LIM homeobox 1 (LHX1)

Confirmed as monogenic cause of MRKH

T box transcription factor (TBX6)

High Mobility Group nucleosome binding (HMGN)

Characteristics

• One of the family group HMG

• Binds to DNA molecules and nucleosome particles

• Influence epigenetic modifications

• Modulate chromatin structure

Therefore

• Affect DNA related activities such as transcription, translation and DNA repair.

5 different proteins HMGN1 through HMGN5

HMGN protein

Member of high mobility group (HMG) family

Binds to DNA and to nucleosomes

Influences epigenetic modification

Modulates chromatin structure

Affects activities including transcription, translation and DNA repair

Five different proteins

HMGN1 – HMGN5

Experimental Outline

• Cell line: UVW Cultured.

• Protein extraction done.

Now,

• Western Blotting

– (+)DSF:Cu / (-)DSF:Cu

– (+)AMS / (-)AMS

Experimental outline

untreated + disulfuram:Cu + ammonium sulfate (known inhibitor of

ribonucleotide reductase)

Protein extract

Western blot using MAb for ribonucleotide reductase

UVW cells (glioma)

Hypertension

• Affects 40% of worldwide population

• Responsible for 7.5 million deaths annually

• Untreated it can result in left ventricular hypertrophy, stroke and heart attack

• Can be inherited

• Cause of hypertension is complex

Affects 40% population worldwide

7.5 million deaths annually

Can lead to left ventricular hypertrophy, stroke and heart attack

Inherited variants Environment

Cause of hypertension is complex

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Facts about MAOA (monoamine oxidase A) Gene

Called warrior gene

The MAOA gene is located on the short arm (p) of the X chromosome at position 11.3.

The MAOA gene regulates the expression of monoamine oxidase A,

Monoamine oxidase A, is enzymatic protein that breaks down important neurotransmitters in the brain

About 5–10% of all severe violent crime in Finland is attributable to MAOA and CDH13 genotypes (Tiihonen et al, 2015)

The MAOA gene predicts happiness in women

MAOA – the “warrior gene”

X chromosome

MAOA MAOB

Monoamine oxidase A

Breaks down neurotransmitters

in brain

~9% of all severe violent crime in Finland is attributable to low expression MAOA genotypes*

Lower expression of MAOA is

linked with happiness in women

*Tiihonen et al, 2015

Sequencing of the transgene

1. Aim to sequence R6/2 transgene from models with larger repeats

=> Modifiers of instability

=> Greater understanding of mechanisms

2. Aim to sequence R6/2 transgene with contraction biased somatic instability

=> Modifiers of biased instability

=> Understanding of mechanisms involved

=> Potential therapeutic target??

Sequencing of the transgene

Sequence R6/2 transgene from models with larger

repeats

Sequence R6/2 transgene with contraction biased

somatic instability

Greater understanding of

mechanisms

Modifiers of biased instability

Modifiers of instability

Potential therapeutic target?

• 35 couples from different families surveyed

• 61 parents wanted children tested

• 9 did not want children tested

Umans-Eckenhausen et al ,2002

• 44.3% said information was the most important factor

• Expectation and experience were 2nd and 3rd respectively

• 42.9% said emotion was least important

Umans-Eckenhausen et al ,2002

What do parents say?

70 parents (35 couples)

61 parents wanted children tested

9 parents did not want children tested

44%: information is most important

factor

43%: emotion is least important

factor

Expectation & experience ranked 2nd & 3rd Umans-Eckenhausen

et al ,2002

Lung Cancer

Small Cell Lung Cancer (SCLC) ~20%

Non- small Cell Lung Cancer (NSCLC) ~80%

Lung Cancer

Small Cell Lung Cancer (SCLC) ~20%

Non- small Cell Lung Cancer (NSCLC) ~80%

Common affected pathways

• Initial gene profiling of 100 PDAC patients uncovered a series of commonly observed variants in several key pathways (Waddell et al. 2015) including: – Known pancreatic cancer genes (KRAS, TP53…)

– DNA damage repair gene (BRCA1/2, MLH1, MSH2...)

– Metabolic cancer genes (ERBB2/HER2, Pi3K…)

• These were used to subdivide the patients into specific groups for targeted treatment.

• Next step -> further characterise patients and develop effective drug therapies.

Common affected pathways

100 PDAC patients

Gene profiling

Common variants in key pathways

Pancreatic cancer genes KRAS, TP53

DNA damage repair genes

BRCA1/2 MLH1, MSH2

Metabolic cancer genes ERBB2/HER2

Pi3K

Subdivide patients by mutation profile

Further characterise & develop effective

therapies

Waddell et al, 2015

Introduction

32

Non invasive prenatal diagnosis method (NIPD) is a prenatal examination that does not required to invade into the body of mother or child. Compared to invasive prenatal diagnoses methods, these procedure does not impose risk to both the foetus and the mother. For example, analysis of cell free foetal DNA (cff-DNA) in maternal plasma.

Prenatal diagnosis (PND)

Conventional, invasive PND

• Amniocentesis; chorionic villus sampling; fetal blood sampling

• Miscarriage risk

• Risk to health of mother

Non-invasive PND

• Eg cell free fetal DNA (cff-DNA) in maternal plasma

• No risk to fetus

• No risk to mother

• Did proband forward information

• Effective & safe medication

• Emotional Support

• Brochures & booklets

• Fear, guilt & blame

• Awareness & education

• The right not to know vs. ethical duty to warn

• Geographical location

• Procedure logistics/recording information

Factors that effect testing

Factors that affect testing

Emotional support

Brochures & booklets

Fear, guilt & blame

Awareness & education

Geographical location

Procedure logistics / recording information

Did proband forward the information?

Effective & safe medication

The right not to know versus the ethical duty to warn

The strategy:

• Collection of retrospective patients results for the last 5 years.

• Data and information from laboratory : • IOE database for result and clinical history • Alamut bioinformatics software for classification &

significance of variants: 1) Grantham score 2) SIFT 3) Polyphen 4) Splicing prediction • Literature review • dbSNP/Ensembl

Strategy for audit

ANALYSIS

Retrospective patient results 2010-2015

Mutation data from laboratory

IOE database for result and clinical history

Alamut software for classification of variants

SIFT PolyPhen Grantham Splice predict

dbSNP/Ensembl

Literature