Lecture 15: NEW BORN SCREENING

Question 1

Early detection: pku

Answer 1

• Phenylketonuria (PKU) Population Screening
• Bacterial Inhibition Assay - 1963
• Dr. Robert Guthrie
• ‘With early detection,
• PKU became a treatable disease
• with excellent outcome’

Question 2

Phenylalanine and Tyrosine

Answer 2

Phenylalanine hydroxylase
PAH gene
PKU

Phenylalanine —BH4 and PAH —> TYROSINE

Question 3

PKU how was the term coined?

Answer 3

1 * 1934 Norwegian physician Ivar Asbjørn Følling

2 * Hyperphenylalaninemia was associated with intellectual disability

3 * Permanent and irreversible
4 * Coined the term phenylketonuria (PKU)

Question 4

Newborn bloodspot screening in aus = 5

Answer 4

1. Population screening program for genetic disease
2. • Targeted and treatable disorders of metabolism
3. • Bloodspot sample at 48 – 72h of age, largely a biochemical approach
4. • Australasia – All newborns, voluntary, consented, no charge
5. • A Worldwide program…

Question 5

Evolution of NBS Technological developments…5

Answer 5

1. PKU 1963
   Bacterial inhibition assay
2. Bacterial inhibition assays
   Amino acid disorders 1960’s

3.Immunoassay
1970-80’s Endocrine

4. Mass Spectrometry
   1990-2000’s
   Multiplex panel
   Metabolomics

5.Genetic
Sequencing,
Array
Targeted

Question 6

Evolution of NBS Technological developments — Future expansion = 5

Answer 6

1. Coupled techniques in
2. Metabolomics, MSMS analyte discovery
3. Enzymology, functional assays
4. Genetic analysis
5. Multiple techniques required

Question 7

NBS Screened Disorders: Western Australia
Births ~35,000 per year

Answer 7

table on slide 8

Question 8

MSMS in NBS

Answer 8

table on slide 9

Question 9

Tandem Mass Spectrometry (MSMS)
in newborn bloodspot screening = 7

Answer 9

1. (LC)MSMS in NBS
2. • Enabled screening for a large number of disorders, difficult to detect
3. • Measures metabolic markers in an extract of a single bloodspot
     4 * Amino acids
     5 * Acylcarnitines

6* Enables reflex (second tier) screening on selected samples

7 * E.g Specific metabolite analysis (homocysteine, steroid profiling…)

Question 10

understanding what pku is

Answer 10

on MSMS
* Increased phenylalanine, decreased tyrosine
* Increased Phe/tyr ratio
Enzyme defect * Phenylalanine hydroxylase
* Biopterin synthesis disorders (GTPCH, DHPR deficiencies)
Inheritance * Autosomal recessive
Incidence in WA * 1 in 15,400 births
Diagnostic tests * Plasma amino acids
* Urine pterin studies
* Bloodspot DHPR assay
Symptoms if untreated * Microcephaly
* Mental retardation
* Seizures
* Autistic-like behaviour
* Fair-light complexion, hair and eye colour
* Musty odour
Treatment * Phenylalanine restricted diet
* Tyrosine supplementation
* Tetrahydrobiopterin cofactor in some
Screening issues * The screening test is almost uniformly abnormal within 48 hours of birth,
whether or not the baby is receiving full milk feeds
* Any infant who is tested before 48 hours must have the test repeated
later for reliable screening

Question 11

understanding what congenital hypothyroidism

Answer 11

Diagnostic metabolites
on screen
* Increased thyroid stimulating hormone (TSH)
Defect * No thyroid gland (athyrosis)
* Small maldescended gland
* Iodination defect
Inheritance * Most cases are sporadic
* About 10% of cases result from inherited enzyme defects within the thyroid
Incidence in WA * 1 in 3,500 births
Diagnostic tests * Plasma TSH and free T4
* A technetium scan of the thyroid gland or an X-ray of the knee for bone age
are also sometimes performed
Symptoms if untreated * Prolonged jaundice
* Failure to thrive
* Lethargy, hypotonia
* Feeding problems, constipation
* Coarse facial features, thick tongue
* Umbilical hernia
* Hoarse cry
* Mental retardation
* Growth retardation
Treatment * Daily treatment with thyroid hormone tablets results in normal growth and
development
Screening issues * The screening results may be affected by maternal thyroid disease and
medication, as well as iodine excess or deficiency

Question 11

the understanding of what ‘cf’ is…

Answer 11

Diagnostic metabolites
on screen
* Increased immunoreactive trypsin (IRT)
* Mutation analysis in those with IRT>99% (F508, G551D, G542X, and 621+1)
Defect * Defective cystic fibrosis transmembrane conductance regulator (CFTR) function
Inheritance * Autosomal recessive
* About 1 in 25 carriers
Incidence in WA * 1 in 2,500 births
Diagnostic tests * Sweat test
* Extended mutation analysis
* Clinical assessment
* Genetic counselling
Symptoms if untreated * Meconium ileus
* Progressive respiratory disease
* Malabsorption
* Liver disease
* Diabetes mellitus
* Impaired fertility (CBAVD)
Treatment * Aims to reduce morbidity by early diagnosis and treatment of lung infections and
attention to good nutrition
Screening issues * In WA, approximately 95% of infants with CF carry one of four common mutations
(F508, G551D, G542X, and 621+1)
* A small number of healthy carriers of CF are detected

Question 12

Medium-chain acyl-CoA dehydrogenase deficiency

MCAD deficiency: Detected by MSMS

= 9

Answer 12

1 * Most common fatty acid oxidation defect

2 * More common than phenylketonuria (PKU)

3 * 1:9800 births in WA (3 per year)

4 * ~38 diagnoses per year in Australasia

5 * Requires MSMS for detection

6 * Hypoketotic hypoglycaemia

7 * Unscreened newborns are at risk of permanent neurological deficit, death

8 * Treated easily once diagnosed

9 * Avoidance of fasting, glucose supplementation, sick day regime

Question 13

MCAD & Fatty acid oxidation flowchart…

Answer 13

Increased energy/glucose demand

flowchart on slide 15

Question 14

NBS- Spinal Muscular Atrophy (SMA) =11

Answer 14

SMA – Autosomal recessive

1 * Affects the central and peripheral nervous system and skeletal muscle

2 * NBS for Chromosome 5 SMN-related SMA

3 * Detection of del exon 7 in SMN1 gene by qPCR of bloodspot DNA

4 * Deficiency of “survival of motor neuron”, SMN required for normal motor neurone function

5 * Variability in age of onset, symptoms, and rate of progression, types 1 through 4

6 * Type 0 prenatal onset. Lowest level of functioning – type 1.

7 * Later-onset SMA with a less severe course (types 2–4)

8 * SMN2 gene – 99% homology, produces ~20% protein

9 * Multiple copies of SMN2; 2 copies = type 1

10 * Other phenotype modifiers, unclear

11* Treatment available – “gene therapy”

Question 15

NBS- Severe Combined Immune Deficiency (SCID) = 11

Answer 15

1. SCID and X-Linked agammaglobulinaemia
2. • > 20 rare genetic disorders of T and B cell immunity
3. • X-Linked (most common), autosomal recessive group of disorders
4. • Absent or low T cells, absent or non-functional B cells
5. • Susceptible to life-threatening infection from pathogens
6. • During T-cell and B-cell development, non-replicating episomal circular DNA is formed
7. • T-cell receptor excision circles – TREC
8. • B-cell kappa-deleting recombination-excision circles – KREC
9. • NBS quantification of bloodspot extracted TREC and KREC by qPCR
10. • SCID, non-SCID T-cell lymphopaenia, syndromes with T-cell impairment
11. • Treatment available – Bone marrow transplant, stem cell transplant
      immunoglobulin therapy, enzyme replacement, gene therapy

Question 16

NBS Process = 2

Answer 16

1. Collection by Midwives
2. On-ward or home review
3. At 48–72h of age

4.Dry bloodspot and transport
to NBS Laboratory QEIIMC
5. Transit <4d transit
~140 sample in lab per day

6. Punch 3.2mm spots
   ~3uL blood
7. 96-well tray format
8. Bloodspot punch indexer

9.PE GSP Automated Immunoassay Analyser

10. Quantitative PCR

11.Tandem mass spectrometer

Question 17

Newborn screening Laboratory Process

Answer 17

Initial lab test –
—positive or normal
- second their test
- clarify initial
- result - value add

if positive
— positive –> recall —> diagnostic test

Question 18

NBS Aims
Pre-symptomatic detection & treatment of metabolic genetic disease = 9

Answer 18

1 * Select babies with increased risk of disease prior to decompensation/pathology

2 * 31 targeted primary disorders in the screening panel

3 * >44 disorders – subtypes

4 * Maximum sensitivity – minimise false negative screens

5 * Minimise false positives – minimise harm
6 * Normal newborns are not patients
7* In-lab reflex testing, second tier selected analysis

8 * Minimise heterozygote detection

9 * Babies at risk referred to clinical specialist for recall & confirmatory testing

Question 19

Screening vs Diagnostic =

Answer 19

1 * Screening aim
2 * Select babies with increased risk of disease

3 * Initial testing – SCREENING test aims
4 * High sensitivity – minimise false negative results
5 * Overlap with normal population – minimal false positive results

6 * Action for borderline results
7 * Second-tier reflex analysis
8* Repeat newborn bloodspot sample – results usually resolve to normal

9 * Recall affected newborn for confirmatory DIAGNOSTIC testing
* CHT TFTs
* CAH Steroid profile (5 steroids)
* CF Sweat test, extended genotyping
* MSMS Amino acids/acylcarnitines/organic acids, genotyping
* Galactosaemia Genotyping
* SMA screen Diagnostic genotyping
* SCID Flow cytometry, CD markers, genotyping

Question 20

Criteria for selecting disorders screened
Newborn Bloodspot Screening: National Policy Framework = 6

Answer 20

1 * The condition should be a serious health problem that leads to significant
morbidity or mortality

2 * There should be a benefit to conducting screening in the newborn period.

3 * There should be a suitable test protocol to identify the presence of the
condition.

4 * The test protocol should, on balance, be socially and ethically acceptable
to health professionals and the public.

5 * Health care services for diagnosis and management should be available
so that these services can be offered if there is an abnormal screening
result.

6 * There should be an accepted intervention for those diagnosed with the
condition.

Question 21

Newborn screening impact: “screen positive”
WA data & 33849 mean annual births 2015-2020)

Answer 21

table on slide 25

Question 21

NBS- Treatment options

Answer 21

1. Spinal Muscular Atrophy

2 * Risdiplam
Small molecule, coumarin derivative, increasing SMN2 protein (splice enhancer)
Daily oral dose, ~$125k annually, on PBS

3 * Nusinersen/Spinrasa
Antisense oligonucleotide (ASO) increasing SMN2 protein (splice enhancer)
Intrathecal, loading doses plus ongoing doses, >$300k per year, on PBS

4* Onasemnogene abeparvovec-xioi/Zolgensma
- AAV9 gene replacement therapy containing SMN1 gene
- Intravenous single dose ~$2.5m, on PBS

Question 22

NBS – Australia =2

Answer 22

• Under review 2023, Australia
• Sickle cell disease, thalassemias, haemoglobinopathies
• X-linked adrenoleucodystrophy
• Active expansion program
• USA and elsewhere
• Duchenne muscular dystrophy
• Lysosomal storage diseases
• Creatine synthesis (GAMT)
• G6PD deficiency
• Infectious diseases: HIV, CMV,
• Toxoplasmosis (South America)

Question 23

NBS & Genetic analysis

• First-tier analysis: Metabolic vs genetic analysis = 5

Answer 23

1 * Metabolic disorders - The existing biochemical technology is preferred

2 * No improvement in sensitivity, speed or cost saving with genetic analysis

3 * Urgency required with metabolic disease

4 * VUS, Late-onset variants, intronic variants, pseudo-deficiency alleles, pseudogenes

5 * Lessons from Pompe disease NBS – 684290 infants, 395 NBS Pos, 3 IOPD, 26 LOPD
- Metabolic targets AND enzymatic analysis AND genetic analysis is needed

Question 24

NBS & Genetic analysis

• Direct genetic analysis

Answer 24

Direct genetic analysis is use with targeted disorders

• SMA qPCR, MLPA, digital droplet PCR
• SCID qPCR

Question 25

NBS & NGS

Answer 25

• NGS has many ELSI considerations
• NBS advances must move at the pace of public trust – or risk a declined test
• Criteria of the National Policy Framework (Wilson & Jungner 1968) essentially remain…
• But are being challenged

Question 26

NBS & cost savings

Answer 26

conomic evaluation of neonatal screening for phenylketonuria and congenital hypothyroidism..
…..A net saving of dollar A2.9 million is attributable to the programme annually. The
economic benefits derive from the prevention of intellectual disability which otherwise incurs costs
throughout the life of the affected individual…..
(Geelhoed et al J Paediatr Child Health. 2005 Nov;41(11):575-9)
Healthcare Use and Costs of Medium-chain Acyl-CoA Dehydrogenase Deficiency in Australia:
Screening Versus No Screening
….. screening children cost an average of $A1676 (US$1297) per year for inpatient, emergency
department, and outpatient visits, compared with $A1796 for children in whom a clinical
diagnosis was made. Forty-two percent of the children who underwent screening were admitted
to the hospital, compared with 71% of children who did not undergo screening. Children who
did not undergo screening used significantly more inpatient services and
cost significantly more in emergency services.
(Haas et al, J Pediatr 2007;151:121-6)
Economic Evaluation of Tandem Mass Spectrometry Screening in California
…..The total estimated, annualized, incremental costs of MS/MS screening of 540,000 births in
California were nearly $5.7 million; 83 affected newborns would be identified…..…..MS/MS
screening produced a benefit/cost ratio of $9.32…..….. We found that the benefits
of MS/MS screening outweighed the costs and that the net benefits were significant and robust
in various scenarios with various conservative underlying assumptions.…..

Question 27

NBS is not a test – it’s a Program

Answer 27

1. Screening
   *Sample collection
   *Sample submission
   *Laboratory testing

2.Follow up
*Report test results
*Repeat test
*Ensure diagnostic testing

3.Diagnosis
*Specialist Assessment
*Results shared with family
*Counselling

4.Management
*Treatment
*Long-term follow-up
*Specimen storage

5.Evaluation
*Quality assurance
*Outcome evaluation
*Cost effectiveness

Question 28

60 years of NBS

Answer 28

• Bacterial Inhibition
  Agar - 1963
• Immunoassay - DELFIA
• MSMS
• DNA Analysis