Lecture 15: NEW BORN SCREENING Flashcards
Early detection: pku
- Phenylketonuria (PKU) Population Screening
- Bacterial Inhibition Assay - 1963
- Dr. Robert Guthrie
- ‘With early detection,
- PKU became a treatable disease
- with excellent outcome’
Phenylalanine and Tyrosine
Phenylalanine hydroxylase
PAH gene
PKU
Phenylalanine —BH4 and PAH —> TYROSINE
PKU how was the term coined?
1 * 1934 Norwegian physician Ivar Asbjørn Følling
2 * Hyperphenylalaninemia was associated with intellectual disability
3 * Permanent and irreversible
4 * Coined the term phenylketonuria (PKU)
Newborn bloodspot screening in aus = 5
- Population screening program for genetic disease
- Targeted and treatable disorders of metabolism
- Bloodspot sample at 48 – 72h of age, largely a biochemical approach
- Australasia – All newborns, voluntary, consented, no charge
- A Worldwide program…
Evolution of NBS Technological developments…5
- PKU 1963
Bacterial inhibition assay - Bacterial inhibition assays
Amino acid disorders 1960’s
3.Immunoassay
1970-80’s Endocrine
- Mass Spectrometry
1990-2000’s
Multiplex panel
Metabolomics
5.Genetic
Sequencing,
Array
Targeted
Evolution of NBS Technological developments — Future expansion = 5
- Coupled techniques in
- Metabolomics, MSMS analyte discovery
- Enzymology, functional assays
- Genetic analysis
- Multiple techniques required
NBS Screened Disorders: Western Australia
Births ~35,000 per year
table on slide 8
MSMS in NBS
table on slide 9
Tandem Mass Spectrometry (MSMS)
in newborn bloodspot screening = 7
- (LC)MSMS in NBS
- Enabled screening for a large number of disorders, difficult to detect
- Measures metabolic markers in an extract of a single bloodspot
4 * Amino acids
5 * Acylcarnitines
- Measures metabolic markers in an extract of a single bloodspot
6* Enables reflex (second tier) screening on selected samples
7 * E.g Specific metabolite analysis (homocysteine, steroid profiling…)
understanding what pku is
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
understanding what congenital hypothyroidism
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
the understanding of what ‘cf’ is…
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
Medium-chain acyl-CoA dehydrogenase deficiency
MCAD deficiency: Detected by MSMS
= 9
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
MCAD & Fatty acid oxidation flowchart…
Increased energy/glucose demand
flowchart on slide 15
NBS- Spinal Muscular Atrophy (SMA) =11
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”
NBS- Severe Combined Immune Deficiency (SCID) = 11
- SCID and X-Linked agammaglobulinaemia
- > 20 rare genetic disorders of T and B cell immunity
- X-Linked (most common), autosomal recessive group of disorders
- Absent or low T cells, absent or non-functional B cells
- Susceptible to life-threatening infection from pathogens
- During T-cell and B-cell development, non-replicating episomal circular DNA is formed
- T-cell receptor excision circles – TREC
- B-cell kappa-deleting recombination-excision circles – KREC
- NBS quantification of bloodspot extracted TREC and KREC by qPCR
- SCID, non-SCID T-cell lymphopaenia, syndromes with T-cell impairment
- Treatment available – Bone marrow transplant, stem cell transplant
immunoglobulin therapy, enzyme replacement, gene therapy
- Treatment available – Bone marrow transplant, stem cell transplant
NBS Process = 2
- Collection by Midwives
- On-ward or home review
- At 48–72h of age
4.Dry bloodspot and transport
to NBS Laboratory QEIIMC
5. Transit <4d transit
~140 sample in lab per day
- Punch 3.2mm spots
~3uL blood - 96-well tray format
- Bloodspot punch indexer
9.PE GSP Automated Immunoassay Analyser
- Quantitative PCR
11.Tandem mass spectrometer
Newborn screening Laboratory Process
Initial lab test –
—positive or normal
- second their test
- clarify initial
- result - value add
if positive
— positive –> recall —> diagnostic test
NBS Aims
Pre-symptomatic detection & treatment of metabolic genetic disease = 9
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
Screening vs Diagnostic =
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
Criteria for selecting disorders screened
Newborn Bloodspot Screening: National Policy Framework = 6
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.
Newborn screening impact: “screen positive”
WA data & 33849 mean annual births 2015-2020)
table on slide 25
NBS- Treatment options
- 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
NBS – Australia =2
- 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)
NBS & Genetic analysis
- First-tier analysis: Metabolic vs genetic analysis = 5
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
NBS & Genetic analysis
- Direct genetic analysis
Direct genetic analysis is use with targeted disorders
- SMA qPCR, MLPA, digital droplet PCR
- SCID qPCR
NBS & NGS
- 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
NBS & cost savings
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.…..
NBS is not a test – it’s a Program
- 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
60 years of NBS
- Bacterial Inhibition
Agar - 1963 - Immunoassay - DELFIA
- MSMS
- DNA Analysis
