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)
