1 newborn screening

UWA PATH3305 lecture 02/04/2015

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Newborn Bloodspot ScreeningAn expanding public health program

Lawrence Greed

Senior Scientist in Charge

Department of Clinical Biochemistry

Biochemical Genetics Unit & WA Newborn Screening Program

PathWest Laboratory Medicine WA

Newborn bloodspot screening

What to take from this presentation

• What newborn bloodspot screening is?

• The process and principles of screening

• The categories of disorders covered

• The impact of screening on health

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Newborn bloodspot screening

• Population screening program for inherited (genetic) disease

• Targeted and treatable disorders of metabolism

• Bloodspot sample taken at 48 – 72h of age (The Guthrie Card)

• Australasia – All newborns, voluntary, consented, no charge

• North & South America, Europe, UK

• Worldwide program…

https://reproductivehealth.perkinelmer.com/for_parents/newborn_screening

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Phenylketonuria (PKU) Population Screening

Bacterial Inhibition Assay - 1963

Dr. Robert Guthrie

Inborn errors of metabolism

• Sir Archibald Garrod 1908

• “disorders that are caused by a deficiency of enzyme catalysis or an enzyme that facilitates the transport of biological substances across membranes”

Phenylketonuria (PKU)• 1934 Norwegian physician Ivar Asbjørn Følling

• Hyperphenylalaninemia was associated with intellectual disability

• Permanent and irreversible

• Coined the term phenylketonuria

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Phenylalanine 500-2500 mol/L [NR <120]

Phenylketonuria – Classical PKU

Phenylketones

Urine, blood

Tyrosine (decreased)

Phenylalanine HydroxylaseClassic PKU

Phenylalanine 500-2500 mol/L [NR <120]

Phenylketonuria – PKU

Phenylketones

Urine, blood

Tyrosine (decreased)

Phenylalanine HydroxylaseClassic PKU

BH4

BH2

DHPR

GTP

Pterin cofactor defects

Atypical PKU

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NBS Aim

• Pre‐symptomatic detection & treatment of metabolic genetic disease

• 25 targeted disorders in the screening panel (varies on jurisdiction)

• Early detection & treatment prior to metabolic decompensation

NBS Aim




Newborn bloodspot screening process

• Bloodspot samples taken (optimally) at 48 – 72h of life by midwife

• The “Guthrie” test – blood spotted onto “filter paper”

• Sample dried & sent by express post/courier

• Analysis by a central NBS laboratory

• Referral to consultant for infant recall & confirmatory testing

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NBS Aim




Newborn bloodspot screening process

• Bloodspot samples taken (optimally) at 48 – 72h of life by midwife

• The “Guthrie” test – blood spotted onto “filter paper”

• Sample dried & sent by express post/courier

• Analysis by a central NBS laboratory – result within 24 (<72h)

• Referral to consultant for recall & confirmatory biochemical/genetic testing

Metabolites vs genetic analysis

• Time/speed, cost, VUSs

• Direct genetic analysis: SCID, SMA…

NBS Process

Dry bloodspot and transport to NBS Laboratory

Collection by Midwives

WA ~140/day

Aust/NZ ~1500/day

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Bloodspot punch indexer

Punch 3.2mm spots~3uL blood

96-well tray format

PE GSP Automated bloodspot immunoassay

analyser

Tandem mass spectrometer

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MSMS in NBS

Group DisorderAmino acids PKU

TyrosinaemiasMSUD (branched chain AAs)

HomocystinuriaCitrullinaemia

Argininosuccinic acidaemiaFatty acid oxidation defects Medium‐chain acyl‐CoA dehydrogenase def

Very long‐chain acyl‐CoA dehydrogenase defMultiple‐CoA dehydrogenase def

Long‐chain hydroxyl‐CoA dehydrogenase defCarnitine transporter defect

Carnitine palmitoyl transferase def I & IICarnitine‐acylcarnitine transferase def

Organic acidaemias Methylmalonic acidaemiasPropionic acidaemiaIsovaleric acidaemiaGlutaric acidaemia

Hydroxymethylglutaric acidaemia

PKU

• Microcephaly, mental retardation, seizures…

CHT

• Prolonged jaundice, FTT, hypotonia, feeding problems, coarse facies, MR…

Galactosaemia

• Vomiting, jaundice, hepatic/renal dysfunction, sepsis, MR, cataracts

CF

• Meconium ileus, respiratory disease, malabsorption/FTT, liver disease…

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Tandem Mass Spectrometry (MSMS)in newborn bloodspot screening

MSMS flow injection analysis (no LC)• Australia 1998 (NSW), 2002 (Vic), and 2004 (WA Qld NZ)

• ACT, NT, Tas covered by other states

• Enabled screening for a large number of disorders, difficult to detect• Measures metabolic markers in an extract of a single bloodspot

• Amino acids, Acylcarnitines• Sequential analysis of a panel of disorders from a bloodspot extract• ~90s sample cycle

LCMSMS in NBS – second tier/reflex selected analysis• Specific analysis on selected screening samples• E.g Specific metabolite analysis (homocysteine, steroid profiling…)

Mass spectrometry panel (MSMS expanded screening)

• Amino acid disorders

• PKU, tyrosinaemias, MSUD, homocystinuria, citrullinaemia, ASAuria

• MR, DD, neurological sequelae (brain, Seizures, MR..)

• Liver dysfunction/failure, renal dysfunction, cardiomyopathy, eye, vascular

• Metabolic acidosis, ketolactic acidosis, hyperammonaemia

• Death

• Fatty acid oxidation – energy supply (oxidation and transport)

• Medium & very long chain FA oxidation, carnitine, ETF cofactor

• Hypoketotic hypoglycaemia, acute encephalopathy

• Neurological sequelae (brain, seizures, MR..)

• Fatty liver (Reye syndrome) & liver dysfunction, cardiomyopathy

• Death

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• Organic acidaemias – amino acid degradation (Val, Leu, Ile + others)

• Methylmalonic, propionic, isovaleric, glutaric acidaemias….

• Hyperammonaemia & metabolic acidosis (ketolactic and organic acids)

• Acute encephalopathy


• Cardiac, liver renal dysfunction


• Organic acidaemias – amino acid degradation (Val, Leu, Ile + others)

• Methylmalonic, propionic, isovaleric, glutaric acidaemias….

• Hyperammonaemia & metabolic acidosis (ketolactic and organic acids)

• Acute encephalopathy


• Cardiac, liver renal dysfunction

Not screened by MSMS (in WA/Aus)

• Urea cycle – OTC deficiency, CPS deficiency

• Mitochondrial lactic acidaemias, peroxisomal disorders

• Storage disorders: glycogen, lysosomal

• CAH (Immunoassay + LCMSMS)

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NBS Screened Disorders: WA Birth rate ~34,000

Disorder Introduced in WAWA

Incidence MethodPKU 1969 1:15400 BIA (Pre MSMS)

Galactosaemia (classical) 1980 1:54000 Enzyme assay

Congenital hypothyroidism 1981 1:2400 Immunoassay

Cystic Fibrosis 2000 1:2600 Immunoassay & DNA

Expanded MSMS panel22 disorders

2004 1:3500 MSMS (includes PKU)

NBS Screened Disorders: WA Birth rate ~34,000

Disorder Introduced in WAWA

Incidence MethodPKU 1969 1:15400 BIA (Pre MSMS)

Galactosaemia (classical) 1980 1:54000 Enzyme assay

Congenital hypothyroidism 1981 1:2400 Immunoassay

Cystic Fibrosis 2000 1:2600 Immunoassay & DNA

Expanded MSMS panel27 disorders

2004 1:3500 MSMS (includes PKU)

Biotinidase deficiency NZ Enzymatic

Congenital adrenal hyperplasia

1984 NZ ~1:15,000 Immunoassay/LCMSMS

Congenital adrenal hyperplasia

2018 NSW2020 Qld, 2021 SA

~1:15,000 Immunoassay/LCMSMS

Spinal muscular atrophy 2018 (NSW pilot) ~1:10,000 Quantitative PCRSMN1 gene

Severe combined immune deficiency

2018 NSW NZ

~1:50,000 Quantitative PCR TREC/KREC DNA

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Criteria for selecting disorders screened

Policy statement on newborn screening: Joint subcommittee of the HGSA and the Division of Paediatrics of the RACP

• There is benefit to the baby from early diagnosis

• The benefit is reasonably balanced against any harms and costs

• There is a reliable test suitable for newborn bloodspot screening

• There is a satisfactory system in place to deal with diagnostic testing and follow-up care of babies with abnormal screening results

These are based on the original screening principles of Wilson & Jungner 1968(Wilson JMG, Jungner G. Principles and practice of screening for disease. Geneva: WHO; 1968. Available from: http://www.who.int/bulletin/volumes/86/4/07‐050112BP.pdf )

Unaffected

Affected

Analyte concentration

Max. sensitivity

Max. specificity

?Cutoff

Sam

ple

s

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Screening vs Diagnostic• Screening aim

• Select babies with increased risk of disease

• Initial testing – SCREENING test aims

• High sensitivity – minimise false negative results

• Overlap with normal population – minimal false positive results

• Action for borderline results

• Second‐tier reflex analysis

• Repeat newborn bloodspot sample – results usually resolve to normal

• Recall affected newborn for confirmatory DIAGNOSTIC testing

• CHT TFTs

• CF Sweat test, extended genotyping

• MSMS Amino acids/acylcarnitines/organic acids, genotyping

• Galactosaemia Genotyping• CAH Steroid profile

MSMS in NBS

Group DisorderAmino acids PKU

TyrosinaemiasMSUD (branched chain AAs)

HomocystinuriaCitrullinaemia

Argininosuccinic acidaemiaFatty acid oxidation defects Medium‐chain acyl‐CoA dehydrogenase def

Very long‐chain acyl‐CoA dehydrogenase defMultiple‐CoA dehydrogenase def

Long‐chain hydroxyl‐CoA dehydrogenase defCarnitine transporter defect

Carnitine palmitoyl transferase def I & IICarnitine‐acylcarnitine transferase def

Organic acidaemias Methylmalonic acidaemiasPropionic acidaemiaIsovaleric acidaemiaGlutaric acidaemia

Hydroxymethylglutaric acidaemia

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Medium-chain acyl-CoA dehydrogenase deficiency

MCAD deficiency: Detected by MSMS

• Most common fatty acid oxidation defect

• More common than phenylketonuria (PKU)

• 1:9800 births in WA (3 per year)

• ~38 diagnoses per year in Australasia

• Requires MSMS for detection

• Hypoketotic hypoglycaemia

• Unscreened newborns are at risk of permanent neurological deficit, death

• Treated easily once diagnosed

• Avoidance of fasting, glucose supplementation, sick day regime

MCAD & Fatty acid oxidation

MCADC6

C8

C2Ketones increased

Triglyceride

C2Acetyl CoA (energy)

Increased energy/glucose demand

C16

C14

C12

C4

C10

Fatty acid-carnitine ester - Mitochondria

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MCAD & Fatty acid oxidation

MCADC6C8

C2 Ketones inadequate

Triglyceride

C2Acetyl CoA (energy)Relative deficit

Increased energy/glucose demand

C16

C14

C12

C4

C10

Fatty acid-carnitine ester - Mitochondria

Blood marker:C8 acylcarnitine

Urine: C6, C8 glycine

Urine marker

deficiency

Newborn screening impact: “screen positive”(Australia - based on incidence, 315,000 births 2018)

Disorder Incidence Screen positiveCongenital hypothyroidism 1:2400 130Cystic fibrosis 1:2600 121Galactosaemia 1:54000 6MSMS, including PKU 1:3500 90CAH 1:15000 21SMA 1:6000 52SCID 1:50000 6

TOTAL 1:608 426

Australia/NZ 315,147 births (2018)Incidence, screen positive 0.14%

4190 screen positive treatable newborns

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Newborn screening impact: “screen positive”(Australia - based on incidence, 315,000 births 2018)

Disorder Incidence Screen positiveCongenital hypothyroidism 1:2400 130Cystic fibrosis 1:2600 121Galactosaemia 1:54000 6MSMS, including PKU 1:3500 90CAH 1:15000 21SMA 1:6000 52SCID 1:50000 6

TOTAL 1:608 426

Australia/NZ 315,147 births (2018)Incidence, screen positive 0.14%

USA, projected incidence 3,788,235 births (2018)5230 screen positive treatable newborns

4190 screen positive treatable newborns

Newborn bloodspot screening tests

• Disorders screened dependent on many factors

• Ethnic variability, cost/benefit analysis

• Sickle cell disease, thalassemia, haemoglobinopathies

• G6PD deficiency

• Infectious diseases: HIV, CMV, Toxoplasmosis (South America)

• Duchenne muscular dystrophy

• Lysosomal storage diseases

• Adrenoleucodystrophy

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Newborn screening - Treatment options• Amino acid disorders

Dietary regulation of protein/amino acid intake, cofactor/vitamin therapy

• Fatty acid oxidation disorders, organic acidemiasSick‐day regime – prevention of hypoglycaemia and decompensationCarnitine supplementation, cofactor/vitamin therapy (B2, B6, B12…)

• Congenital hypothyroidismThyroxine supplementation

• GalactosaemiaDietary control of lactose/galactose intake

• Cystic fibrosisKalydeco/Ivacaftor for G551D genotypeVX‐445 Triple drug therapy (Symdeko, tezacaftor/ivacaftor)

• CAHHydrocortisone

Newborn screening - Treatment options• SMA

Nusinersen/Spinrasaantisense oligonucleotide (ASO)$367,000 per dose (per year), $41 on Aust PBS

Onasemnogene abeparvovec‐xioi/ZolgensmaAAV9 gene therapy containing SMN gene$US 2.1m per single dose, Aust PBS deferred, listed on UK NHS

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Newborn screening - Treatment options• SCID

Bone marrow transplant

• Lysosomal storage diseasesEnzyme replacement therapy, BMT

• Duchenne muscular dystrophyExon skipping & stop codon read‐through

• PEG-ylated enzymese.g. Phenylalanine ammonia lyase for PKU

• CRISPR/CAS Gene editingClinical trials active: Beta thalassemia

• Gene therapyVectors, viral vectors, retroviruses, AAV

• Prenatal genetic diagnosis & pre-implantation genetic diagnosis

NBS & cost savings

Economic 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.…..

(Feuchtbaum Pediatrics 2006;117;S280‐S286)

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Newborn screening: Impact on servicesScreening

•Sample collection

•Sample submission

•Laboratory testing

Education

feedback

Diagnosis

•Specialist Assessment

•Results shared with family

•Counselling

Management

•Treatment

•Long-term follow-up

•Specimen storage

Evaluation

•Quality assurance

•Outcome evaluation

•Cost effectiveness

Follow up

•Report test results

•Repeat test

•Ensure diagnostic testing

Australian National Governance

http://www.cancerscreening.gov.au/internet/screening/publishing.nsf/Content/newborn‐bloodspot‐screening

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50 years of NBS

Bacterial Inhibition Agar ‐ 1963

Immunoassay ‐ DELFIA MSMS

50 years of NBS


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Immunoassay ‐ DELFIA MSMS

DNA Quantification

50 years of NBS


Thank you for listening!

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1 newborn screening

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