Follow up ofFollow up of

Abnormal Neonatal ScreeningAbnormal Neonatal Screening

For Inborn Errors of For Inborn Errors of MetabolismMetabolism

James D. Shoemaker MD PhDJames D. Shoemaker MD PhD

Director, Metabolic Screening Director, Metabolic Screening LabLab

St. Louis University School of St. Louis University School of MedicineMedicine

From:

NNSGRC

National

Neonatal

Screening

And

Genetics

Resource

Center

as of

9/25/06

Dr. David S. Millington

Duke University, Department of Genetics

85 dalton fragment

Preparation of blood spots for tandem mass spectrometry:

1) Punch out spot and put in test tube

2) Swish blood spot in methanol

3) Remove paper and dry methanol under nitrogen stream

4) Add butanol and acetylchloride to make butyl esters of amino acids

5) Transfer to sample plate for automated injection

6) Compile the molecular weights of all ions that yield a fragment weighing 85 daltons

7) Subtract weight of carnitine to identify “acyl” portion of acylcarnitine

8) Scan for substances with a “neutral loss” of 102: amino acids

Note: Disorders above detected as the amino acid precursor

Formerly only 1 “organic acidemia” among screened disorders

Biotinidase and MSUD were not screened for in MO and IL

Current Screening Protocol:

All MS/MS results go to Dr. Jinks

312/793-1053

Who makes the determination:

Presumptive Positive or Borderline

Straight to specialist To ordering MD

recommend repeat MS/MS on new

blood spot

Instead of 1/9624

2005

½ year

Of Tandem MS/MS

Missouri 2006 Expanded Newborn Screening Results

Inborn errors detected: 18 42 “high risk” cases over 8 months

Inborn Errors Confirmed:

20 39 since inception

0.05% vs. 0.032 % for

Illinois

H2N – C – NH2

||

O

2 NH3 + CO2

urease

CF3-COO - +H3N- CH2-COO - +HN-[CH2CH3]3

| Trifluoroacetate R Triethylammonium

CF3-COO - +H3N[CH2CH3]3

Triethylammonium Trifluoroacetate

H2N-CH-COOH TMS2N-CH-COO-TMS | |

R R R-COOH R-COO-TMS

R-CH2-OH R-CH2-O-TMS

R-CH2-SH R-CH2-S-TMS

Trimethylsilylation

Creatinine Mass Spectra; top: native, bottom: d3

How the Metabolic Screening Lab

and Expanded Newborn Screening

Work Together:

I. MS/MS cut-offs for “significance” are set for screening purposes, that is, low, to be most sensitive, but not specific. Physiological significance may be indicated on other signs of metabolic stress available on the MSL panel.

From first year medical school lecture on MCAD deficiency:

MSL panel reports acylglycines, not acylcarnitines.

Acylglycines are made only when pathological amounts of medium chain derivatives are present and are therefore “more abnormal”

DNA Probe by PCR: A985G [same as K329E in the peptide], confirmed by Piero Rinaldo MD at Yale University; the most common MCAD mutation ascertained from symptomatic patients.

Medium Chain Acyl-CoA-Dehydrogenase (MCAD) is a nuclear encoded (chromosome 1p31), mitochondrial, tetrameric protein specific for the initial beta-oxidation of fatty acids of chain length 6 through 12, primarily. MCAD deficiency is the most common genetic defect of fatty acid oxidation and is one of the most common genetic disorders of people of Northern European origins. The most common mutation, A985G in exon 11, causes lysine 329 to be replaced by glutamate (K329E). The substitution of the negatively charged glutamate for the positively charged lysine disrupts the folding and assembly of the tetrameric subunits and the conformation of the FAD-binding active site. High doses of riboflavin may help to correct the folding and assembly defects (ref 24,35 ) The heterozygous frequency of the A985G mutation has been estimated as follows in various ethnic groups: Netherlands 1/59, United Kingdom 1/68, Belgium 1/77, Bulgaria 1/91, Poland 1/98, Denmark 1/101, Germany 1/116, US 1/122, (North Carolina 1/84, Pennsylvania 1/95), Sweden 1/127, France 1/140, Hungary 1/168, Turkey, 1/216, Czech Republic 1/240, Italy 1/333, Spain 1/200, Spanish gypsies 1/17, Japan: none.

How the Metabolic Screening Lab

and Expanded Newborn Screening

Work Together:

II. MS/MS provides molecular weights that are consistent with various classes of compounds, but not positive compound identification.

By contrast, GC/MS used by MSL, provides positive compound ID by retention time and mass fragmentogram.

Leucine C6H13NO2 MW = 131.17 Hydroxyproline C5H9NO3 MW = 131.13

How the Metabolic Screening Lab

and Expanded Newborn Screening

Work Together:

III. False positives are the hallmark of the newborn screening program in another way: the cut-offs are set so low that the heterozygous state of the newborn of a parent with a benign inborn error is detectable, leading to late-life diagnosis of the parent.

How the Metabolic Screening Lab

and Expanded Newborn Screening

Work Together:

IV. At present, expanded newborn screening does not pick up some of the most life-threatening categories of inborn errors.

Of 120Inborn Errors: [multiple cases]

• 12 Urea cycle• 10 S-MCAD-LCHAD• 12 MMA• 6 MSUD• 15 Tyrosinemia

(many neonatal)

• 8 Galactosemia

• 4 Cystinuria

(all over 1 y.o.)

• 3 Propionic

• 2 HMG

• 2 Renal Fanconi

• 2 Hyperoxaluria

• 3 Molybdenum Cofactor Deficiency

MS/MS techniques for detection of orotic acid have been published, but have not been incorporated into the

panel of Illinois or Missouri.

How the Metabolic Screening Lab

and Expanded Newborn Screening

Work Together:

V. At present, expanded newborn screening does not pick up some of the most rare types of inborn errors such as

Hawkinsinuria

Of 74 Inborn Errors: [multiple cases]

• 9 Urea cycle• 6 MCAD-LCHAD• 6 MMA• 5 MSUD• 7 Tyrosinemia

(5 neonatal)

• 4 Galactosemia

• 4 Cystinuria

(all over 1 y.o.)

• 3 Propionic

• 2 HMG

• 2 RenalFanconi

• 2 Hyperoxaluria

• 2 Molybdenum Cofactor Deficiency

Of 120Inborn Errors: [multiple cases]

• 12 Urea cycle• 11 S-MCAD-LCHAD• 12 MMA• 6 MSUD• 15 Tyrosinemia

(many transient)

• 8 Galactosemia

• 4 Cystinuria (all over 1 y.o.)

• 3 Propionic

• 2 HMG

• 2 Renal Fanconi

• 2 Hyperoxaluria

• 3 Molybdenum Cofactor Deficiency

• 2 Cystathioninuria

Pink: not likely picked up by standard organic acid screening

Of 120 Inborn Errors: (continued)

• 6 Glutaric I • 2

hypophosphatasia

• 4 Adrenoleukodystrophy

• 4 3-methylglutaconic

• Fumaric• 2 Pyroglutamic• 2 IminoAciduria• 2 3-met-crot

CO2ase• 2 Biotinidase

• Pyruvate Dehydrogenase,

• Proline Oxidase, • Succinic Semi-

aldehyde dehydrogenase Deficiencies

• Hartnups & Canavan’s Diseases

• Renal Glycosuria• Hawkinsinuria • FIGLU uria, • PKU • 2 Alkaptonuria• 2-Hydroxyglutaric

aciduria

Of 11724 Samples, from 10061 Of 11724 Samples, from 10061 patientspatients

1993 Abnormal 17%1993 Abnormal 17%

120 confirmed Inborn Errors120 confirmed Inborn Errors

Total: 1.17% of patientsTotal: 1.17% of patients

49 involved non-acidic 49 involved non-acidic metabolitesmetabolites

68% increase due to urease 68% increase due to urease methodmethod compared to standard compared to standard extraction or extraction or column clean-up.column clean-up.

SourceSource # of# of

patientspatients

% %

AbnormalAbnormal

# of Inborn # of Inborn

ErrorsErrors

% Inborn% Inborn

ErrorsErrors

GlennonGlennon

InptsInpts

23062306 24.824.8 4848 2.12.1

Glennon Glennon

Outpt.Outpt.

15101510 9.29.2 1111 0.70.7

CH OaklandCH Oakland 13201320 17.717.7 2727 1.791.79

Spring-Spring-

field, ILfield, IL

738738 9.89.8 44 0.50.5

SpecialtySpecialty

LabsLabs

29912991 5.35.3 1212 0.40.4

CarleCarle

ClinicClinic

160160 8.18.1 11 0.60.6

Of 120 Inborn Errors: [more common cases]How many would be picked up by

MO-IDPH neonatal screen?• 12 Urea cycle• 11 MCAD-LCHAD• 12 MMA• 6 MSUD• 15 Tyrosinemia

(many transient)

• 8 Galactosemia

• 6 Glutaric I

• 4 Adrenoleukodystrophy

• 4 3-methylglutaconic

• 4 Cystinuria

(all over 1 y.o.)

• 3 Propionic

Answer: 65/85 of more common disorders

Of rarer disorders: 8/35 TOTAL: 73/120 = 60.8%

Of 120 Inborn Errors: (continued)

• 3 Molybdenum Cofactor Deficiency

• 2 HMG• 2 Renal Fanconi• 2 Hyperoxaluria

• 2 hypophosphatasia• 2 Pyroglutamic• 2 IminoAciduria• 2 3-met-crot CO2ase• 2 Biotinidase • 2 Alkaptonuria

• Fumaric• Pyruvate

Dehydrogenase,• Proline Oxidase, • Succinic Semi-

aldehyde dehydrogenase Deficiencies

• Hartnups & Canavan’s Diseases

• Renal Glycosuria• Hawkinsinuria • FIGLU uria, • PKU • HHH, 2

Cystathioninuria• 2-Hydroxyglutaric

Aciduria

Overall rate of vitamin deficiency detection

500 / 2726 = 18.3%Of very ill infants and children suspected of having inborn errors of metabolism

Will Neonatal Screening Eliminate the need for Labs like mine?

Methionine

Homocysteine

S-Adenosyl-methionine

S-Adenosyl-homocysteine

Cystathionine

Alpha-ketobutyrate Cysteine

Serine

Tetrahydrofolate

Methyl-THF

B-6

B-6

B-12

Methylene-THF

B-6

FAD

Recommendation:

• If an infant requires intravenous fluids, add MVI to the first bag

• Or give oral equivalent .

Indications for ordering Organic Compound Screening

• HIGH YIELD LOW YIELD

• but sometimes necessary

• Unexplained or Recurrent:

• Acidosis Seizure Disorder

• Hypoglycemia Failure to Thrive

• Hyperammonemia Developmental Delay

Saint Louis University School of Medicine

E.A.Doisy Department of Biochemistry and Molecular Biology

*quinolinic acid nicotinic acid

NADH NAD+

2-acroleyl-3-aminofumarate ketoadipate CO2 + H2O

3-hydroxyanthranilic acid

pyridoxal phosphate

3-hydroxykynurenine xanthurenic acid

*kynurenine *kynurenic acid

tryptophan

MS/MS answers the question:

What was the molecular weight of the parent ion that gave rise to this daughter ion weighing 85 (carnitine) or 102 (amino complex)

Although MS/MS happens “instantaneously” it is still a separative technique: the computer records the millisecond at which the parent ion giving rise to “daughters of 85” entered the second source and can derive its molecular weight.

So you know the molecular weight of the precursor (parent) and can identify the weight of the acyl group (acylcarnitines) or side chain (amino acids) of this molecule

Illinois Experience in the First Year of Expanded MS/MS TestingIllinois Experience in the First Year of Expanded MS/MS Testing

How many newborns were tested July 1, 2002 – June 30, 2003?How many newborns were tested July 1, 2002 – June 30, 2003?

Over 400,000 Over 400,000

How many confirmed cases of inborn errors were found?How many confirmed cases of inborn errors were found?

73 (about 1/5500)73 (about 1/5500)

From what main category?From what main category?

All categories: amino acid, organic acid, fatty acid.All categories: amino acid, organic acid, fatty acid.

Thanks to: Dr. David Jinks PhD, Chief of Newborn ScreeningThanks to: Dr. David Jinks PhD, Chief of Newborn Screening

Illinois Department of Public Health, Chicago, ILIllinois Department of Public Health, Chicago, IL

First Complete Year: 7/1/2002 - 6/30/2003 MS/MS Results

Analytes detected in Missouri Expanded Screening

Yellow shading = Possibly diagnostic, not definitive

Years before MS/MS screening in Missouri

Year 2004

2006 January-March