Neonatal Screening for Inborn Errors of Metabolism

Dr. Anil B. Jalan
MD DCH MCPS

INTRODUCTION :- According to WHO figures, about 140 million children are born every year. 5 million children die in the first month of life in developing countries and 4 million children are born with some congenital anomaly. Thousands of children die of no difinable reason e.g. S.I.D.S. ( Almost 27 - 30 % of these babies are now proved to be having some I.E.M. e.g. MCAD deficiency, Organic acidemia like Propionic acidemia or Isovaleric Acidemic ). About 5 to 15 % of all sick neonates in NICU are expected to have some Inborn Error of Metabolism, which may be transient or permanent. The advances in medical sciences has led to reduction in infectious diseases, especially in neonates, the genetic and metabolic disorders are emerging as a major group of disorders responsible for morbidity and mortality. The ultimate of such IEMs is mental retardation. It is a wellknown fact which is also endorsed by WHO that in India about 4 % of population suffere from mental handicap. Of all live births approximately 2 % babies will develop significant mental retardation and many more children will develop - mild grade mental retardation, learning disabilities, autism, dyslexia, behavioural abnormalities, scholastic backwardness, later in life and we are hardly able to diagnose the exact etiology. More dificult is the task of treating them and rehabilitating them.

The most rational and cost effective way of preventing such tragedies would be to have a Neonatal Screening Programme . ( N.S.P. ), which will detect most of the preventable or treatable, if not all IEMs and other disorders, like thalassemia or Toxoplasma infection which are associated with high morbidity and mortality. Even WHO has considered NSP to be important for prevention. This must be accepted as prevention programme and not as research programme. Now in USA and Europe, NSP is mandatory by law for each and every newborn . NSP is the only feasible population based study of genetic disorders which is really cost effective .

It is calculated and proved that to manage a child with PKU successfully an annual expenditure would be approximately 360,000 $ or 57,000 pounds in UK. From our own experience any child with mental retardation, the expenditure to a family is approximately not less than 25,000 to 50,000/- Rs. Without any special diets etc. , which are anyway very difficult to arrange in India. Obviously the best approach for country like ours, would be NSP. Since there are no government funding and non govt. Organisations ( NGO ) are really not willing for such investigations, the only option left is to select a high risk group to begin with .

The diseases needing neonatal screening :- ( most commonly accepted )

1. Hypothyroidism
2. Phenylketonuria
3. M.S.U.D.
4. Biotinidase deficiency
5. Sickle cell disease
6. Galactosemia
7. Tyrosinemia
8. Histidinemia
9. G6 PD deficiency
10. Cystic fibrosis

At our centre , we have identified two high risk groups :-

1 . Critically ill newborns - Many of whom may be having Galactosemia, M.S.U.D., Propionic acidemia, Multiple Carboxylase def., Urea cycle defects etc. etc.

2 . Families with history of :-

a.Previous child with mental retardation or C.P. or congenital anomaly .
b.Past history of recurrent abortions .
c.History of sudden infant death in previous sib .
d.Mother with significantly low intelligence and microcephaly .
e.Family history of haemoglobinopathy .
f.Significant degree of consanguinity .

3 . Routine Neonatal screening :- Recently from 24th Feb. 2000 we have started neonatal screening programme for all the babies born at our centre, which is approximately 2000 babies per annum. We collect either cord blood ( from Vashi Hospital ) or blood by heel prick after 48 hrs. of birth ( from Kalamboli Hospital ). We use special filter paper approved by CDC of USA for neonatal screening S & S 903. After the collection, papers are air dried for 2 hrs. and then preserved in a plastic envelop or paper envelop at 2 to 8 degrees centigrade in fridge. Samples are processed on alternate days and the abnormal reports are communicated to the concerned physicians the same day by telephone. At our hospital we have adopted neonatal screening for

1. Hypothyroidism : T 4 by EIA method .
2. Galactosemia : GALT activity by EIA method.
3. Phenylketonuria : Phenylalanine by EIA method.
4. G 6 PD deficiency : G 6 PD activity by EIA method
5. Toxoplasmosis : Toxo Ig M titre by EIA method

We have decided to have a recheck on 10 % samples as internal Quality control and we are also participating in Neonatal Screening Quality Assuarance and Performance Analysis programme by CDC of USA. Any abnormal result is confirmed with the more specifictests like T 3 , T 4 , TSH by RIA for suspected hypothyroidism cases.

At present the entire oprogramme has been conceieved as a preventive programme and patients are not charged for any tests. The NSP ( Neonatal Screening Programme has been entirely funded by the MGM Hospital?esearch centre ). Further details and protocols are discussed at length in the last chapter of the book.

For the high risk group we offer :-

1. Genetic counselling .
2. Antenatal diagnostic facilities if they are willing -

a ] TORCH titre
b ] USG with doppler and anomaly scan .
c ] C.V.Bx. with Karyotyping .
d ] Amniotic fluid studies for -
e ] Karyotype / F.I.S.H.
f ] A.F.P. & beta - HCG
g ] Biochemical analysis
i ] G.C.M.S.
h ] SOS enzyme assay or DNA studies

3. Haemoglobinopathy workup .
4. Neonatal Screening and Neonate? management in NICU with regular followup .

Our team consists of :-

1. Paediatric geneticist :- responsible for tests and treatment of IEMs.
2. Neonatologists :- responsible for management of newborns in NICU.
3. Paediatrician :- responsible for programme management , funding, publicity and public relations, arranging lectures for paediatricians, under graduate medical students etc .
4. Paediatric Nutritionist :- Responsible for the dietary management and treatment of babies with I.E.M.
5. Neurodevelopmental Physiotherapist :- Responsible for N.D.P.T. of diagnosed patients and arranging rehabilitation programme .
6. Genetic nurse or Parent counseller :- Responsible for giving background information to relatives .
7 . Gynecologists

8 . Other team members :-

a ] Orthopaedic surgeons
b ] Plastic surgeons
c ] Neuro - surgeons
d ] Paediatric - surgeons
e ] E.N.T.- surgeons
f ] Ophthalmic surgeons
g ] Endocrinologists
h ] Cardiologists
i ] Nephrologists
j ] Clinical - psychologists
k ] Speech - therapists
l ] Physiotherapists
m ] Child - psychiatrist
n ] Child - counseller

9 . Ultrasonologist .

Neonatal screening in NICU for Inborn Errors of Metabolism :- Though individually IEMs are rare, collectively they are quite common. It is quite unfortunate that we still have the general impression that I.E.M.s do not exist in India especially in the N.I.C.U. setups ! If we investigate thoroughly all the sick newborns, we may find quite a significant number of cases amongst them. The word IEM carries such a notorious stigma that most of us are still quite allergic to such disorders and we donot attempt to diagnose these conditions in quite early stages when they are really quite mangiable and we can definitely prevent mortality and reduce the morbidity to certain extent .

DEFINITION OF GENETIC SCREENING :- Search in the population for persons with genetic characteristics likely to be harmful to themselves or their descendants .

A ] First Test Described :-

1 . Guthrie dried blood spot test for phenylketonuria. It is a bacteriological test, described in 1962 .

2 . Thyroid hormones test :- on dried blood spot by R.I.A. for congenital hypothyroidism .

B ] Goals of neonatal screening :-

1 . Medical intervention .
2 . Genetic counselling .
3 . Prenatal diagnosis .

C ] Criteria for disease selection :-

1 . Treatable disease .
2 . Difficult to diagnose
3 . Requires immediate therapy to prevent disability and mortality .
4 . Reasonably frequent in population

D ] Recommended screening for :-

1 . Hypothyroidism
2 . Galactosemia
3 . Phenyl ketonuria
4 . Maple Syrup Urine Disease
5 . Tyrosinemia
6 . Other aminoacidopathies
7 . Sickle cell disease

E ] Other recommended disorders :-

1 . Congenital adrenal hyperplasia : ( C.A.H. )
2 . Histidinemia
3 . Methioninemia
4 . Thalassemia
5 . Duchenne Muscular Dystrophy : ( D.M.D. )

Prerequisite for Screening :-

Test should be :-
1 . Simple
2 . Specific
3 . Sensitive
4 . Cost effective
5 . Easy for collection and transport .

Incidence of I.E.M. in India :- Ref. Dr. Radha Ramadevi , M.D. , D.C.H.

No Disdeases screened by Neonatal Screening 1 in population

1 Generalised aminoaciduria 1 in 1,605
2 Congenital Hypothyroidism 1 in 3,500
3 Galactosemia 1 in 4,000
4 Tyrosinemia 1 in 6,234
5 M.S.U.D. 1 in 10,215
6 Phenyl ketonuria 1 in 18,728
7 Hyperglycinemia 1 in 26,053
8 Histidinemia 1 in 37,456
9 Methioninemia 1 in 112,369

Incidence of other disorders in world .

No Diseases 1 in gen. Population

1 Cystic fibrosis 1 in 2,500
2 Cystinuria 1 in 7,000
3 Alfa - 1 - Anti trypsin deficiency 1 in 8,000
4 Iminoglycinuria 1 in 20,000
5 Hartnup disease 1 in 26,000
6 Hyperprolinemia 1 in 40,000
7 Biotidinase def. 1 in 60,000
8 Adenosine deaminase def. 1 in > 100,000

The remarkable progress in the control of infectious diseases has led to the emergence of Genetic and Metabolic diseases as important causes of morbidity and mortality. The socio economic burden makes couple desire 1 - 2 children, free from handicaps. They would readily accept screening tests during pregnancy or in the neonatal period to ensure normality of the child . : I.C.Verma

Incidence of Aminoacid metabolism disorders ( Data from various Newborn screening programmes ) by Dr. Vivian Shih , M.D.

No Disorders 1 in gen. Population

1 Phenylketonuria 1 in 11,5000
2 M.S.U.D. 1 in 220,000
3 Hypermethioninemia & Homocystinuria 1 in 220,000
4 Histidinemia 1 in 24,000
5 Hyperprolinemia ?
6 Hyperlysinemia 1 in 245,000
7 Hereditary Tyrosinemia ?
8 Non ketotic - Hyperglycinemia
Ketotic - Hyperglycinemia
1 in 245,000
1 in 245,000
9 Arginiosuccinic aciduria 1 in 245,000
10 Hyper ornithinemia 1 in 245,000
11 Hartnup disease 1 in 26,000
12 Cystinuria 1 in 7,000
13 Iminoglycinuria 1 in 20,000
14 Fanconi?syndrome 1 in 245,000

Incidence of various I.E.M.s in neonatal screening programme in the state of Georgia ( U.S.A.). ( personal communication with the director Dr. M. Ramachandran, Ph.D. - 13 th Oct. 1999 at Hinduja Hospital Symposium on Neonatal Screening .) Data accumulated by screening over 2 million children .

Disease Total Incidence 1 in gen.population
1 Phenylketonuria

Classic 75 26,149
HyperPHA 40 47,723
Total 115 16,893

2 Galactosemia

Classic 49 38,957
Variant 206 9,266
Total 255 7,486

3 M.S.U.D. 16 119,308

4 Homocystinuria

Classic 5 381,857
Methionine abnormality 41 46,559

5 Tyrosinemia 6 318,156

Transient Tyro.of N.B. 2046 933

6 Congenital Hypothyroidism

Primary 427 4,470
Secondary 33 57,846
Transient 24 79,539
T.B.G. deficiency 595 3,200
Total 1,079 1,769

7 Congenital Adrenal Hyperplasia
Salt losing variety 31 27,044
Non salt losing type 8 104,795
Total 39 21,496

Now let us analysis the situation in the Asian countries in general. Probably Thailand was the first country to introduce mass screening for neonatal screening nationwide. Today the situation is that barring India and probably Pakistan, Bangladesh and Shri-Lanka, almost all the Asian countries have national Neonatal Screening Programme for atleast 2 or 3 diseases common in their countries. Countries like Japan have much less incidence of PKU, MSUD, CHT etc., where as Thialand has a higher incidence of CHT & PKU as compared to other Asian countries. India is no different in this respect and we have a high incidence of CHT, Galactosemia, PKU and MSUD.

Load of such genetic diseases is bound to be higher in India for several reasons,

1 ] The very high population itself .
2 ] Higher incidence of consanguinity .
3 ] Malaria infested zone - giving rise to higher incidence of Thalassemia and
sickle cell anemia .

Status of Neonatal Screening in Asian Countries. Neonatal Screening Programme began in

no. Countries Year

1 China 1981
2 Hong kong 1984
3 India Not yet
4 Indonesia 1993
5 Japan 1976
6 Korea 1991
7 Philippines 1996
8 Singapore 1964
9 Taiwan 1984
10 Thialand 1960
11 New zealand 1969
12 Bangladesh ?
13 Pakistan ?
14 Shrilanka ?

Table No. 2 Inidence of Various Genetic disorders in some of the Asian Countries .

Country Cong. Hypothyroidism Phenyl Ketonuria Galacto-
semia Congenital Adrenal Hyperplasia
1 China 1 : 6,255 1 : 15,503 NIL ? ?

2 Hong kong 1 : 2,834 ? ? ?

3 India 1 : 3,500 1 : 18,728 1 : 4,000 ?

4 Indonesia ? ? ? ?

5 Japan 1/10th of china,USA, UK ? ? ?
6 Korea 1 : 4,413 1 : 56,488 ? ?

7 Philippines 1 : 4,834 1 : 62,481 1 : 31,421 1 : 12,568

8 Singapore 1 : 2,500 ? ? ?

9 Taiwan 1 : 2,840 1 : 40,000 ? ?

10 Thialand 1 : 1,792 1 : 10,752 ? ?

11 New Zea land 1 : 4,680 1 : 21,250 1 : 132,900 1 : 25.400


Test Methods :-

1 B.I.A. Bacterial Inhibition Test .
2 E.A.A. Enzyme Auxotrophic Assay .
3 F.S.T. Fluorescence Spot Test .
4 R.I.A. Radio Immuno Assay
5 E.P.P. Electro Phoretic Pattern
6 T.L.C. Thin Layer Chromatography
7 A.R.G. Auto Radio Graphy .
8 P.C.R. Polymerase Chain Reaction .

Test Methods :-

1. Bacterial Inhibition Tests are used for :-
1 . Phenylalanine 2 . Leucine
3 . Methionine 4 . Tyrosnine
5 . Histidine 6 . Lysine
7 . Glutamine 8 . Glycine
9 . Proline

2 . E.A.A. :Enzyme Auxotrophic Assay :-
1 . Argininosuccinic aciduria .
2 . Citrullinemia
3 . Argininemia
4 . Ornithinemia
5 . Orotic Aciduria

3 . F.S.T. : Flurosecence Spot Test :-
1 . Gal - 1 - Put enzyme
2 . Alfa - 1 - Antitrypsin enzyme
3 . Adenosine Deaminase enzyme
4 . G - 6 - PD enzyme
5 . C - 1 - Esterase inhibitor ( for Angioneurotic edema ) .

4 . E.P.P. : Electrophoretic Pattern :-
1 . Haemoglobinopathies .
2 . Thalassemia .
3 . Sickle cell anemia .

5 . R.I.A : Radio Immuno Assay :-
1 . T 3 , T 4 , T.S.H.
2 . Trypsin for Cystic Fibrosis .
3 . Pregnandiole - ( for adrenogenital syndrome ) .

6. T.L.C : Thin Layer Chromatography :- ( single & two dimensional ) .
1 . Aminoacids in urine, blood, C.S.F. etc .
2 . Sugars .
3 . Organic acids .

7 .A.G.R. : Auto Radio Graphy :-
1 . Enzyme - H.G.P.R.T. ( for Lesch Nyhan Syndrome )

8 .Colorimetric Method :-
1 . Biotidinase enzyme for Multiple Carboxylase Def. Syndrome .

9. Tandem Mass Spectrophotometry :-

1 . Organic acid analysis , M.C.A.D.
2 . Fatty acid oxidation defects .
3 . Aminoacid metaboism disorders .

10 . Micro - chip technology :- 1 cm x 1 cm chip ; DNA sequencing for about
30 disorders.

11. ELISA :-

1. Phenylalanine
2. Methionine
3. Homocystine
4. Lucine & isoleucine
5. T 4
6. GALT

False Negative Results :-

1 . When child has not been fed with milk for sufficient time .
2 . On I.V. fluids etc .
3 . After blood transfusion from a healthy donor .

False Positive Results :-

1 . Antibiotics .
2 . Contamination with urine and stools .
3 . Prematurity .
4 . Baby on ventilator .
5 . Blood transfusion with affected donor .

Precautions :-

1 . Collect cord blood ( best method )
2 . Collect urine after 48 hrs. after initiation of milk feeding .
3 . Collect blood and urine sample before blood transfusion or 48 - 72
hrs. after blood transfusion .
4 . Use standard Whatman filter paper no . 3 or ( S & S - 903 ).
5 . Diameter of each spot should be 6 mm minimum .
6 . Blood should soak through to the other side of the paper .
7 . Proper details of the baby .
8 . Repeat the test after 1 - 2 wks. , if the results are abnormal .
9 . Use better methods like , H..P.T.L.C. , H.P.L.C. , G.L.C. , G.C.M.S. , R,I.A. , E.P.P. & D.N.A. / R.N.A. study for confirmatory diagnosis .
10.Use specific enzyme asays for the diagnosis & carrier state studies
11.Use specific mutation studies for the confirmed diagnosis and carrier state studies .

Positive Consequences :-

1 . Early intervention , prevents disability and deaths .
2 . Family counselling possible .
3 . Prenatal diagnosis for next pregnancy .
4 . Avoids frustration for both parents and physicians .

Negative consequences :-

1 . Parental agony and anxiety .
2 . Over protection or neglect of the child .
3 . Feeling of guilt .
4 . Family breakup and divorce .

Neonatal sereening which was considered as a part of research in the past, can now be applied to the field of health as preventive programme just like immunization .

Some of the ethical questions raised for Neonatal Screening programme are

1 . Will presymptomatic diagnosis benefit the baby ?
2 . Will the benefit out - weigh the possible harm ?
3 . Is there an effective test ?
4 . Can we afford it ?
5 . Whether screening tests can be made available to all ?
6 . Whether they should be made mandatory ?
7 . And if not, to what extent informed consent is needed ?
8 . How far the screening laboratory? responsiblity extends in ensuring
that the patient receives appropriate management ?

Results of one study of Neonatal screening for Cystic fibrosis :-

New born screening of 650,341 babies Trypsinogen with DNA study was undertaken. 74 babies were diagnosed as Cystic Fibrosis. These babies were followed up for next 10 yrs. and compared with children diagnosed by std. methods at a later age group. These babies diagnosed earlier by Neonatal Screening had better anthropometric indices. These babies also had better lung compliance and lesser inflammation than the control group .

Ref . Farrell P M , for the Wisconsin Cystic Fibrosis Neonatal Screening Study Group ( University of Wisconsin , Madison et. al. ) New England Journal of Medicine : 337 : 963 - 969 : 1997 .

When diagnosis of metabolic disorder is made in one mamber of the family , a survey of siblings and parents is indicated . Other relatives should be included when their medical history suggests that they may be similarly affected . It should be emphasized that when all siblings are mentally retarded and no biochemical abnornmality can be found in any of them , it is important to screen the parents , particularly the mother , for any metabolic abnormalities , since maternal PKU as a cause of neonatal retardation and congenital anomalies is well recognized .

N.B. :- A facility to which patients can be referred for further study , treatment , and genetic counselling is an integral part of a screening programme without which purpose of neonatal screening would be defected .

Normal Values in Neonatal Screening Programme :-

No. Diseases Normal ranges Alert ranges

1 Phenyl alanine < 4.00 mgm / dL > 6.00 mgm / dL

2 Leucine < 4.00 mgm / dL > 4.00 mgm / dL

3 Methionine < 2.00 mgm / dL > 4.00 mgm / dL

4 Tyrosine < 6.00 mgm / dL > 6.00 mgm / dL

5 Gal - 1 - PUT 100 % fluorescence Low or no fluorescence
GALT quantitative assay
Units / Gm of Haemoglobin Above 2.4
2.4 - 1.3 equivocal Less than 1. 3

Gal - 1 - Phosphate < 11.0 mgm / dL > 12.0 mgm / dL

6. T 4 ( Thyroxine hormone )

a. for < 1 wk. age baby ( A.G.A. )
Normal range > 7.5 ugm / dL
Alert range < 7.5 ugm / dL

b. for > 1 wk age baby ( A.G.A. )
Normal range > 5.4 ugm / dL
Alert range < 5.4 ugm / dL

c. for L.B.W. baby ( < 2500 gms )
Normal range > 4.5 ugm / dL
Alert range < 4.5 ugm / dL

7 T.S.H. ( Thyroid Stimulating Hormone )

Normal range < 48 hrs. age up to 30 u I.U. / ml
Normal range > 48 hrs. age up to 25 u I.U. / ml