j . Inher. Metab. Dis. 1 (1978) 67-70
Phenylketonuria in Indian Children GERTRUDE E. JOSHUA,* S. CHANDY,~ A. N. RADttAKRISHNAN,$D. MAMMEN¶ and K. V. MATHAI~ Departments of Child Health,* Yathology,t Neurological Sciences,¶ and the Welleome Laboratory,+ Christian Medical College Hospital, VdIore, S. India T h r e e untreated phenylketonuric Indian children aged respectively 3} years, t½ years and t year showed rapid neurological deterioration. Plasma, cerebrospinal fluid and urine phenylalanine concentrations were significantly raised and the phenylalanine-tyrosine ratio was high. Analysis of a biopsy of the right fi'ontal lobe of the brain in one case showed the myeline Iipids - c e r e b r o s i d e and sulphatide - t o be decreased. T h e total cerebroside in white matter was low-. Light microscopy showed marked pallor of the white matter of the brain and extensive spongy degeneration. Uhrastructurally these spongy vesicles are located between the lameltae of the myelin sheath. Centerwai1 and tttyerah, 1966; J o s h u a et at., 1968, 1973; R a m a R a o Sridhara el al., 1977). This paper presents the clinical and biochemical features of P K U in three children, and morphological and neurochemical findings in biopsied brain tissue of one, and is the first detailed report of classical P K U in Indian children.
In the developing countries, malnutrition and infectious diseases are the major health p r o b l e m and screening of the newborn and the mentally retarded for metabolic disease has not been given due importance. Phenylketonuria is believed to be a rare metabolic disorder in Indian children and hitherto only 25 cases have been reported (Krupanidhi and Punekar, t963;
Table 1 Clinical data on three phenylketonuric children
Case n o . , sex and age
Consanguinity j?~milyhisto~2),
Clinical signs
Symptoms
EEG (sleep)
1. Female, 3½ years
Parents: first cousins once removed. Mother's three siblings had similar disease
Delayed milestones, grand-mal seizures, myoclonic seizures, adventitious movements of limbs and face, intermittent tremor of hands
Fair complexion with brown hair and irides. Decreased muscle tone, hyperactive tendon reflexes. Optic atrophy. Severely mentally retarded (weight: 9kg; head circumference : 44 cm ; chest circumference : 46 cm)
2. Female, I year
Sibling of Case 1
Delayed miIestones, grand-real seizures
Fair complexion, brown Sharp spike discharge hair and irides. Generalized bilaterally mild hypotonia. Hyperactive tendon reflexes (weight: 8.6kg; head circumference : 43 cm ; chest circumference : 43 cm) Clinging musty odour; generalized seborrhoeic dermatitis. Severely retarded
Myoclonic seizures at 2½ years 3. Male, 1} years
Parents: first cousins
Delayed milestones, grand-real seizures, adventitious movements of limbs and face
One sibling mentally retarded
At 3 years-bedridden, frequent myoclonic seizures
67
Fair complexion. Disoriented brown hair and irides. Primitive reflexes (moro, sucking, rooting) active (weight : 8 kg ; head circumference : 42 cm ; chest circumference : 45 cm) Severely retarded ; decreased muscle tone. Hyperactive tendon reflexes, musty odour. Seborrhoeic dermatitis. Optic atrophy
Frequent bursts of spikes, spike and wave and polyspikes bilaterally and synchronously. Frequent burst suppression pattern
Single and multiple spike discharge bilaterally. Paroxysmal slow activity. Slow polyspikes and wave discharge from parieto-occipitat region bilaterally
68
Joshua, Chandy, Radizakrishnan, Mammen and Mathai
MATERIAL AND METHODS
RESULTS
The pertinent family history, clinical data and electroencephalographic findings in the three cases are given in Table 1. Urine was subjected to ferric chloride and dinitrophenylhydrazine (DNPH) tests, and both blood and urine were examined for amino acids by paper chromatography and for orthohydroxyphenyl acetic acid (OHPA). Right frontal lobe biopsy was performed under general anaesthesia in Case 3, and as a control, tissue which became available from a subject without neurological disease was used and the tissue examined by light and electron microscopy (EM) and a portion stored at - 18°C prior to neurochemical analysis.
Clinical data The three children were mentally retarded and lighter in complexion than the rest of the family and they had brown hair and irides. They all had myoclonic and grand-mal seizures. Follow-up over a period of two years showed progressive neurological deterioration and they were bedridden and severely mentalty retarded. The EEG showed single and multiple loci of spike, polyspike and slow waves and diffuse slow background activity bilaterally. Biochemistry The ferric chloride, D N P H and O H P A tests were positive in all three patients. The phenylalanine concentration in plasma, cerebrospinal fluid and urine was significantly increased and the phenylalanine-tyrosine ratio in these fluids was very high (Table 2).
Biochemical methods Quantitative determination of amino acids in plasma, urine and cerebrospinal fluid was made using an automatic amino acid analyser. Plasma was first deproteinized by ultrafiltration according to Benson and Patterson (1965). Sphingolipids were extracted and identified by thinlayer chromatography (TLC), using the methods described by Kokrady et aL (1971).
Brain lipids The myelin lipids cerebroside and sulphatide--were decreased in both grey and white matter and the ratio of cerebroside to sulfatide was decreased. Cerebroside in white matter was much lower in the patient than in the normal control (2 and 10.5 mg/g wet tissue respectively). The total ganglioside content and the ganglioside pattern were essentially normal (Table 3).
Histopathological methods Frozen and formalin-fixed tissue was stained with haematoxylin and eosin, periodic acid Schiff, Sudan I I I , oil red O, Holmes and Luxol Fast Blue prior to light microscopy. For electron microscopy, brain tissue containing grey and white matter was immediately cut into blocks, fixed in chilled 3.5()/o buffered glutaraldehyde and prepared for EM study using the technique described by Estable-Puig et aL (1965). Ultra-thin sections stained with lead citrate and uranyl acetate were examined using a Philips E M 200 electron microscope. Table 2
Histopathology Light microscopy showed marked pallor and spongy degeneration of the white matter (Figure 1). Very fi:w well-preserved myelin sheaths were present and in most areas they had a vacuolated, fibrillary and disorganized appearance. Electron microscopy showed large spongy vacuoles with dark lamellae forming their walls (Figure 2). These lamellae appeared to separate into vesicles in some areas
Phenylalanine and tyrosine concentration in body fluids of three children with PKU P/zenylalanine mg/ lO0 m!
Tyrosine mg/ l O0 m[
Pheny[alanine-lyrosine ratio
Case No. plasma
1 2 3 Normal control
urine
27.10 22.60 18.20 1.04
CSF*
plasma
urine
CSF
plasma
urine
CSF
---3.88 5.86 0. i 0
1.35 0.87 0.40 0.89
1.88 0.97 0.29 0.81
0.10 0.26 0.17
20 26 45 1
23 13 26 0.7
39 23 0.6
44.0 12.95 7.53 0.60
* The plasma and CSF were obtained on different days
Table 3 Sphingolipids in brain tissue of a phenylketonuric child (Case 3 aged four years) and a control subject without neurological disease Cerebroside + sulphatide (mg/g wet tissue)
Patient: grey matter Patient: white rnatter Control: grey matter Control: white matter
3 4 6.5 14
Cerebroside Sulphatide (mg/g wet (mg/g wet tissue) tissue)
1.6 2 5.5 10.5
1.5 1.5 1.4 3.5
Tota! Cerebroside/ sulphatide ganglioside (t~mol .~CddVA/g ratio < wet tissue) GTI
1:1 1:0.8 4:1 3:I
* The control tissue was noe separated into grey and white matter prior to ganglioside analysis
2 0.93 1.3"
Individual gangliosides < °/o of total N A N A + GDIb
23.4 28.8 22.7*
GDla
GMI
58.4 18.2 60.0 11.
Phenylketonuria in Indian Children GERTRUDE E. JOSHUA,* S. CHANDY,~ A. N. RADttAKRISHNAN,$D. MAMMEN¶ and K. V. MATHAI~ Departments of Child Health,* Yathology,t Neurological Sciences,¶ and the Welleome Laboratory,+ Christian Medical College Hospital, VdIore, S. India T h r e e untreated phenylketonuric Indian children aged respectively 3} years, t½ years and t year showed rapid neurological deterioration. Plasma, cerebrospinal fluid and urine phenylalanine concentrations were significantly raised and the phenylalanine-tyrosine ratio was high. Analysis of a biopsy of the right fi'ontal lobe of the brain in one case showed the myeline Iipids - c e r e b r o s i d e and sulphatide - t o be decreased. T h e total cerebroside in white matter was low-. Light microscopy showed marked pallor of the white matter of the brain and extensive spongy degeneration. Uhrastructurally these spongy vesicles are located between the lameltae of the myelin sheath. Centerwai1 and tttyerah, 1966; J o s h u a et at., 1968, 1973; R a m a R a o Sridhara el al., 1977). This paper presents the clinical and biochemical features of P K U in three children, and morphological and neurochemical findings in biopsied brain tissue of one, and is the first detailed report of classical P K U in Indian children.
In the developing countries, malnutrition and infectious diseases are the major health p r o b l e m and screening of the newborn and the mentally retarded for metabolic disease has not been given due importance. Phenylketonuria is believed to be a rare metabolic disorder in Indian children and hitherto only 25 cases have been reported (Krupanidhi and Punekar, t963;
Table 1 Clinical data on three phenylketonuric children
Case n o . , sex and age
Consanguinity j?~milyhisto~2),
Clinical signs
Symptoms
EEG (sleep)
1. Female, 3½ years
Parents: first cousins once removed. Mother's three siblings had similar disease
Delayed milestones, grand-mal seizures, myoclonic seizures, adventitious movements of limbs and face, intermittent tremor of hands
Fair complexion with brown hair and irides. Decreased muscle tone, hyperactive tendon reflexes. Optic atrophy. Severely mentally retarded (weight: 9kg; head circumference : 44 cm ; chest circumference : 46 cm)
2. Female, I year
Sibling of Case 1
Delayed miIestones, grand-real seizures
Fair complexion, brown Sharp spike discharge hair and irides. Generalized bilaterally mild hypotonia. Hyperactive tendon reflexes (weight: 8.6kg; head circumference : 43 cm ; chest circumference : 43 cm) Clinging musty odour; generalized seborrhoeic dermatitis. Severely retarded
Myoclonic seizures at 2½ years 3. Male, 1} years
Parents: first cousins
Delayed milestones, grand-real seizures, adventitious movements of limbs and face
One sibling mentally retarded
At 3 years-bedridden, frequent myoclonic seizures
67
Fair complexion. Disoriented brown hair and irides. Primitive reflexes (moro, sucking, rooting) active (weight : 8 kg ; head circumference : 42 cm ; chest circumference : 45 cm) Severely retarded ; decreased muscle tone. Hyperactive tendon reflexes, musty odour. Seborrhoeic dermatitis. Optic atrophy
Frequent bursts of spikes, spike and wave and polyspikes bilaterally and synchronously. Frequent burst suppression pattern
Single and multiple spike discharge bilaterally. Paroxysmal slow activity. Slow polyspikes and wave discharge from parieto-occipitat region bilaterally
68
Joshua, Chandy, Radizakrishnan, Mammen and Mathai
MATERIAL AND METHODS
RESULTS
The pertinent family history, clinical data and electroencephalographic findings in the three cases are given in Table 1. Urine was subjected to ferric chloride and dinitrophenylhydrazine (DNPH) tests, and both blood and urine were examined for amino acids by paper chromatography and for orthohydroxyphenyl acetic acid (OHPA). Right frontal lobe biopsy was performed under general anaesthesia in Case 3, and as a control, tissue which became available from a subject without neurological disease was used and the tissue examined by light and electron microscopy (EM) and a portion stored at - 18°C prior to neurochemical analysis.
Clinical data The three children were mentally retarded and lighter in complexion than the rest of the family and they had brown hair and irides. They all had myoclonic and grand-mal seizures. Follow-up over a period of two years showed progressive neurological deterioration and they were bedridden and severely mentalty retarded. The EEG showed single and multiple loci of spike, polyspike and slow waves and diffuse slow background activity bilaterally. Biochemistry The ferric chloride, D N P H and O H P A tests were positive in all three patients. The phenylalanine concentration in plasma, cerebrospinal fluid and urine was significantly increased and the phenylalanine-tyrosine ratio in these fluids was very high (Table 2).
Biochemical methods Quantitative determination of amino acids in plasma, urine and cerebrospinal fluid was made using an automatic amino acid analyser. Plasma was first deproteinized by ultrafiltration according to Benson and Patterson (1965). Sphingolipids were extracted and identified by thinlayer chromatography (TLC), using the methods described by Kokrady et aL (1971).
Brain lipids The myelin lipids cerebroside and sulphatide--were decreased in both grey and white matter and the ratio of cerebroside to sulfatide was decreased. Cerebroside in white matter was much lower in the patient than in the normal control (2 and 10.5 mg/g wet tissue respectively). The total ganglioside content and the ganglioside pattern were essentially normal (Table 3).
Histopathological methods Frozen and formalin-fixed tissue was stained with haematoxylin and eosin, periodic acid Schiff, Sudan I I I , oil red O, Holmes and Luxol Fast Blue prior to light microscopy. For electron microscopy, brain tissue containing grey and white matter was immediately cut into blocks, fixed in chilled 3.5()/o buffered glutaraldehyde and prepared for EM study using the technique described by Estable-Puig et aL (1965). Ultra-thin sections stained with lead citrate and uranyl acetate were examined using a Philips E M 200 electron microscope. Table 2
Histopathology Light microscopy showed marked pallor and spongy degeneration of the white matter (Figure 1). Very fi:w well-preserved myelin sheaths were present and in most areas they had a vacuolated, fibrillary and disorganized appearance. Electron microscopy showed large spongy vacuoles with dark lamellae forming their walls (Figure 2). These lamellae appeared to separate into vesicles in some areas
Phenylalanine and tyrosine concentration in body fluids of three children with PKU P/zenylalanine mg/ lO0 m!
Tyrosine mg/ l O0 m[
Pheny[alanine-lyrosine ratio
Case No. plasma
1 2 3 Normal control
urine
27.10 22.60 18.20 1.04
CSF*
plasma
urine
CSF
plasma
urine
CSF
---3.88 5.86 0. i 0
1.35 0.87 0.40 0.89
1.88 0.97 0.29 0.81
0.10 0.26 0.17
20 26 45 1
23 13 26 0.7
39 23 0.6
44.0 12.95 7.53 0.60
* The plasma and CSF were obtained on different days
Table 3 Sphingolipids in brain tissue of a phenylketonuric child (Case 3 aged four years) and a control subject without neurological disease Cerebroside + sulphatide (mg/g wet tissue)
Patient: grey matter Patient: white rnatter Control: grey matter Control: white matter
3 4 6.5 14
Cerebroside Sulphatide (mg/g wet (mg/g wet tissue) tissue)
1.6 2 5.5 10.5
1.5 1.5 1.4 3.5
Tota! Cerebroside/ sulphatide ganglioside (t~mol .~CddVA/g ratio < wet tissue) GTI
1:1 1:0.8 4:1 3:I
* The control tissue was noe separated into grey and white matter prior to ganglioside analysis
2 0.93 1.3"
Individual gangliosides < °/o of total N A N A + GDIb
23.4 28.8 22.7*
GDla
GMI
58.4 18.2 60.0 11.
