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Neonatal screening for alpha1-antitrypsin deficiency
1006
June 1978 TheJournalofPEDIATRICS
Neonatal screeningfor alphal-antitrypsin deficiency The Oregon State Pubfic Health Laboratory screened 107,038 newborn infants between 1971 and 1974 to determine th e frequency and clinical characteristics of alphal-antitrypsin deficiency. The screening program was based upon an assay of the total trypsin inhibitory activity in dried blood specimens collected on filter paper from infants during the first week of life and again from 75% of the same infants at four to six weeks of age. Twenty-one homozygous-defieient (PiZ) infants were identified, an incidence of one in 5,000. Of the 18 infants studied and followed, only one had neonatal hepatitis;five had hepatomegaly or biochemical abnormalities or both, indicating hepatic damage. Presently, the children range from three to six years of age; all are asymptomatic. Four of them have mild hepatomegaly and biochemical evidence of liver damage. As a result of family studies, four homozygous-deficient (PiZ) siblings were identified. One child had evidence of mild hepatic dysfunction, but the other three were cfinically and bioehemically normal. Nine of the 21 PiZ infants detected were missed on the initial sample, but identified on the four to six week sample. I f a screening method based upon TIA is to be utilized, these results indicate that a repeat screening specimen should be obtained at four to six weeks of age. Newborn screening for alpha~-antitrypsin deficiency is not warranted at this time in view of the low frequency of ~ignificant pulmonary or hepatic involvement in childhood and the absence of specific therapy for this condition.
Mary Lynn O'Brien, Neil R. M. Buist, and William H. Murphey,* Portland, Ore.
ALPHA~-ANTITRYPSIN deficiency was first described as a clinical entity by Laurell and Eriksson, 1 who noted its association with the onset of pulmonary emphysema in early middle life. It was subsequently discovered to be associated with liver disease in children ~' ~ and in adults. 4-~ Over 20 distinct genetic variants of a 1-AT protein are recognized; these variants are identified by electrophoresis.7 The most common (normal) homozygote is designated as PiM. The genetic variant which is most closely From the Department of Pediatrics, University of Oregon Health Sciences Center, and the Oregon State Public Health Laboratory. Supported in part by grants from the National Foundation (No. CE-14), and from the National Institutes of Health (No. HD 03967-10), and from Maternal ChiM Health (Grant No. 435). *Reprint address: Oregon State Public Health Laborator)~ P.O. Box 275, Portland, OR 9720Z
VoL 92, No. 6, pp. 1006-1010
associated with lung and liver disease is PiZ; the other variants (e.g., S,F,) and MZ heterozygotes are spared most, if not all, of the hepatic and pulmonary disease. Although a l - A T deficiency has a high frequency compared to other inherited biochemical disorders that can be detected in the newborn period, such as phenylkeAbbreviations used: Pi: proteaseinhibitor a 1,AT: alphal-antitrypsin PKU: phenylketonuria TIA Trypsin inhibitorY activity SGOT: serum glutamic oxalacetic transaminase SGPT: serum glutamic pyruvic transaminase GGT: gamma glutamyl transpeptidase trypsin inhibitory capacity TIC: NAD: nicotinamide adenine dinucleotide tonuria, galactosemia, and maple syrup urine disease, widespread screening for a l - A T deficiency has not been adopted in this country. Most studies to date have been in
0022-3476/78/0692-1006500.50/0 9 1978 The C. V. Mosby Co.
Volume 92 Number 6
Neonatal screening for alphal-antitrysin
children with liver disease, 8 of whom up to 35% may have a l - A T deficiency. In adults, studies have been on individuals in whom pulmonary disease has been identified, or on relatively small populations of hospitalized patients? lo The purposes of this study were to evaluate: (1) a new screening test for a 1-AT deficiency and (2) the clinical status of infants with PiZ variant detected by such screening. METHODS
AND MATERIALS
Mass screening of newborn infants. Between 1971 and 1974, the Oregon State Public Health Laboratory screened 107,038 infants with blood specimens routinely collected on Schleicher and Schuell No. 903 filter paper. The blood specimens were obtained by heel-prick from infants upon the day of discharge from the hospital, usually at one to three days of age; a second sample was obtained from 80,344 (75%) of these same infants at four to six weeks of age. Except for 26 infants who had moved, died, were not located, or whose parents declined to participate in the study, all abnormal screening test results were followed up by repeating the screening test on another dried blood paper specimen. For every infant with more than one abnormal screening test, acid-starch gel electrophoresis of serum was performed to determine the Pi phenotype. Methods. Trypsin inhibitory capacities were determined as described by Homer et a111 and by Lieberman. 1~ Routine cellulose acetate electrophoresis of serum proteins wa performed at pH 8.6 with barbital buffers. Pityping of a l - A T proteins was performed by means of acid-starch gel electrophoresis as described by Fagerhol and LaurelF using potato starch (Connaught Medical Laboratory, Toronto, Canada) and a 1-AT antiserum (Behring Diagnostic Inc., Woodbury, N. Y.). Screening test for alphal-antitrypsin deficiency. The screening test was based upon the hydrolysis of a-NbenzoylTarginine ethyl ester to ethanol by a known amount of trypsin. The ethanol was then detected by means of an enzyme-linked reduction of nicotinamide adenine dinucleotide to yield a fluorescent final product (NADH) which can be observed visually ~3 or measured quantitatively. One-eighth-inch discs were punched from the dried blood specimens and incubated for 30 minutes at room temperature in 0.10 ml of sodium phosphate buffer (0.05M, pH 7.0) containing sodium chloride (0.05M) and trypsin (100 /~g). Then 0.05 ml of a freshly prepared solution containing sodium pyrophosphate (0.075M), yeast alcohol dehydrogenase, (0.1 IU), alcohol-free NAD
10 0 7
(25 /~g) (PL-Biochem Inc., Milwaukee, Wis.), and a-Nbenzoyl-arginine ethyl ester (0.25 rag) was added; the incubation was maintained for 20 minutes at room temperature. A drop of this solution was then dried on Whatman No. 1 filter paper, and viewed under long wavelength ultraviolet light. Phenotyping by means of acid starch gel electrophoresis was performed on serum specimens obtained by venipuncture. 7 Evaluation of screening test. To evaluate the accuracy of the trypsin inhibitory activity screening method, cord blood specimens were collected with EDTA anticoagulant during routine deliveries at the Children's Hospital of Buffalo (New York). Aliquots were spotted on filter paper and the remainder of the sample was centrifuged to obtain the plasma. These plasma specimens were stored frozen, as were other plasma samples from routine blood bank donations. Comparisons of trypsin inhibitory capacities and cellulose acetate electrophoresis with the TIA screening test were made using 111 cord blood and 131 blood bank specimens. The TIA screening test using dried blood samples detected all specimens with TIA values less than 0.5 mg/ml (mean _ SEM = 0.91 _+ 0.08), as well as those with less than normal amounts of alphal-globulin protein, as determined by electrophoresis. In the course of these trials, a positive screening test was noted on one cord blood specimen in which a TIC value less than 0.40 m g / m l and a low alphal-globulin peak were observed on serum protein electrophoresis. This sample came from a child who was subsequently diagnosed at three months of age to be PiZ. The mother and father were each typical MZ heterozygotes; however, no M-type protein was detectable in the original cord blood specimen. In a study of 698 other blood bank specimens, 37 had low TIA by the screening test. These 37 plasma samples had TIC values less than 0.45 mg/ml, but none were of the PiZ phenotype, In a single-blind control study using dried blood specimens, the screening test accurately identified each of four normal (MM), two PiZ, one homozygous-variant PiS, and five of eight heterozygousdeficient PiMZ specimens randomly placed among 956 specimens from normal newborn infants. Clinical studies of anti-trypsin deficient children. Families with an infant confirmed to be PiZ were contacted and informed consent to examine the children was obtained by the author. Family pedigrees and medical histories were obtained, and physical examinations were performed at the University of Oregon Health Sciences Center. Liver function was assessed by means of biochemical tests including SGOT, SGPT, GGT, alkaline phosphatase, and serum protein electrophoresis. Individ-
10 0 8
O'Brien, Buist, and Murl)hey
The Journal of Pediatrics June 1978
Table I. Summary of screening infants for alphajantitrypsin deficiency
Total specimens tested Abnormal result Repeat filter paper on abnormals Lost to follow-up Normal Abnormal Serum samples on infants with an abnormal screening test Unobtainable PiZ phenotypes Variant phenotype
Newborn specimens
4-to6week specimens
107,083 83
80,344 87*
8 53 22
17 33 15
5 12 5
0 9 6
*Includes the 22 infants already known tO have an a b n o r m a l screening test on the newborn specimen.
uals who had an abnormality of one of these tests or who had hepatomegaly were retested. Liver biopsies were not considered justified in view of the good clinical condition of these children. Follow-up evaluations are being made at yearly intervals. RESULTS Among the 107,038 children screened with blood specimens collected during the newborn period, 75% had a second screening test performed at four to six weeks of age. Abnormal test results were obtained on 0.08 and 0.11% of the specimens, respectively. Follow-up was attempted on all infants having abnormal screening results; however, 25 had moved or could not be located, or their parents declined to participate in the study; one child had died. Repeat screening tests were performed on 144 such infants. Definitive serologic studies were carried out on the 32 children with two or more abnormal screening tests. Homozygous-deficient (PiZ) phenotypes were identified in 21 of the 32 children; the other 11 infants were heterozygous for variant genotypes (PiMZ, PiSZ, etc.). Twelve of the 21 homozygous-deficient (PiZ) infants had an abnormal test on the specimen collected during the newborn period, but 9 (43%) infants had a normal screening test at that time. The latter children were detected by abnormal results obtained on the specimens collected at four to six weeks of age (Table I). The incidence of homozygous-deficient (PiZ) infants detected in this population was 21 among 107,038, or one in 5,097 (0.02%). J Eighteen of the 21 homozygous-deficient (PiZ) in~ants were examined initially at the ages of two to 30 months;
three were lost to follow-up after having been serologically diagnosed as having PiZ phenotype. Fifteen children were asymptomatic; all of these had normal growth and development. One child had spastic diplegia probably related to prematurity. One child had a history of prolonged neonatal jaundice. One child had a history of frequent attacks of bronchitis and asthma. The other children did not have an unusual number of respiratory infections. Pulmonary function studies have not yet been assessed, but will be included in subsequent examinations. Six children had hepatic abnormalities manifested by hepatomegaly or elevations of serum enzymes (SGOT, GGT, or alkaline phosphatase). One child had hepatomegaly, but had normal biochemical values at 20 months of age; this child then had hepatomegaly and elevations of serum alkaline phosphatase and SGOT v l a u e s at 32 months of age but there was no evidence for an intercurrent hepatic infection. Four of the five children with liver function abnormalities also had mild hepatomegaly on physical examination at 16, 17, 21, and 32 months of age, respectively. One infant had neonatal hepatitis. At three days of age he had a serum total bilirubin concentration of 9 mg/dl. The jaundice cleared and he was clinically normal at three weeks of age. At two months he again became jaundiced; the serum bilirubin was 6 mg/dl, the SGOT was 70 IU, and serum GGT was 850 IU/I. There was moderate hepatomegaly and mild splenomegaly. At 21 months of age he was clinically and biochemically normal. In six pedigrees there was a family history of early onset of emphysema in one or more adults. As a result of family studies, four additional children with PiZ phenotype were identified. One had a mild e!r of SGOT and alkaline phosphatase; the others were clinically and biochemically normal. DISCUSSION Laurell and Sveger TM have used the same type of blood specimens dried on filter paper to screen over 200,000 infants in Sweden by means of an immunologic screening test based upon ~l-AT protein concentrations instead of antitrypsin activities, as used in our screening test. They found an incidence of homozygous deficiency of one in 1,714. This is greater than the one in 5,000 found in our study. The discrepancy could be due to a difference in the gene frequency between the two countries, or it could be due to false negative results in our screening test. The question of why our test might give a high incidence of false negative results remains unanswered, but substances with an anti-trypsin activity other than the ~ 1-AT protein
Volume 92 Number 6
might be present or transferred across the placenta. Although false negative results could be due to partial transfer of maternal M protein across the placenta, this does not seem likely because no M-type was detected in the cord blood specimen from the one proven PiZZ infant whose mother had the MZ phenotype. The screening provided us with an unbiased sample of PiZ infants for evaluation of the clinical effects of this genetic abnormality in infants and children. In view of the lack of therapy for the condition, we f e l t that more aggressive evaluation was unwarranted unless we could show that the known homozygotes were developing significant disease during childhood. When first reported, neonatal hepatitis associated with a l - A T deficiency was felt to carry an ominous prognosis because it was followed by progressive cirrhosis? Subsequent reports revealed that most infants with the deficiency and with clinical evidence of liver disease improved with time? 16 At the present time, all of our patients ranging from three to six years of age are asymptomatic, although some have biochemical evidence of hepatic disease. The reports of the incidence of overt neonatal hepatitis associated with phenotype PiZ are conflicting. Moroz et al TM . reported the deficiency in 29% of patients with neonatal hepatitis, whereas Odievre et aP found 4.5% of children with liver disease to be a l - A T deficient. These studies had the bias that the population studied consisted of patients with liver disease. Based upon mass screening of a newborn population, Sveger 17 found prolonged obstructive jaundice in 7% of PiZ infants; an additional 3% had subclinical jaundice, and another 6% had variable symptoms of liver disease. Approximately 50% had biochemical abnormalities indicative of liver damage..In our study, only one of 18 (5%) had neonatal hepatitis, and five of 18 (28%) had either hepatomegaly or biochemical abnormalities indicating damage to the hepatocytes or bile ducts or both. It would be interesting to follow the hepatic abnormalities by biopsy, but we feel this is not warranted in these children. Although the prognosis for children with the PiZ phenotype appears to be relatively good, the long-term outlook is not known. It seems probable that some will develop emphysema in adult life, but it is not known what factors determine which individuals will be affected. It is not known how many will have liver damage. Clearly, all children with fiver disease should be tested for a I-AT deficiency. Johnson and Alper 3 reported that in their patients presenting with liver disease, those with cO-AT deficiency had severe, often progressive cirrhosis, and two
Neonatal screening f o r alphal-antitrysin
1009
of three died within a few months. Our findings and those of Sveger1~ suggest that Johnson and Alper's patients represent one extreme end of the spectrum of hepatic disease in patients with a 1-AT deficiency. The primary reason for initiating this screening program was to determine the incidence and severity of disease in patients with this condition during childhood and to provide genetic counseling to those at risk. If the hepatic and pulmonary abnormalities had proved to be more frequent, Or more serious than has been observed, a case could be made for screening for this disorder in the newborn period. As there is currently no specific therapy, and the prognosis is uncertain, we do not recommend routine neonatal screening for ~I-AT deficiency at the present time. The technical assistance of Adam Orfanos, Elizabeth Lncowski, and the staff of the Metabolic Disorders section of the Oregon State Public Health Laboratory are greatly appreciated. REFERENCES
1. Laurell C-B, and Eriksson S: The electrophoretic ~lglobulin pattern of serum in al-antitrypsin deficiency, Scand J Clin Lab Invest 15:132, [963. 2. Sharp HL, Bridges RA, Drivit W, and Freier ER: Cirrhosis associated with alpha 1-antitrypsindeficiency: A previously unrecognized inherited disorder, J Lab Clin Med 74"934, 1969. 3. Johnson AM, and Alper CA: Deficiency and Alpha-1antitrypsin in childhood liver disease, Pediatrics 46:921, 1970. 4. BergNO, and Eriksson S: Liver disease in adults with alpha 1-antitrypsin deficiency~N Engl J Med 287:1264, 1972. 5. Babb RR, Lillington GA, and Kempon RL: Cirrhosis in an adult with emphysema and alpha 1-antitrypsin deficiency, Am J Dig Dis 18:803, 1973. 6. Palmer PE, Wolfe HJ, and Gherardi GJ: Hepatic changes in adult alpha 1-antitrypsin deficiency, Gastroenterology 65:284, 1973. 7. Fagerhol MK, and Laurell C-B: The Pi System-inherited variants of serum 1-antitrypsin, Prog Med Genet 7:96, 1970. 8. Odievre MD, Martin J-P, Hadchouel M, and Atagille D: Alpha l-antitrypsin deficiency and liver disease in children: Phenotypes, Manifestations, and Prognosis, Pediatrics 57:226, 1976. 9. Mittman C, editor: Pulmonary emphysema and proteolysis, Academic Press i87-305, 1972. 10. Lieberman J, Mittman C, and Gordon HW: Alpha 1antitrypsin in the livers of patients with emphysema, Science 175:63, 1972. 11. Homer GM, Katchman BJ, Zipf RE: A spectrophotometric method for measuring serum trypsin inhibitor capacity, Clin Chem 9:428, 1963. 12. Lieberman J: Heterozygous and homozygous alpha-1 anti-
10 10
O'Brien, Buist, and.Murphey
trypsin deficiency in patients with pulmonary emphysema, N Engl J Med 281"279, 1969. 13. Beutler E, and Baluda: A simple spot screening test for galactosemia, J Lab Clin Med 68:137, 1966. 14. Laurell C-B, and Sveger T: Mass screening of newborn Swedish infants for 1-antitrypsin deficiency, Am J Hum Genet 27:213, 1975. 15. Talamo RC, and Feingold M; Infantile cirrhosis with
The Journal of Pediatrws June 19'78
hereditary alpha l-antitrypsin deficiency, Am J Dis Child 125"845, 1973. 16. Moroz SP, Autz E, Cox DW, and Sass-Kortsak A: Liver disease associated with alpha 1-antitrypsin deficiency in childhood, J PEDIATR88:19, 1976. 17. Sveger T: Liver disease in alpha l-antitrypsin deficiency detected by screening of 200,000 infants, N Engl J Med 294:1316, 1976.
June 1978 TheJournalofPEDIATRICS
Neonatal screeningfor alphal-antitrypsin deficiency The Oregon State Pubfic Health Laboratory screened 107,038 newborn infants between 1971 and 1974 to determine th e frequency and clinical characteristics of alphal-antitrypsin deficiency. The screening program was based upon an assay of the total trypsin inhibitory activity in dried blood specimens collected on filter paper from infants during the first week of life and again from 75% of the same infants at four to six weeks of age. Twenty-one homozygous-defieient (PiZ) infants were identified, an incidence of one in 5,000. Of the 18 infants studied and followed, only one had neonatal hepatitis;five had hepatomegaly or biochemical abnormalities or both, indicating hepatic damage. Presently, the children range from three to six years of age; all are asymptomatic. Four of them have mild hepatomegaly and biochemical evidence of liver damage. As a result of family studies, four homozygous-deficient (PiZ) siblings were identified. One child had evidence of mild hepatic dysfunction, but the other three were cfinically and bioehemically normal. Nine of the 21 PiZ infants detected were missed on the initial sample, but identified on the four to six week sample. I f a screening method based upon TIA is to be utilized, these results indicate that a repeat screening specimen should be obtained at four to six weeks of age. Newborn screening for alpha~-antitrypsin deficiency is not warranted at this time in view of the low frequency of ~ignificant pulmonary or hepatic involvement in childhood and the absence of specific therapy for this condition.
Mary Lynn O'Brien, Neil R. M. Buist, and William H. Murphey,* Portland, Ore.
ALPHA~-ANTITRYPSIN deficiency was first described as a clinical entity by Laurell and Eriksson, 1 who noted its association with the onset of pulmonary emphysema in early middle life. It was subsequently discovered to be associated with liver disease in children ~' ~ and in adults. 4-~ Over 20 distinct genetic variants of a 1-AT protein are recognized; these variants are identified by electrophoresis.7 The most common (normal) homozygote is designated as PiM. The genetic variant which is most closely From the Department of Pediatrics, University of Oregon Health Sciences Center, and the Oregon State Public Health Laboratory. Supported in part by grants from the National Foundation (No. CE-14), and from the National Institutes of Health (No. HD 03967-10), and from Maternal ChiM Health (Grant No. 435). *Reprint address: Oregon State Public Health Laborator)~ P.O. Box 275, Portland, OR 9720Z
VoL 92, No. 6, pp. 1006-1010
associated with lung and liver disease is PiZ; the other variants (e.g., S,F,) and MZ heterozygotes are spared most, if not all, of the hepatic and pulmonary disease. Although a l - A T deficiency has a high frequency compared to other inherited biochemical disorders that can be detected in the newborn period, such as phenylkeAbbreviations used: Pi: proteaseinhibitor a 1,AT: alphal-antitrypsin PKU: phenylketonuria TIA Trypsin inhibitorY activity SGOT: serum glutamic oxalacetic transaminase SGPT: serum glutamic pyruvic transaminase GGT: gamma glutamyl transpeptidase trypsin inhibitory capacity TIC: NAD: nicotinamide adenine dinucleotide tonuria, galactosemia, and maple syrup urine disease, widespread screening for a l - A T deficiency has not been adopted in this country. Most studies to date have been in
0022-3476/78/0692-1006500.50/0 9 1978 The C. V. Mosby Co.
Volume 92 Number 6
Neonatal screening for alphal-antitrysin
children with liver disease, 8 of whom up to 35% may have a l - A T deficiency. In adults, studies have been on individuals in whom pulmonary disease has been identified, or on relatively small populations of hospitalized patients? lo The purposes of this study were to evaluate: (1) a new screening test for a 1-AT deficiency and (2) the clinical status of infants with PiZ variant detected by such screening. METHODS
AND MATERIALS
Mass screening of newborn infants. Between 1971 and 1974, the Oregon State Public Health Laboratory screened 107,038 infants with blood specimens routinely collected on Schleicher and Schuell No. 903 filter paper. The blood specimens were obtained by heel-prick from infants upon the day of discharge from the hospital, usually at one to three days of age; a second sample was obtained from 80,344 (75%) of these same infants at four to six weeks of age. Except for 26 infants who had moved, died, were not located, or whose parents declined to participate in the study, all abnormal screening test results were followed up by repeating the screening test on another dried blood paper specimen. For every infant with more than one abnormal screening test, acid-starch gel electrophoresis of serum was performed to determine the Pi phenotype. Methods. Trypsin inhibitory capacities were determined as described by Homer et a111 and by Lieberman. 1~ Routine cellulose acetate electrophoresis of serum proteins wa performed at pH 8.6 with barbital buffers. Pityping of a l - A T proteins was performed by means of acid-starch gel electrophoresis as described by Fagerhol and LaurelF using potato starch (Connaught Medical Laboratory, Toronto, Canada) and a 1-AT antiserum (Behring Diagnostic Inc., Woodbury, N. Y.). Screening test for alphal-antitrypsin deficiency. The screening test was based upon the hydrolysis of a-NbenzoylTarginine ethyl ester to ethanol by a known amount of trypsin. The ethanol was then detected by means of an enzyme-linked reduction of nicotinamide adenine dinucleotide to yield a fluorescent final product (NADH) which can be observed visually ~3 or measured quantitatively. One-eighth-inch discs were punched from the dried blood specimens and incubated for 30 minutes at room temperature in 0.10 ml of sodium phosphate buffer (0.05M, pH 7.0) containing sodium chloride (0.05M) and trypsin (100 /~g). Then 0.05 ml of a freshly prepared solution containing sodium pyrophosphate (0.075M), yeast alcohol dehydrogenase, (0.1 IU), alcohol-free NAD
10 0 7
(25 /~g) (PL-Biochem Inc., Milwaukee, Wis.), and a-Nbenzoyl-arginine ethyl ester (0.25 rag) was added; the incubation was maintained for 20 minutes at room temperature. A drop of this solution was then dried on Whatman No. 1 filter paper, and viewed under long wavelength ultraviolet light. Phenotyping by means of acid starch gel electrophoresis was performed on serum specimens obtained by venipuncture. 7 Evaluation of screening test. To evaluate the accuracy of the trypsin inhibitory activity screening method, cord blood specimens were collected with EDTA anticoagulant during routine deliveries at the Children's Hospital of Buffalo (New York). Aliquots were spotted on filter paper and the remainder of the sample was centrifuged to obtain the plasma. These plasma specimens were stored frozen, as were other plasma samples from routine blood bank donations. Comparisons of trypsin inhibitory capacities and cellulose acetate electrophoresis with the TIA screening test were made using 111 cord blood and 131 blood bank specimens. The TIA screening test using dried blood samples detected all specimens with TIA values less than 0.5 mg/ml (mean _ SEM = 0.91 _+ 0.08), as well as those with less than normal amounts of alphal-globulin protein, as determined by electrophoresis. In the course of these trials, a positive screening test was noted on one cord blood specimen in which a TIC value less than 0.40 m g / m l and a low alphal-globulin peak were observed on serum protein electrophoresis. This sample came from a child who was subsequently diagnosed at three months of age to be PiZ. The mother and father were each typical MZ heterozygotes; however, no M-type protein was detectable in the original cord blood specimen. In a study of 698 other blood bank specimens, 37 had low TIA by the screening test. These 37 plasma samples had TIC values less than 0.45 mg/ml, but none were of the PiZ phenotype, In a single-blind control study using dried blood specimens, the screening test accurately identified each of four normal (MM), two PiZ, one homozygous-variant PiS, and five of eight heterozygousdeficient PiMZ specimens randomly placed among 956 specimens from normal newborn infants. Clinical studies of anti-trypsin deficient children. Families with an infant confirmed to be PiZ were contacted and informed consent to examine the children was obtained by the author. Family pedigrees and medical histories were obtained, and physical examinations were performed at the University of Oregon Health Sciences Center. Liver function was assessed by means of biochemical tests including SGOT, SGPT, GGT, alkaline phosphatase, and serum protein electrophoresis. Individ-
10 0 8
O'Brien, Buist, and Murl)hey
The Journal of Pediatrics June 1978
Table I. Summary of screening infants for alphajantitrypsin deficiency
Total specimens tested Abnormal result Repeat filter paper on abnormals Lost to follow-up Normal Abnormal Serum samples on infants with an abnormal screening test Unobtainable PiZ phenotypes Variant phenotype
Newborn specimens
4-to6week specimens
107,083 83
80,344 87*
8 53 22
17 33 15
5 12 5
0 9 6
*Includes the 22 infants already known tO have an a b n o r m a l screening test on the newborn specimen.
uals who had an abnormality of one of these tests or who had hepatomegaly were retested. Liver biopsies were not considered justified in view of the good clinical condition of these children. Follow-up evaluations are being made at yearly intervals. RESULTS Among the 107,038 children screened with blood specimens collected during the newborn period, 75% had a second screening test performed at four to six weeks of age. Abnormal test results were obtained on 0.08 and 0.11% of the specimens, respectively. Follow-up was attempted on all infants having abnormal screening results; however, 25 had moved or could not be located, or their parents declined to participate in the study; one child had died. Repeat screening tests were performed on 144 such infants. Definitive serologic studies were carried out on the 32 children with two or more abnormal screening tests. Homozygous-deficient (PiZ) phenotypes were identified in 21 of the 32 children; the other 11 infants were heterozygous for variant genotypes (PiMZ, PiSZ, etc.). Twelve of the 21 homozygous-deficient (PiZ) infants had an abnormal test on the specimen collected during the newborn period, but 9 (43%) infants had a normal screening test at that time. The latter children were detected by abnormal results obtained on the specimens collected at four to six weeks of age (Table I). The incidence of homozygous-deficient (PiZ) infants detected in this population was 21 among 107,038, or one in 5,097 (0.02%). J Eighteen of the 21 homozygous-deficient (PiZ) in~ants were examined initially at the ages of two to 30 months;
three were lost to follow-up after having been serologically diagnosed as having PiZ phenotype. Fifteen children were asymptomatic; all of these had normal growth and development. One child had spastic diplegia probably related to prematurity. One child had a history of prolonged neonatal jaundice. One child had a history of frequent attacks of bronchitis and asthma. The other children did not have an unusual number of respiratory infections. Pulmonary function studies have not yet been assessed, but will be included in subsequent examinations. Six children had hepatic abnormalities manifested by hepatomegaly or elevations of serum enzymes (SGOT, GGT, or alkaline phosphatase). One child had hepatomegaly, but had normal biochemical values at 20 months of age; this child then had hepatomegaly and elevations of serum alkaline phosphatase and SGOT v l a u e s at 32 months of age but there was no evidence for an intercurrent hepatic infection. Four of the five children with liver function abnormalities also had mild hepatomegaly on physical examination at 16, 17, 21, and 32 months of age, respectively. One infant had neonatal hepatitis. At three days of age he had a serum total bilirubin concentration of 9 mg/dl. The jaundice cleared and he was clinically normal at three weeks of age. At two months he again became jaundiced; the serum bilirubin was 6 mg/dl, the SGOT was 70 IU, and serum GGT was 850 IU/I. There was moderate hepatomegaly and mild splenomegaly. At 21 months of age he was clinically and biochemically normal. In six pedigrees there was a family history of early onset of emphysema in one or more adults. As a result of family studies, four additional children with PiZ phenotype were identified. One had a mild e!r of SGOT and alkaline phosphatase; the others were clinically and biochemically normal. DISCUSSION Laurell and Sveger TM have used the same type of blood specimens dried on filter paper to screen over 200,000 infants in Sweden by means of an immunologic screening test based upon ~l-AT protein concentrations instead of antitrypsin activities, as used in our screening test. They found an incidence of homozygous deficiency of one in 1,714. This is greater than the one in 5,000 found in our study. The discrepancy could be due to a difference in the gene frequency between the two countries, or it could be due to false negative results in our screening test. The question of why our test might give a high incidence of false negative results remains unanswered, but substances with an anti-trypsin activity other than the ~ 1-AT protein
Volume 92 Number 6
might be present or transferred across the placenta. Although false negative results could be due to partial transfer of maternal M protein across the placenta, this does not seem likely because no M-type was detected in the cord blood specimen from the one proven PiZZ infant whose mother had the MZ phenotype. The screening provided us with an unbiased sample of PiZ infants for evaluation of the clinical effects of this genetic abnormality in infants and children. In view of the lack of therapy for the condition, we f e l t that more aggressive evaluation was unwarranted unless we could show that the known homozygotes were developing significant disease during childhood. When first reported, neonatal hepatitis associated with a l - A T deficiency was felt to carry an ominous prognosis because it was followed by progressive cirrhosis? Subsequent reports revealed that most infants with the deficiency and with clinical evidence of liver disease improved with time? 16 At the present time, all of our patients ranging from three to six years of age are asymptomatic, although some have biochemical evidence of hepatic disease. The reports of the incidence of overt neonatal hepatitis associated with phenotype PiZ are conflicting. Moroz et al TM . reported the deficiency in 29% of patients with neonatal hepatitis, whereas Odievre et aP found 4.5% of children with liver disease to be a l - A T deficient. These studies had the bias that the population studied consisted of patients with liver disease. Based upon mass screening of a newborn population, Sveger 17 found prolonged obstructive jaundice in 7% of PiZ infants; an additional 3% had subclinical jaundice, and another 6% had variable symptoms of liver disease. Approximately 50% had biochemical abnormalities indicative of liver damage..In our study, only one of 18 (5%) had neonatal hepatitis, and five of 18 (28%) had either hepatomegaly or biochemical abnormalities indicating damage to the hepatocytes or bile ducts or both. It would be interesting to follow the hepatic abnormalities by biopsy, but we feel this is not warranted in these children. Although the prognosis for children with the PiZ phenotype appears to be relatively good, the long-term outlook is not known. It seems probable that some will develop emphysema in adult life, but it is not known what factors determine which individuals will be affected. It is not known how many will have liver damage. Clearly, all children with fiver disease should be tested for a I-AT deficiency. Johnson and Alper 3 reported that in their patients presenting with liver disease, those with cO-AT deficiency had severe, often progressive cirrhosis, and two
Neonatal screening f o r alphal-antitrysin
1009
of three died within a few months. Our findings and those of Sveger1~ suggest that Johnson and Alper's patients represent one extreme end of the spectrum of hepatic disease in patients with a 1-AT deficiency. The primary reason for initiating this screening program was to determine the incidence and severity of disease in patients with this condition during childhood and to provide genetic counseling to those at risk. If the hepatic and pulmonary abnormalities had proved to be more frequent, Or more serious than has been observed, a case could be made for screening for this disorder in the newborn period. As there is currently no specific therapy, and the prognosis is uncertain, we do not recommend routine neonatal screening for ~I-AT deficiency at the present time. The technical assistance of Adam Orfanos, Elizabeth Lncowski, and the staff of the Metabolic Disorders section of the Oregon State Public Health Laboratory are greatly appreciated. REFERENCES
1. Laurell C-B, and Eriksson S: The electrophoretic ~lglobulin pattern of serum in al-antitrypsin deficiency, Scand J Clin Lab Invest 15:132, [963. 2. Sharp HL, Bridges RA, Drivit W, and Freier ER: Cirrhosis associated with alpha 1-antitrypsindeficiency: A previously unrecognized inherited disorder, J Lab Clin Med 74"934, 1969. 3. Johnson AM, and Alper CA: Deficiency and Alpha-1antitrypsin in childhood liver disease, Pediatrics 46:921, 1970. 4. BergNO, and Eriksson S: Liver disease in adults with alpha 1-antitrypsin deficiency~N Engl J Med 287:1264, 1972. 5. Babb RR, Lillington GA, and Kempon RL: Cirrhosis in an adult with emphysema and alpha 1-antitrypsin deficiency, Am J Dig Dis 18:803, 1973. 6. Palmer PE, Wolfe HJ, and Gherardi GJ: Hepatic changes in adult alpha 1-antitrypsin deficiency, Gastroenterology 65:284, 1973. 7. Fagerhol MK, and Laurell C-B: The Pi System-inherited variants of serum 1-antitrypsin, Prog Med Genet 7:96, 1970. 8. Odievre MD, Martin J-P, Hadchouel M, and Atagille D: Alpha l-antitrypsin deficiency and liver disease in children: Phenotypes, Manifestations, and Prognosis, Pediatrics 57:226, 1976. 9. Mittman C, editor: Pulmonary emphysema and proteolysis, Academic Press i87-305, 1972. 10. Lieberman J, Mittman C, and Gordon HW: Alpha 1antitrypsin in the livers of patients with emphysema, Science 175:63, 1972. 11. Homer GM, Katchman BJ, Zipf RE: A spectrophotometric method for measuring serum trypsin inhibitor capacity, Clin Chem 9:428, 1963. 12. Lieberman J: Heterozygous and homozygous alpha-1 anti-
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trypsin deficiency in patients with pulmonary emphysema, N Engl J Med 281"279, 1969. 13. Beutler E, and Baluda: A simple spot screening test for galactosemia, J Lab Clin Med 68:137, 1966. 14. Laurell C-B, and Sveger T: Mass screening of newborn Swedish infants for 1-antitrypsin deficiency, Am J Hum Genet 27:213, 1975. 15. Talamo RC, and Feingold M; Infantile cirrhosis with
The Journal of Pediatrws June 19'78
hereditary alpha l-antitrypsin deficiency, Am J Dis Child 125"845, 1973. 16. Moroz SP, Autz E, Cox DW, and Sass-Kortsak A: Liver disease associated with alpha 1-antitrypsin deficiency in childhood, J PEDIATR88:19, 1976. 17. Sveger T: Liver disease in alpha l-antitrypsin deficiency detected by screening of 200,000 infants, N Engl J Med 294:1316, 1976.