Scott Mandel

Follow-up of newborns with low thyroxine and nonelevated thyroid-stimulating hormone–screening concentrations: Results of the 20-year experience in the Northwest Regional Newborn Screening Program

OBJECTIVES To determine the type and frequency of thyroid disorders detected in infants with low thyroxine (T4) and nonelevated thyroid-stimulating hormone (TSH) screening test results in the Northwest Regional Newborn Screening Program (NWRNSP) over the 20-year period from May 1975 to May 1995 and to determine the effect of follow-up of these infants on the overall recall rate. STUDY DESIGN The NWRNSP requests a serum specimen in infants with an absolute T4 level < 38.6 nmol/L (< 3 mg/dl) and in infants with two filter paper T4 concentrations less than the 3%, regardless of the TSH concentration. We conducted a retrospective analysis of infants who were followed up because of low T4 and nonelevated TSH concentrations on newborn screening. To determine the effect of follow-up of infants with low T4 levels, nonelevated TSH concentrations on the recall rate, we selected 1 year (1994) for review. Serum sample requests were evaluated to determine the reason for the request. RESULTS Over this 20-year period, the NWRNSP detected 450 infants with primary hypothyroidism among 1,747,805 infants screened (1:3,884). Of these, 416 were detected on the basis of low T4 levels and nonelevated TSH screening test results, whereas an additional 34 infants with primary hypothyroidism and 29 infants with hypopituitary hypothyroidism were detected as a result of follow-up of low T4 levels and nonelevated TSH screening test results. This included 25 infants with delayed TSH rise (1:67,226), 9 infants with mild hypothyroidism (TSH levels < 25 mU/L) (1:194,212), 29 infants with hypopituitary hypothyroidism (1:60,269), and 434 infants with T4-binding globulin deficiency (1:4,027). Excluding those with T4-binding globulin deficiency, the false-positive rate was 43.5:1. This compares with an overall false-positive rate of 12:1 for our screening program. CONCLUSION Follow-up of infants with low T4 and nonelevated TSH concentration on screening led to the detection of 63 additional infants with hypothyroidism, for an overall frequency of 1:27,743. We believe this yield justifies continued follow-up of infants with low T4 levels, nonelevated (TSH) screening test results in our program.

Thyroxine-binding globulin deficiencydetected by newborn screening

We examined the results of the Northwest Regional Screening Program from May 1975 to June 1991 to determine the prevalence of inherited thyroxine-binding globulin (TBG) deficiency and its effect on thyroid hormone concentrations in infants. Serum thyroxine (T4), triiodothyronine resin uptake (T3RU), and thyrotropin values were requested of physicians caring for all infants with a single filter paper T4 level < 38.6 nmol/L (3 micrograms/dl) or a T4 level < 3rd percentile on two filter paper tests (at birth and 2 to 6 weeks of age). From 1,367,724 infants screened in five states, TBG deficiency, an X-linked disorder, was identified in 317 infants (285 boys). For the entire screening program the calculated frequency of TBG deficiency was 1:4315 infants (1:2400 for boys). In Oregon, where 95% of infants have two screening tests performed, the calculated frequency was somewhat higher (1:3080 infants; 1712 boys) and is probably more accurate. The mean serum T4 concentration for TBG-deficient boys was 41.9 nmol/L (3.26 micrograms/dl); 31% had values < 25.7 nmol/L (2.0 micrograms/dl). The mean serum T4 concentration for TBG-deficient girls was 60.2 nmol/L (4.68 micrograms/dl), with none < 2.0 micrograms/dl. The mean T3RU value was 0.472 in TBG-deficient boys, and 0.412 in TBG-deficient girls; the T3RU value was > 0.55 in 24% of TBG-deficient boys but was > 0.55 in only one girl. Free serum T4 levels were normal in all 56 TBG-deficient infants studied, and TBG levels were low in all 20 infants studied. Inherited TBG deficiency is common in boys in the Northwest, with a frequency of 1:1700 and a male/female ratio of 8.9:1. Boys with TBG deficiency have mild, moderate, or severe alterations in total T4 and T3RU values, but severe deficiency is rare in girls.

Massive Levothyroxine Ingestion Conservative Management

The clinical course of a 29-month-old girl who was referred for evaluation after ingesting ninety 0.2-mg tablets of levothyroxine is reported. Despite an initial thyroxine (T4) level of 282 μg/dl and a triiodothyronine (T3) level of 1,837 ng/dl at 48 hours postingestion, her symptoms were mild and included irritability, vomiting, tremor, and tachycardia. Treatment was limited to activated charcoal and propranolol. Thyroid hormone levels fell to normal by 13 days postingestion. The childs clinical course was benign. Even after massive acute ingestions of levothyroxine, childrens symptoms are usually mild and may be controlled with propranolol. This conservative approach should be considered before expensive and potentially dangerous therapies are undertaken.

The effect of GH therapy on the immunoreactive forms and distribution of IGFBP-3, IGF-I, the acid-labile subunit, and growth rate in GH-deficient children

We have previously shown that the major correlates of growth following growth hormone (GH) therapy in growth hormone-deficient (GHD) children are changes in circulating insulin-like growth factor-I (IGF-I) and IGF binding protein-3 (IGFBP-3), suggesting a synergistic interaction between IGF-I and IGFBP-3 (1). The first aim of this project was to examine the molecular forms of IGFBP-3 and the acid-labile subunit (ALS), and to assess the changes in these molecular forms during GH administration to GHD children. Plasma samples from prepubertal GHD patients, prior to therapy and during the first year of GH treatment, were subjected to Western ligand and immunoblot analysis. Densitometric analysis of Western ligand blotting (WLB) showed a 76% increase in IGFBP-3 (p=0.02), but a 56% decrease in 36-kDa IGFBP-2 (p=0.03) during GH therapy. Western immunoblot (WIB) analysis of IGFBP-3 revealed the presence of intact (40- to 45-kDa doublet) as well as a proteolyzed (28-kDa) form of IGFBP-3 in the serum of GHD and healthy children. Both immunoreactive forms of IGFBP-3 increased by 64% during GH therapy (intactp=0.003; proteolyzedp=0.0001). WIB analysis of the ALS showed an 84-to 86-kDa doublet, which increased by 41% with GH therapy (p=0.01). The response to GH therapy, as measured by the height velocity standard deviation score (SDS) adjusted for bone age, correlated with the percent change in total IGFBP-3 (r=0.772,p=0.002 by WIB), intact IGFBP-3 (r=0.845,p=0.0005 by WLB;r=0.541,p-0.05 by WIB), and proteolyzed IGFBP-3 (r=0.703,p=0.007), as well as with the percent change in ALS (r=0.813,p=0.014).The second aim of this project was to assess the changes in distribution of the immunoreactive forms of IGFBP-3 and IGF-I among the ternary (ALS/IGFBP-3/IGF) complex, the binary (IGFBP-3/IGF) complex, and uncomplexed IGF during the first year of GH therapy, and to explore further the correlation with growth response to GH. Plasma samples, prior to therapy and after the first year of GH treatment, were separated by neutral size-exclusion chromatography and then subjected to IGFBP-3 immunoradiometric assay (IRMA), IGFBP-3 WIB, and IGF-I IRMA analysis. IGFBP-3 increased in both the ternary (p<0.0001) and binary (p=0.01) complexes, but there was a shift in the percentage of IGFBP-3 from the binary to the ternary complex during GH therapy. Both intact and proteolyzed forms of IGFBP-3 were found in both the ternary and binary complexes, but the shift occurred primarily for the proteolyzed (28-kDa) form (p=0.001). There was a significant increase in IGF-I in the ternary (p=0.001) and binary (p=0.005) complexes, but not in uncomplexed IGF-I. The percentage of IGF-I in the ternary complex increased (p=0.006), whereas the percentage of uncomplexed IGF-I decreased (p=0.02), during GH therapy. Growth rate, assessed by the height velocity SDS for bone age, correlated best with the changes in ternary complex IGFBP-3 (r=0.72,p=0.01) and ternary complex IGF-I (r=0.56,p=0.10).In conclusion, GH treatment of GHD children results in significant increases of intact, proteolyzed, and total IGFBP-3, as well as an increase in ALS, which all correlate with the growth response to GH therapy. In addition, GH treatment results in increases in ternary complex IGFBP-3 and IGF-I, which also correlate with the response to therapy. We suggest that the formation of the ternary complex may be a determining factor in the somatic growth response.

Diminished thyroid-stimulating hormone secretion associated with neonatal thyrotoxicosis

lobacter may stimulate their production/ Alternatively, Campylobacter antibodies that penetrate the C S F may be responsible for neural damage. Kaldor and Speed have shown that patients with Guillain-Barr6 syndrome associated with Campylobacter gastroenteritis are more likely to have elevated C S F IgG titers than are similar patients with no history of Campylobacter infection. Another possibility is that the choleralike enterotoxin produced by C. jejum ~ may bind to neural tissue in a similar way to cholera toxin, causing neural damage. I~ Erythromycin therapy was not used initially because the child had made an uneventful recovery from her original illness. It was introduced later in her illness when she developed a febrile illness with exacerbation of diarrhea and was known to be still excreting Campylobacter. There is no evidence to suggest that erradication of the organism limits or prevents the neurologic syndrome2.5 We believe pediatricians should be more aware of the Campylobacter-Guillain-Barr6 syndrome association and that in patients with Guillain-Barr6 syndrome, particularly when associated with a gastrointestinal tract illness, C. jejuni should be sought as a possible etiologic agent.

Neonatal hypopituitary hypothyroidism associated with maternal thyrotoxicosis

Hypopituitary hypothyroidism was found in an infant born to a mother with inadequately treated thyrotoxicosis. The infant had low thyroxine (T4), low free T4, and diminished thyroid-stimulating hormone (TSH) levels with a blunted response to thyrotropinreleasing hormone (TRH) stimulation until 6 weeks of age when thyroid hormone replacement was started. Excess fetal thyroid hormone may have caused an alteration in TSH secretory dynamics leading to the development of neonatal hypopituitary hypothyroidism.

Preoperative diagnostic evaluation of children with Cushing's syndrome

Recent advances in biochemical and imaging studies have improved the diagnostic accuracy of Cushings syndrome. To better define roles for these studies in children, the authors reviewed their experience with this rare group of patients. Fifteen children, aged 11 weeks to 17 years, were treated for noniatrogenic Cushings syndrome over a 33-year period. All children presented with signs of hypercortisolism. Nineteen different diagnostic tests were used, reflecting changes in how these patients are evaluated. Pathological diagnoses were adrenal cortical carcinoma (3), primary adrenocortical nodular dysplasia (PAND) (2), and pituitary adenoma (10). Children with adrenal cortical carcinoma presented with an adrenal mass and at a younger age (mean, 22.3 months). Key diagnostic features of patients with PAND were a low plasma adrenocorticotrophin hormone (ACTH) and no suppression with high-dose dexamethasone. Children with a pituitary cause of Cushings syndrome presented at an older age (mean, 15.7 years) and were diagnosed using a combination of high-dose dexamethasone testing, simultaneous inferior petrosal sinus sampling, and/or ovine corticotrophin-releasing hormone stimulation test. A strategy for the diagnosis of Cushings syndrome in children is presented.

Collection of blood in heparinized tubes does not alter the molecular distribution or forms of IGFBP-3 and IGF.

The major serum carrier of the insulin-like growth factors (IGFs) is IGF-binding protein-3 (IGFBP-3) that exists in the circulation associated with IGF and an acid labile subunit to form a ternary (158-kDa) complex. It has been reported that heparin disrupts the IGF carrying capacity of the ternary complex and is a potent inhibitor of ternary complex reformation (Clemmons et al., 1983; Baxter, 1990). Thus, the aim of this study was to determine if, in a clinical setting where blood may be collected in both nonheparinized and heparinized tubes, heparin alters the molecular distribution or immunoreactive measurement of IGFBP-3 and IGF-I. Two different collection modalities were examined: protocol 1, blood was drawn and immediately centrifuged and aliquotted; and protocol 2, blood was drawn, left at room temperature for 2 h and then at 4°C overnight prior to centrifugation. Samples were drawn from a normal adult and from a growth hormone-deficient (GHD) child and subjected to neutral size-exclusion chromatography to separate the ternary 158-kDa complex from the binary IGFBP-3-IGF (approx 50 kDa) complex. Fractions were then subjected to Western ligand blot (WLB), western immunoblot (WIB), and measurement of IGFBP-3 by immunoradiometric assay (IRMA), while the IGF distribution was measured by radioimmunoassay (RIA) following acidic size-exclusion chromatography. In both serum and plasma of a normal adult, WLB detected a 45–40-kDa IGFBP-3 doublet eluting primarily within the 158-kDa IGFBP region (i.e., ternary complex). Similarly, assessment of immunoreactive IGFBP-3 by WIB showed a 45–40-kDa IGFBP-3 doublet, as well as a 29 kDa immunoreactive form primarily eluting in the 158-kDa IGFBP region of the chromatography. Measurement of IGFBP-3 by IRMA confirmed these findings. No difference between serum and plasma was detected in either collection protocol. RIA of IGF-I revealed that the ternary complex carried the majority of the circulating IGF-I and that there was no difference between serum and plasma. Assessment of serum and plasma of a GHD child showed reduced serum concentrations of IGFBP-3 but no difference in the IGFBP profiles between serum and plasma. These data demonstrate that the collection of blood in heparinized tubes does not alter the molecular distribution or forms of IGFBP-3 and IGF-I.

ACTIVATING MUTATION IN THE STIMULATORY G PROTEIN GENE IN AN INFANT WITH ADRFNOCORTICONODULAR DYSPLASIA

Infantile Cushings Syndrome is a rare condition caused by excess cortisol production. We describe an activating mutation in the gene coding for the stimulatory G protein (Gsalpha) in DNA from adrenal tissue in an infant with ACTH independent Cushings Syndrome. Activating mutations in exons 8 and 9 of the Gsalpha gene have been associated with growth hormone secreting pituitary tumors and McCune-Albright syndrome. Specifically, point mutations coding for an arginine to cysteine or an arginine to histidine substitution at site 201 in exon 8 have been described.The patient in this study was Cushingoid in appearance with poor linear growth by 3 months of age. Evaluation revealed elevated serum cortisol despite low and high dose dexamethasone suppression and undetectable baseline ACTH levels (<3 pg/ml). He underwent bilateral adrenalectomy with subsequent steroid replacement. Adrenal histology revealed bilateral adrenalcortical dysplasia with nodular elements.Genomic DNA was extracted from adrenal tissue and amplified by PCR with primers for Gsalpha exon 8. Using allele specific oligonucleotide hybridization, the amplified DNA was probed for the presence of point mutations which correspond to the known mutations in exon 8. An oligonucleotide probe corresponding to the cysteine substitution at site 201 hybridized with amplified DNA from the patients adrenal tissue. A “wild type” probe coding for arginine bound to both leukocyte DNA from a normal human control and patient DNA while the probe for the arginine to hisiidine mutation bound to neither patient nor control DNA.We conclude that the arginine to cysteine activating mutation in exon 8 of Gsalpha is present in the adrenal tissue of ihis infant with Cushings Syndrome. This mutation may be involved in the pathogenesis of adrenocorticonodular dysplasia in some patients with infantile Cushings Syndrome.

SCREENING FOR CONGENITAL HYPOTHYROIDISM: IS FOLLOW-UP OF NEWBORNS WITH LOW T4 AND “NON-ELEVATED” TSH CONCENTRATIONS INDICATED? 525

Most newborn screening programs using a primary T4 screen, with TSH measurement in specimens below a T4 cutoff, choose not to follow-up infants with low T4 and “non-elevated” (typically <25) TSH concentrations. The Northwest Regional Screening Program (NWRSP), which obtains two routine filter paper (FP) specimens in infants born in Oregon and in most infants in Idaho, Alaska, and Nevada, requests a serum specimen in infants with two FP T4 concentrations <3% and in infants with absolute T4< 3ug/dl even when the FP TSH is “not elevated”. To determine the type and frequency of thyroid disorders detected in infants with low T4 and “non-elevated” TSH concentrations, we examined the results of the NWRSP from May 1975 to May 1995. Over this 20 year period, the NWRSP screened 1,747,805 infants. Follow-up of infants with low T4s and“non-elevated” TSHs on screening detected 25 infants with delayed TSH rise (1:67,226), 9 infants with mild hypothyroidism (TSH<25)(1:194,212), 26 infants with hypopituitary hypothyroidism (1:67,223), and 434 infants with TBG deficiency (1:4027). Mean thyroid values were as follows:Table In 1994, this approach led to a recall of 116 infants, including 29 with TBG deficiency and 2 with hypopituitary hypothyroidism. Excluding those with TBG deficiency, the false positive rate was 1:43.5. Newborns who were premature or ill were found with disproportionate frequency among infants with these laboratory values. Thus, the majority of these infants are normal or have TBG deficiency; therefore the psychological harm in creating the “vulnerable” child vs. identifying the rare child with delayed TSH rise, mild hypothyroidism, or hypopituitary hypothyroidism (overall detection rate 1:29, 130) must be evaluated. The NWRSP continues to follow-up infants with low T4 and“non-elevated” TSH levels.