ABSTRACT
The pathogenesis of very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is highly heterogeneous and still unclear. Additional novel variants have been recently detected in the population. The molecular and cellular effects of these previously unreported variants are still poorly understood and require further characterization. To address this problem, we have evaluated the various functions and biochemical consequences of six novel missense variants that lead to mild VLCAD deficiency. Marked deficiencies in fatty acid oxidation (FAO) and other mitochondrial defects were observed in cells carrying one of these six variants (c.541C>T, c.863T>G, c.895A>G, c.1238T>C, c.1276G>A, and c.1505T>A), including reductions in mitochondrial respiratory-chain function and adenosine triphosphate (ATP) production, and increased levels of mitochondrial reactive oxygen species (ROS). Intriguingly, higher apoptosis levels were found in cells carrying the mutant VLCAD under glucose-limited stress. Moreover, the stability of the mutant homodimer was disturbed, and major conformational changes in each mutant VLCAD structure were predicted by molecular dynamics (MD) simulation. The data presented here may provide valuable information for improving management of diagnosis and treatment of VLCAD deficiency and for a better understanding of the general molecular bases of disease variability.
ABSTRACT
OBJECTIVE@#To investigate the epidemiological characteristics, phenotype, genotype, and prognosis of medium-chain acyl-CoA dehydrogenase deficiency (MCADD) in the Chinese population.@*METHODS@#A retrospective analysis was performed for the clinical data of the neonates who underwent screening with high-performance liquid chromatography-tandem mass spectrometry from January 2009 to June 2018 and were diagnosed with MCADD by gene detection.@*RESULTS@#A total of 2 674 835 neonates underwent neonatal screening, among whom 12 were diagnosed with MCADD. Gene detection was performed for 10 neonates with MCADD and found 13 mutation types at 16 mutation sites of the ACADM gene, among which there were 7 reported mutations (p.T150Rfs*4, p.M1V, p.R206C, p.R294T, p.G310R, p.M328V, and p.G362E), 5 novel mutations (p.N194D, p.A324P, p.N366S, c.118+3A>G, and c.387+1del G), and 1 exon 11 deletion; p.T150Rfs*4 was the most common mutation (4/16). The detection rate of mutation sites in the ACADM gene was 80%. No phenotype-genotype correlation was observed. Dietary guidance and symptomatic treatment were given after confirmed diagnosis. No acute metabolic imbalance was observed within 4-82 months of follow-up. All neonates had good prognosis except one who had brain dysplasia.@*CONCLUSIONS@#MCADD is relatively rare in southern China, and p.T150Rfs*4 is a common mutation in the Chinese population. Cases with positive screening results should be evaluated by octanoylcarnitine C8 value and gene detection.
Subject(s)
Humans , Infant, Newborn , Acyl-CoA Dehydrogenase , Carnitine , China , Follow-Up Studies , Lipid Metabolism, Inborn Errors , Mutation , Neonatal Screening , Retrospective StudiesABSTRACT
Primary carnitine deficiency is an autosomal recessive hereditary disease caused by the mutation of SLC22 A5 gene,which leads to increased carnitine excretion in urine and low level of carnitine in blood,tissues and cells. Due to the heterogeneity and non-specificity of the clinical manifestations of PCD,it is easy to be misdiagnosed or missed and it is potentially fatal without timely treatment. This disease can be detected early through the newborn screening. Maternal carnitine deficiency and the secondary carnitine deficiency caused by other diseases should be excluded. Genetic test can give a clear diagnosis. Avoiding hunger and use of oral L-carnitine supplementation to maintain normal plasma carnitine concentrations are effective treatments.
ABSTRACT
<p><b>BACKGROUND</b>It has been 11 years since newborn screening started in Zhejiang in 1999. The aim of this study was to analyze and summarize the status of newborn screening in Zhejiang from 1999 to 2009.</p><p><b>METHODS</b>Blood samples were collected from the heels of newborns 72 hours after birth. We have conducted laboratory tests that the congenital hypothyroidism (CH) and circulating levels of thyroid-stimulating hormone (TSH) was detected. Blood phenylalanine (Phe) was detected for phenylketonuria (PKU). Dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) was used for detection.</p><p><b>RESULTS</b>From 1999 to 2009, 3 875 228 newborns were screened and 2309 cases were confirmed as CH and 155 cases were confirmed as PKU. The incidence of CH and PKU were 1:1678 and 1:25 001 respectively.</p><p><b>CONCLUSION</b>In 11 years, the Zhejiang newborn screening center screened more than 3.8 million newborns, and helped more than 2000 CH and PKU patients to obtain early treatment in order to prevent physical disability and mental retardation.</p>
Subject(s)
Humans , Infant, Newborn , China , Congenital Hypothyroidism , Diagnosis , Neonatal Screening , Methods , Phenylketonurias , DiagnosisABSTRACT
<p><b>OBJECTIVE</b>To determine the impact of expanded newborn screening using tandem mass spectrometry (MS/MS) on the overall detection rate of inborn errors of metabolism in Zhejiang province and to assess the outcome of the patients who were diagnosed.</p><p><b>METHOD</b>Blood spots were collected between days 3 and 6 of life from the newborns. All samples were subjected to MS/MS analysis using Waters Quattro API. Confirmation tests included amino acid analysis, urinary organic acids by GC-MS, routine blood analysis, biochemistry, blood gas analysis, blood glucose and ammonia tests, blood homocysteine, lactate and pyruvate tests, urine acetone tests, biotin and biotin enzyme profile and DNA analysis. Standard treatment protocol was given to the patients. Protein restricted diet, special powdered formula and medicines recommended for the patients with amino acidemias. Protein restricted diet and L-carnitine, folic acid and Vitamin B12 supplementation were given for the patients with organic acidemia. L-carnitine was given to the patients with primary carnitine deficiency. The overall epidemiology, prognosis, follow-up of the screening program were also investigated in the neonates.</p><p><b>RESULT</b>A total of 129 415 neonates were investigated for 26 inborn errors of metabolism during the period. Twenty-three newborns were confirmed as having inborn errors of metabolism, including 13 with amino acidemias, 6 with organic acidemias and 4 with fatty acid oxidation disorders. The prevalence was 1:5626. Positive predictive value was 2.10%, specificity was 99.72% and sensitivity 100%. Seventeen children remain asymptomatic during the follow-up. Five patients had motor and mental developmental delay. One patient presented metabolic disorders during the follow-up. No death occurred in this series of patients.</p><p><b>CONCLUSION</b>This strategy represents a valuable preventive medicine approach by enabling diagnosis and treatment before the onset of symptoms.</p>
Subject(s)
Female , Humans , Infant, Newborn , Male , Follow-Up Studies , Metabolism, Inborn Errors , Diagnosis , Metabolism , Neonatal Screening , Methods , Spectrometry, Mass, Electrospray Ionization , Tandem Mass SpectrometryABSTRACT
<p><b>OBJECTIVE</b>To study the under-five mortality rate and the causes of death in Zhejiang Province between 2000 and 2009 in order to provide a basis for reducing the mortality rate in the region.</p><p><b>METHODS</b>By stratified random cluster sampling, all the children under 5 years old from 30 sampling areas of Zhejiang Province between 2000 and 2009 were enrolled. The under-five mortality rate and the leading causes of death were investigated by descriptive analysis and Chi-square test.</p><p><b>RESULTS</b>The under-five mortality rate demonstrated a decreased trend in Zhejiang Province between 2000 and 2009, with the under-five mortality rate of 14.83‰ in 2000 compared to 9.49‰ in 2009. In 2009, the under-five mortality rate in rural regions was significantly higher than that in urban regions (9.14‰ vs 6.50‰; P<0.01). Compared with the resident population, there were an increased under-five mortality rate in floating population (12.12‰ vs 6.42‰; P<0.01). Preterm delivery/low birth weight was the top death cause in children under 5 years old. The top three causes of death in infants less than 1 year old were preterm delivery/low birth weight, congenital heart disease and birth asphyxia compared to drowning, traffic accident and falling in children aged 1-4 years.</p><p><b>CONCLUSIONS</b>There are differences in the under-five mortality rate between rural and urban children as well as between the floating and resident population in 2009. The leading causes of death in different age groups are different. It is essential to reduce the mortality rate by preventing preterm delivery, low birth weight and congenital malformations to infants and preventing accidental injuries to children aged 1-4 years.</p>
Subject(s)
Child, Preschool , Humans , Infant , Infant, Newborn , Cause of Death , Child Mortality , China , Epidemiology , Infant Mortality , Time FactorsABSTRACT
<p><b>OBJECTIVE</b>To study the best observation time for drug administration and withdrawal in the treatment of children with transient congenital hypothyroidism,seeking an objective basis for the safe drug withdrawal.</p><p><b>METHODS</b>Levothyroxine was prescribed for 1 144 children diagnosed with congenital hypothyroidism (CH) and according to the results levothyroxine was adjusted to a maintenance dosage. Examinations were performed periodically including physical and mental development, thyroid ultrasonography,and blood levels of T3, T4, TSH. For the patients with a small maintenance dosage of levothyroxine (15.0 - 16.6 g/d) and all the examinations normal, levothyroxine was withdrawn at 2 - 3 years, and the children were followed up and reexamined after 1 month, 2 months, and 10 months, respectively. Permanent drug withdrawal was determined for children with all the examinations normal. Once abnormal TSH occurred, levothyroxine was prescribed again, and followed up continuously.</p><p><b>RESULT</b>Levothyroxine was withdrawn from 157 children. During the follow up, for 15 children (9.55%) levothyroxine were prescribed continuously, and for 142 children permanent drug withdrawal (confirmed with transient CH) was determined. Abnormal TSH of various degrees was detected in 48 cases: 25.48 % (40/157),4.46 % (7/157), and 0.64 % (1/157) were detected at 1, 2 and 10 months after drug withdrawal, respectively. In 15 children levothyroxine was prescribed again for the remarkably high TSH, and the other 33 with mildly abnormal TSH finished the treatment since TSH normalized during follow-up.</p><p><b>CONCLUSION</b>After 2 - 3 years of regular treatment, levothyroxine can be withdrawn from children with normal T3, T4, TSH, physical and mental development, and thyroid function. The best observation time for drug withdrawal should be 2 - 3 months. If T3, T4 and TSH levels are in the normal range, drug can be withdrawn safely. Once abnormal results were detected during follow-up, levothyroxine should be administrated continuously.</p>
Subject(s)
Child , Child, Preschool , Female , Humans , Infant , Male , Congenital Hypothyroidism , Blood , Drug Therapy , Drug Administration Schedule , Follow-Up Studies , Thyrotropin , Blood , Thyroxine , Blood , Therapeutic Uses , Time Factors , Triiodothyronine , Blood , Withholding TreatmentABSTRACT
<p><b>OBJECTIVE</b>To investigate the correlation of congenital hypothyroidism (CH) with birth weight and gestational age in newborn infants.</p><p><b>METHODS</b>The screening of CH was conducted in all the live births in 2005 of Zhejiang Province, the blood samples were collected from heel stick. The thyroid stimulating hormone (TSH) was measured by time-resolved fluorescence immunoassay (TRFIA). If TSH was>9.0 mU/L, the infant was recalled for further examination.</p><p><b>RESULTS</b>A total of 387,926 infants were screened in 2005, of whom 289 cases were identified with CH. The prevalence rate was 1/1,342. Among the 289 CH cases, the prevalence of premature infants, term infants and post term infants were 1/1,454, 1/1,469 and 1/246, respectively. The CH prevalence of post term infants was significantly higher than that in other two groups (P<0.01). The prevalence of low birth weight infants, normal weight infants and macrosomia was 1/575, 1/1,505 and 1/473, respectively. The prevalence of low birth weight infants and macrosomia was significantly higher than that of normal weight group (P<0.01).</p><p><b>CONCLUSION</b>The prevalence of CH is associated with the birth weight and gestational age. To reduce the prevalence of CH in children, it is crucial to prevent post term, low birth weight, and macrosomia cases.</p>
Subject(s)
Female , Humans , Infant, Newborn , Male , Birth Weight , China , Epidemiology , Congenital Hypothyroidism , Blood , Epidemiology , Gestational Age , Neonatal Screening , Prevalence , Thyrotropin , BloodABSTRACT
<p><b>OBJECTIVE</b>To know the blood lead levels (BLL) of preschool children in Zhejiang Province between 1997 to 2003.</p><p><b>METHODS</b>The BLL of preschool children was investigated in this province with cluster sampling in 2003, and compared with the results with that of 1997. The BLL was determined with graphite oven atom absorption spectrography method.</p><p><b>RESULT</b>The average BLL of 2013 preschool children in 2003 was (0.34 +/- 0.13) micromol/L, similar with that of 1997 [(0.35 +/- 0.26)micromol/L (P >0.05)]. There were 274 children with BLL >or= 0.483 micromol/L, and the rate of lead poisoning was 13.61 %, lower than that of 1997 (when there were 448 children with BLL >or= 0.483 micromol/L, and the rate of lead poisoning was 23.84%). In 2003, the rates of lead poisoning among preschool children of Hangzhou, Shangyu, Zhoushan and Changshan were 7.69%, 15.37%, 10.87% and 20.15 %, respectively. There were significant differences among the four areas (P < 0.01). In comparison with the results of 1997, the lead poisoning rates of Hangzhou and Shangyu children decreased significantly, while that of Changshan children increased significantly (P <0.01), and that of Zhoushan children didn't change significantly. The highest rate of lead poisoning was found in children of 4-6 years group as 6 years ago.</p><p><b>CONCLUSION</b>The rate of lead poisoning among preschool children has decreased significantly now in Zhejiang Province. But the condition of BLL in preschool children is still far from optimistic. The environmental lead pollution in remote mountain areas and island areas should be highly concerned by the government.</p>
Subject(s)
Child , Child, Preschool , Female , Humans , Infant , Male , China , Epidemiology , Environmental Exposure , Incidence , Lead , Blood , Lead Poisoning , Blood , Epidemiology , Mass ScreeningABSTRACT
<p><b>OBJECTIVE</b>To analyze the data of screening for congenital hypothyroidism (CH) newborns in Zhejiang Province during 1999-2004.</p><p><b>METHODS</b>The dried blood samples were collected on filter paper. The levels of thyroid-stimulating hormone (TSH) were measured by time-resolved fluoroimmunoassay, and the serum levels of TSH, triiodothyronine (T(3)) and thyroxine (T(4)) were detected by chemiluminescence. Infants with CH confirmed by neonatal screening were treated with levothyroxine (L-T(4)) initiated with 4-6 g/kg x d(-1 )for 2-3 years. Growth, development and intelligence status, scintigraphy or ultrasonography of thyroid, and bone age were investigated to evaluate the efficacy of therapy during follow-ups.</p><p><b>RESULT</b>A total of 1112784 neonates were screened for CH during 1999-2004 with a coverage rate of 63.5%. Of the 6750 suspected CH cases, 6335 (93.8%) were recalled. 764 cases of CH were confirmed with an average incidence rate of 1 case CH per 1457 newborns (1/1457). 244 of 764 patients were followed-up for more than 1.5-2 years. All of them received thyroid by scintigraphy or ultrasonography. Among them 189 cases were found with normal gland, 35 with hypogenetic gland, 11 with ectopic gland, and the remaining 9 didn't show any image of thyroid. The average score of development quotient (DQ) was 106. 9. Among them, the DQ score was less than 85 in 2 cases, less than 90 in 9 cases, less than 100 in 28 cases, and in 68 cases the DQ was greater than 100. The bone age of 122 CH infants was evaluated with the X-ray radiography. In 90 cases of them,the bone age was normal, and 32 cases had progressed from development delay to normal. The height and weigh measured in all 106 cases had reached the related age criteria. The evaluation indicated that 55 cases were found with primary CH, 169 with temporary CH and 20 with subclinical CH.</p><p><b>CONCLUSION</b>Neonatal screening for CH and regular treatment for CH patients are important for attaining normal body development and intelligence development of patients.</p>
Subject(s)
Female , Humans , Infant, Newborn , Male , China , Epidemiology , Congenital Hypothyroidism , Epidemiology , Incidence , Neonatal Screening , Thyrotropin , Blood , Thyroxine , Blood , Triiodothyronine , BloodABSTRACT
<p><b>OBJECTIVE</b>To investigate the mechanism for the apoptosis of hippocampus neuron induced by hypothyroidism in perinatal rats.</p><p><b>METHODS</b>Hypothyroidism was induced by administration of propylthiouracil (PTU, 50 mg/d) solution to the dams from gestational day 15 by gavage. Pups from both hypothyroid and control groups were harvested at 1, 5, 10 and 15d, respectively. Blood samples were collected at the time of death for the determination of thyroid hormone. Serum free triiodothyronine (FT(3)) and free thyroxine (FT(4)) were measured by chemoluminescence. Hippocampus specimens were collected from the control and hypothyroid pups.Mitochondia was examined under transmission electron microscopy. Translocation of apoptogenic molecules (Bax, cytochrome C and AIF) and activation of caspase-3 were analyzed by Western Blotting.</p><p><b>RESULT</b>Significantly low circulating FT(3) and FT(4) levels confirmed the hypothyroid status of the experimental pups. Electron microscopy showed that altered morphology of mitochondria significantly increased under hypothyroid conditions. The expression of Bax in the cytosol of hypothyroid pups was higher than that of control pups at all stages of development (P<0.05),and significantly higher in mitochondria (P<0.001). The expression of cytochrome c in the cytosol of hypothyroid pups was significantly higher than that of control pups at all stages of development (1,10 and 15 d:P<0.05, 5d: P<0.001), and lower in mitochondria (P<0.05). The expression of AIF in the cytosol of hypothyroid pups was higher than that of control pups at all stages of development (P<0.001), and significantly lower in mitochondria (1, 5d: P<0.001, 10, 15 d: P<0.01). he expression of caspase-3 P20 in the cytosol of hypothyroid pups was significantly higher as compared with that of the age-matched controls (1, 15d: P<0.01, 5,1 0 d: P<0.001).</p><p><b>CONCLUSION</b>The intrinsic death pathway in mitochondria may be one of the mechanisms with which hypothyroid induces apoptosis of hippocampus neuron in developing rats.</p>
Subject(s)
Animals , Female , Pregnancy , Rats , Animals, Newborn , Apoptosis , Physiology , Hippocampus , Pathology , Hypothyroidism , Pathology , Neurons , Pathology , Pregnancy Complications , Pathology , Propylthiouracil , Rats, WistarABSTRACT
<p><b>OBJECTIVE</b>To evaluate the results of treatment of infants with congenital hypothyroidism (CH) with a low initial dosage of levothyroxine.</p><p><b>METHODS</b>138 newborns with primary CH detected by neonatal screening were divided into 3 groups according to levels of serum TSH, TT(3) and TT(4): sub-clinical CH (TSH >50 mU/L), mild CH (TT(4) <54 nmol/L), severe CH (TT(4)<54 nmol/L and TT(3)<1.2 nmol/L). The initial dose of levothyroxine was (3.5 +/-1.0) microg/(kg.d) for sub-clinical CH group, (4.3 +/-0.7)microg/(kg.d) for mild CH group and (4.7 +/- 0.6)microg/(kg.d) for severe CH group. Follow-up evaluation was carried out at 1, 2 and 3 months of age by measuring serum levels of TT(3), TT(4) and TSH. The time, when clinical signs and symptoms were eliminated and serum levels of TT(3), TT(4) and TSH normalized, was recorded. Development Quotient (DQ) testing was performed when CH cases were about 2 years old.</p><p><b>RESULTS</b>The mean initial dose of levothyroxine in 138 cases was (4.3 +/-0.9)microg/(kg.d). In one month the serum TT(3) and TT(4) levels returned to normal, while for TSH levels 67.4 % cases reached normal range in 2 months and 84.1 % in 3 months. Two months after therapy, the levels of TT(3) and TT(4) reached to the upper half of normal range and there were no signs or symptoms of hypothyroidism. The time for all cases in 3 groups to reach the normal clinical and biochemical indicators was similar (P=0.925). The dosage for cases with low circulating thyroxine before treatment was higher than that of the other groups (P<0.01). The average DQ score of 18 cases after treatment was 116.7 +/- 17.0.</p><p><b>CONCLUSION</b>he levothyroxine dosage of (4.3 +/- 0.9)microg/(kg.d) is appropriate for the initial treatment of the majority of infants with CH. However it is better to individualize the dosage for each case.</p>
Subject(s)
Female , Humans , Infant, Newborn , Male , Congenital Hypothyroidism , Hypothyroidism , Drug Therapy , Thyrotropin , Blood , Thyroxine , Blood , Triiodothyronine , BloodABSTRACT
<p><b>OBJECTIVE</b>To investigate effects of developmental lead exposure on nitric oxide synthase (NOS) activity in different brain regions and on N-methyl-D-aspartate (NMDA) receptor mRNA expression in the hippocampus of rats. On the basis of these observations, we explored possible mechanisms by which lead exposure leads to impaired learning and memorizing abilities in children.</p><p><b>METHODS</b>A series of rat animal models exposed to low levels of lead during the developing period was established (drinking water containing 0.025%, 0.05% and 0.075% lead acetate). NOS activities in the hippocampus, the cerebral cortex, the cerebellum and the brain stem were determined with fluorescence measurement and levels of mRNA expression of the NMDA receptor 2A (NR2A) subunit and NMDA receptor 2B (NR2B) subunit in the rat hippocampus were measured with Retro-translation (RT-PCR).</p><p><b>RESULTS</b>There were no differences in the body weight of rat pups between any of the groups at any given time (P>0.05). The blood lead level of Pb-exposed rat pups showed a systematic pattern of change: at 14 d of age, it was lower than that at 7 d of age, then rising to the peak level at 21 d and finally falling to lower levels at 28 d. The hippocampal NOS activities of lead-exposed groups were all lower than that of the control group on the 21st and 28th day (P<0.01). NOS activities in the cerebellum of lead-exposed groups were all lower than that of the control group on the 21st and 28th day (P<0.001) and the NOS activity of the 0.025% group was significantly lower than that of the 0.05% and 0.075% groups on the 28th day (P<0.05). NOS activity in the cerebral cortex of the 0.075% group was significantly lower than that of the control, 0.025% and 0.05% groups on the four day spans (P<0.001). There was no significant difference of NOS activity in the brain stem between any lead-exposed group and the control group on the four day spans. In the 0.05% and the 0.075% groups, the level of NR2A mRNA expression was higher than that in the control group at 7 d and 14 d of age (P<0.05). In the 0.025% group, the level of NR2A was found to be higher than that in the control group at 7 d of age only (P<0.05). No significant differences were found for the levels of NR2B mRNA expression between any of the groups at any given time.</p><p><b>CONCLUSIONS</b>NOS activity in the hippocampus, the cerebral cortex and the cerebellum are inhibited by lead exposure. The degree of the inhibitory effect depends on the time span of exposure and the lead concentration. Developmental low-level lead exposure was found to raise the level of NR2A mRNA expression in the hippocampus of rats. Developmental low-level lead exposure does not affect the level of NR2B mRNA expression in the hippocampus.</p>
Subject(s)
Animals , Female , Male , Rats , Animals, Newborn , Brain , Metabolism , Environmental Exposure , Enzyme Activation , Lead , Toxicity , Neurotoxins , Toxicity , Nitric Oxide Synthase , Metabolism , Receptors, N-Methyl-D-Aspartate , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To study the clinical therapy and prognosis in children with transient congenital hypothyroidism (CH).</p><p><b>METHODS</b>Fifty-seven children with CH diagnosed after neonatal screening were treated with low-dosage levothyroxine (L-T4). Follow-up evaluation included the determination of TT3, TT4 and TSH serum levels and the assessment of thyroid gland morphology, bone age, growth development and development quotients (DQ). A full check-up was performed at age 2, when the affected children first discontinued the L-T4 treatment for 1 month, and one year later. Development quotients were compared with a control group of 29 healthy peers.</p><p><b>RESULTS</b>The initial L-T4 dosage administered was 3.21-5.81 microg/(kg.d) with an average of (16.25+/-3.87) microg/d. Mean duration of therapy was (28.09+/-9.56) months. No significant difference was found between study group and control group in the DQ test (average score (106.58+/-14.40) vs (102.4+/-8.6), P>0.05) and 96.49% of the CH children achieved a test score above 85. Bone age, 99mTc scans and ultrasonographic findings were all normal, and evaluation of physical development was normal too, as were the serum levels of TT3, TT4 and TSH after one year of follow-up.</p><p><b>CONCLUSION</b>A L-T4 dosage of 3.21-5.81 microg/(kg.d) was found sufficient for the treatment of transient CH. The treated children showed satisfactory overall mental and physical development at age 2. So it is possible for CH children to stop taking medicine if their laboratory findings and physical development are all normal after regular treatment and 2-3 years of follow-up.</p>