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Persistent pulmonary hypertension of the newborn (PPHN) results from disruption of the normal fetal-neonatal circulatory transition and may be associated with meconium aspiration, group B streptococcal sepsis, pneumonia, respiratory distress syndrome, congenital diaphragmatic hernia and pulmonary hypoplasia. Seventeen percent of cases are considered idiopathic since there is no identifiable cause. Although it is recognized that acidosis and hypoxia from any cause in neonates may produce pulmonary vasoconstriction and maintain pulmonary hypertension, PPHN has not been reported in inborn errors of metabolism (IEM) associated with metabolic acidosis like methyl malonic acidemia (MMA). We report the first case in the literature of MMA presenting concomitantly with PPHN. Undiagnosed IEMs, like MMA, could represent a subset of idiopathic cases of PPHN. Infants and neonates have a limited repertoire with which to respond to an overwhelming illness. Because metabolic diseases are rare, they are considered only after excluding more common causes of neonatal distress. PPHN is therefore more likely to be attributed to meconium aspiration, sepsis, pneumonia or respiratory distress syndrome than to an IEM. The advent of expanded newborn screening has made pre-symptomatic diagnosis of several IEMs including MMA possible. However, not all IEMs are identified, and in some instances, an infant who has an IEM may become ill before the results of the newborn screen become available. Early diagnosis of IEM is crucial to prevent catastrophic consequences and the awareness of an association with PPHN would lead to an aggressive search of an underlying IEM and its management.

More than 900 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been reported to the cystic fibrosis (CF) consortium. A missense mutation, S1235R, was originally reported in a CF patient with a second mutation (G628R) on the same chromosome. The clinical significance of S1235R was not clear. S1235R is not among the commonly reported mutations, and it is not routinely screened for in most laboratories. However, we have detected the S1235R allele at a frequency that is significantly higher than that of many other CF mutations. Among more than 3,000 patients tested for either a possible diagnosis of CF or to determine CF carrier status, we identified 51 patients heterozygous for S1235R. No patients were homozygous for S1235R. Five patients were compound heterozygotes for a second CFTR mutation: two cases (one family) were N1303K/S1235R and three unrelated cases were deltaF508/S1235R. Our data suggest that S1235R, when combined with a second CF mutation, may be pathogenic, although phenotypic manifestations appear to be variable. The possibility that this represents a rare polymorphism cannot be discounted completely. Genetic counseling is difficult when S1235R is identified, even in the presence of a second known mutation, especially in prenatal cases.

Cationic trypsinogen and cystic fibrosis mutations have been identified in pancreatitis patients, although no study has looked for mutations in both genes in the same patient. Pancreatitis can be induced by alcohol, although not all alcoholics develop pancreatitis. We hypothesize that this phenomenon is due to a genetic predisposition in persons with alcohol-related pancreatitis. We performed sequence analysis of the cationic trypsinogen-coding region in 46 alcohol-related pancreatitis patients and 16 patients with pancreatitis due to causes other than alcohol. We also screened for 40 cystic fibrosis mutations including the 5T allele. No cationic trypsinogen mutations were identified. Cystic fibrosis mutation screening identified the DeltaF508 mutation in two Caucasian alcoholic patients (P0.025). The cystic fibrosis mutation carrier frequency in African-American alcoholic patients was 3%, which was not significantly increased compared with the normal carrier frequency. The frequency of the 5T allele was not significantly increased compared with the normal population carrier frequency in either racial group. These results may suggest a role for the cystic fibrosis gene in alcohol-related pancreatitis but indicate that cationic trypsinogen mutations are not a common predisposing risk factor for alcohol-related pancreatitis. A multicenter study is necessary to attain sufficient numbers to come to a conclusion.

Fetal echogenic bowel has been reported as a normal variant in the second trimester, and has also been associated with an adverse fetal outcome, including cystic fibrosis (CF), an autosomal recessive genetic disease. Previous studies have reported that 3.3 to 13.3 per cent of fetuses with echogenic bowel discovered during the second trimester were affected with CF. Between 1994 and 1998 our laboratory tested 159 cases with echogenic bowel detected during a routine ultrasound examination. The ethnic/racial background of cases included Caucasian, African-American, Middle Eastern, Hispanic, Ashkenazi Jewish and Asian. We identified two CF fetuses (1.3 per cent) and eight fetuses with a single identifiable CF mutation (5 per cent) within this diverse population. These data indicated that the risk of CF in a fetus with echogenic bowel in this population was less than the 3.3 to 13.3 per cent prior risk currently used in most Bayesian calculations. Furthermore, the results suggested that specific risks for couples should be calculated using data specific for their ethnic or racial background. Based on our results, we recommend either amniocentesis for fetal CF studies or CF carrier screening of both parents when fetal echogenic bowel is detected as a 1.3 per cent risk of CF is considered high enough to warrant further testing.

Clinical and molecular studies are reported on a family with X-linked mental retardation (XLMR) in which there are eight affected males in three generations. Although the males have somatic manifestations, these are variable and in most cases do not allow clear distinction of affected and unaffected males. Affected males are shorter and have a smaller head circumference. Several also have a sloping forehead (5/8), hearing loss (3/8), cupped ears (2/8), and small testes (4/6). An LOD score of 4.41 with zero recombination was obtained at locus DXS1166 in Xq13.2. This family highlights the difficulty in classifying XLMR conditions as either nonsyndromic or syndromic because of the variable somatic manifestations observed in the affected males.

We report on the prenatal diagnosis of a fetus with 46,XY and 46,XX cell lines with a normal male phenotype. Cytogenetic and molecular studies ruled out the possibility of maternal cell contamination and showed that all the X chromosomes present in both fetal cell lines were derived from a single maternal X chromosome. This suggests 46,XY/46,XX mosaicism.

We report on a child with congenital heart disease (atrial septal defect, ventricular septal defect, pulmonic stenosis), submucosal cleft palate, hypernasal speech, learning difficulties, and right fifth finger anomaly manifestations, consistent with velocardiofacial syndrome (VCFS); however, cytogenetic analysis demonstrated a small terminal deletion of the segment 4q34.2 to 4qter. Fluorescent in situ hybridization did not identify a deletion of the critical region associated with VCFS. In previously reported 4q deletions with a breakpoint distal to 4q34.2, no cardiac defects or cleft of palate were reported. Our patient has a deletion of 4q34.2 to 4qter and has palate and cardiac involvement and minor learning difficulties, which implies that genes involved in heart and palate development lie distal to 4q34.2, and that the critical region for more severe mental retardation on 4q may reside proximal to 4q34.2. These results suggest that a distal 4q deletion can lead to a phenotype similar to VCFS and emphasizes the importance of searching for other karyotype abnormalities when a VCFS-like phenotype is present and a 22q deletion is not identified.

We report on a 24-year old woman with an Xq duplication and findings suggestive of Prader-Willi syndrome (PWS). Her birth weight was at the 3rd centile and her birth length was less than the 3rd centile. She was hypotonic and had a weak cry as an infant. There were no feeding difficulties, although her mother reports that as an infant, she was "small for her age." Excessive weight gain began between 3 and 4 years. The patient's development was delayed and she received special education. She has a history of hiding food. She has a sleep disturbance disorder and inappropriate social behavior. At the age of 24 years her height was below the 5th centile and weight95th centile. She has physical findings typical of PWS, skin picking, and speech articulation defects. Cytogenetic analysis showed a 46,X,dup(X)(q23q25) karyotype. Fluorescent in situ hybridization (FISH) studies using a chromosome X painting probe demonstrated that the rearrangement was intrachromosomal. The X-chromosome fold scoring technique was used to determine the X inactivation pattern and indicated that some cells expressed the abnormal X chromosome. Results of FISH studies using the SNRPN probe localized to 15q11q13 and DNA studies using the PW71B and SNRPN probes were normal. The duplicated X chromosome, random X inactivation pattern, and the negative molecular studies for PWS indicate that the abnormal X chromosome is the basis of this patient's phenotype. This patient emphasizes the importance of obtaining a karyotype even when a syndrome diagnosable by molecular methods is strongly suspected.

Homozygosity for the mutation Cys282Tyr in the HFE gene has recently been identified as a cause of hereditary hemochromatosis, a disorder resulting in the inappropriate absorption of iron. Approximately 10% of Caucasians are heterozygous for this mutation; however, the gene frequency in African Americans is unknown. A study of a control population of African Americans was performed to determine the frequency of the Cys282Tyr and His63Asp alleles in this ethnic group. The carrier frequency for each mutant allele in our African American population was 3.0%. DNA studies of four African-American hemochromatosis patients did not identify any individuals with the Cys282Tyr allele. These findings suggest that if the Cys282Tyr mutation confers susceptibility to hemochromatosis in Caucasians (as suggested by recent studies) there is an alternative mechanism for hemochromatosis in the American black population.

The most commonly reported manifestations of 16q deletions are severe growth and developmental disorders and anomalies of the craniofacial, visceral, and musculoskeletal systems. We reviewed the findings of patients reported with 16q- syndrome and compared them to our patient, a 4 1/2-year-old boy with a deletion of 16q23.1. Findings include psychomotor retardation, hypotonia, high forehead, hypertelorism, upslanting palpebral fissures, low-set abnormally modeled ears, and talipes equinovarus. Anomalies present in our patient not reported in others with 16q- syndrome include bilateral cataracts, iris coloboma, and autistic-like behavior. It is of note that a locus for autosomal dominant congenital cataract, known as Marner cataract, was mapped previously to 16q22. Because our patient has bilateral cataracts and a unilateral iris coloboma, it seems likely that a gene involved in ocular development is located within 16q23.1. Our patient's deletion may also include the gene involved in Marner cataract and may further assist in the isolation of this gene.

The RSH/Smith-Lemli-Opitz syndrome (RSH/SLOS) is an autosomal recessive malformation syndrome associated with increased levels of 7-dehydro-cholesterol (7-DHC) and a defect of cholesterol biosynthesis at the level of 3 beta-hydroxy-steroid-delta7-reductase (7-DHC reductase). Because rats exposed to inhibitors of 7-DHC reductase during development have a high frequency of holoprosencephaly (HPE) [Roux et al., 1979], we have undertaken a search for biochemical evidence of RSH/SLOS and other possible defects of sterol metabolism among patients with various forms of HPE. We describe 4 patients, one with semilobar HPE and three others with less complete forms of the HPE sequence, in whom we have made a biochemical diagnosis of RSH/SLOS. The clinical and biochemical spectrum of these and other patients with RSH/SLOS suggests a role of abnormal sterol metabolism in the pathogenesis of their malformations. The association of HPE and RSH/SLOS is discussed in light of the recent discoveries that mutations in the embryonic patterning gene, Sonic Hedgehog (SHH), can cause HPE in humans and that the sonic hedgehog protein product undergoes autoproteolysis to form a cholesterol-modified active product. These clinical, biochemical, and molecular studies suggest that HPE and other malformations in SLOS may be caused by incomplete or abnormal modification of the sonic hedgehog protein and, possible, other patterning proteins of the hedgehog class, a hypothesis testable in somatic cell systems.

Familial predisposition to neuroblastoma, a common embryonal cancer of childhood, segregates as an autosomal dominant trait with high penetrance. It is therefore likely that neuroblastoma susceptibility is due to germ line mutations in a tumor suppressor gene. Cytogenetic, functional, and molecular studies have implicated chromosome band 1p36 as the most likely region to contain a suppressor gene involved in sporadic neuroblastoma tumorigenesis. We now demonstrate that neuroblastoma predisposition does not map to any of eight polymorphic markers spanning 1p36 by linkage analysis in three families. In addition, there is no loss of heterozygosity at any of these markers in tumors from affected members of these kindreds. Furthermore, there is strong evidence against linkage to two Hirschsprung disease (a condition that can cosegregate with neuroblastoma) susceptibility genes, RET and EDNRB. We conclude that the neuroblastoma susceptibility gene is distinct from the 1p36 tumor suppressor and the currently identified Hirschsprung disease susceptibility genes.

Multiple endocrine neoplasia type 2 [MEN 2] is an autosomal dominant cancer syndrome with two subtypes, 2A and 2B. MEN 2A and medullary thyroid cancer [MTC] are caused by25 different point mutations in exons 10, 11, and 13 of the RET proto-oncogene, whereas MEN 2B is caused by a single exon 16-point mutation. Various molecular methods have been used to identify the different mutations, including DNA sequencing, restriction enzymatic analyses, chemical cleavage mismatch, Single Stranded Conformational Polymorphism [SSCP], and Denaturing Gradient Gel Electrophoresis [DGGE]. These techniques, although useful and accurate, are labor intensive and some involve the use of radioactivity. We have developed a multiplex PCR assay simultaneously to amplify exons 10, 11, and 13 of the RET proto-oncogene. The multiplex PCR product is then analyzed on a modified Mutation Detection Enhancement [MDE] matrix for heteroduplex identification and visualized with ethidium bromide. Distinct heteroduplexes were detected for each known RET proto-oncogene mutation available in our laboratory (nine in exon 10, five in exon 11, one in exon 13, and the single exon 16 mutation). Presymptomatic DNA diagnosis of MEN 2 is essential since pentagastrin-stimulated calcitonin studies can occasionally produce false positive results and lead to unnecessary thyroidectomies. Prophylactic thyroidectomy is recommended by age 5 or 6 once a mutation is identified in a patient, since penetrance is very high. MDE heteroduplex detection provides a quick, efficient, and inexpensive method of screening for RET mutations in MTC patients with unknown mutations, or for presymptomatic diagnosis in individuals at risk for inheriting a known RET mutation. Confirmation of the specific mutation can be achieved by restriction enzymatic digestion (if feasible) or by DNA sequencing.

The multiple endocrine neoplasia type 2A gene is the RET proto-oncogene located on the long arm of chromosome 10, and many mutations within this gene have been reported.Peripheral blood DNA was analyzed from 95 members of twelve families with multiple endocrine neoplasia type 2A and known mutations in codon 634 (of exon 11) of the RET proto-oncogene. This region was amplified by the polymerase chain reaction, followed by digestion with Cfo I, which detects restriction sites created by the most common TGC-CGC mutation and by a TGC-TGG mutation or with Rsa I, which detects a restriction site created by a TGC-TAC mutation.Diagnoses were confirmed in 39 patients; 15 of 56 at-risk persons were gene carriers and 41 were noncarriers. The noncarriers included seven persons who had previously undergone thyroidectomies for suspected C-cell hyperplasia but were negative for the RET mutation present in affected members of their families.Identification of the specific gene alterations within families permits direct DNA diagnosis of at-risk family members. The 41 noncarriers will not require further testing nor need to be concerned about transmitting multiple endocrine neoplasia type 2A to their descendants. The normal DNA findings in seven of these persons emphasize the importance of DNA studies in patients with C-cell hyperplasia but no medullary thyroid cancer at operation.

The common mutation causing cystic fibrosis (CF) is deletion of phenylalanine 508 (deltaF508). This mutation was analyzed in over 200 North American families using the polymerase chain reaction and hybridization with allele specific oligonucleotides. The deltaF508 mutation was present on 75.8% of non-Ashkenazic Caucasian chromosomes; 96% of the deltaF508 chromosomes carried the B haplotype for XV-2c and KM-19. Only 30.3% of Ashkenazic CF chromosomes carried the deltaF508 mutation, although the B haplotype for XV-2c and KM-19 was found on 97% of Ashkenazic CF chromosomes.

The anomalous eμ and 2-prong μx events produced in e + e − annihilation are used to determine the properties of the proposed τ charged lepton. We find the τ mass is 1.90 ± 0.10 GeV/ c 2 ; the mass of the associated neutrino, ν τ , is less than 0.6 GeV/ c 2 with 95% confidence; V - A coupling is favored over V + A coupling for the τ − ν τ current; and the leptonic branching ratios are 0.186 ± 0.010 ± 0.028 from the eμ events and 0.175 ± 0.027 ± 0.030 from the μx events where the first error is statistical and the second is systematic.

ONLY one specific study on the respiratory system of the turkey has been found. This consisted of two papers published in 1953 by Cover. Cover reviewed the observations of previous investigators on the chicken. He pointed out that in general all reports agree on the number of air sacs in the chicken (4 paired and 1 single) but they often have been named differently. Disagreement centers around the cervical or thoraco-cervical and the posterior thoracic air sacs. Cover (1953b) observed in the turkey that “the paired posterior thoracic and thoraco-cervical as well as the single anterior thoracic air sacs are combined into a single large compartment, the aggregate sac, which communicates with the air passageways of the lung at its anterior ventral border. There are two unions, one with a ventral bronchus and another with an area of several parabronchi. The combined sacs have the same location and visceral relations . . .

IN PREVIOUS reports of studies on embryonic metabolism carried out in this laboratory (Feldman et al., 1958; Feldman and Couch, 1960) a system was defined for the stimulation of oxygen uptake of chick embryo preparations. It was pointed out that the maximal oxidative rate required a balance between the breakdown of adenosine triphosphate (ATP) and its synthesis. Furthermore, it was found that oxygen uptake by chick embryo preparations proceeded slowly unless magnesium and ATP were added in vitro. The addition of low concentrations of dinitrophenol in vitro stimulated the oxygen consumption of cell-free extracts of 10-day chick embryos (Feldman et al., 1958). The stimulatory effect of dinitrophenol was also observed with cell-free extracts of embryos which had been removed from eggs injected with the drug. Very little is known of the enzymatic activity of older chick embryos, particularly the enzyme systems involved in energy metabolism. It was felt that a …

THE hatchability of turkey eggs has been reported by numerous workers to exhibit a marked decline under practical conditions in a period from about the 8th week to the 12th week. Earlier reports have indicated that the decline in hatchability can be delayed by supplementing the diet with sources of unidentified factors. Some of the supplements reported to increase hatchability are pork liver meal, desiccated meat meal, fish meal, dried buttermilk, dried skimmilk, meat scraps, dehydrated alfalfa meal, distillers dried solubles, dried whey, and liver “L” (Nestler et al., 1936; Dickens et al., 1941; Atkinson et al., 1951, 1953, 1955b; Couch et al., 1954; and Ferguson et al., 1956). The fat soluble vitamins E and D, as well as pantothenic acid, have been shown to be necessary in the maintenance of hatchability (Stadelman et al., 1950; Jensen et al., 1953; Jensen, 1953; Jensen et al., 1956; Atkinson and Couch, 1954; . . .

MANY observations have been made on the respiratory tract of the fowl (Locy and Larsell, 1916; McLeod and Wagers, 1939; Bradley, 1951; Hazelhoff, 1951; Cover, 1953; Sturkie, 1954; and Rigdon et al., 1957). The air sacs in the turkey have been described recently by Rigdon and associates (1957). The different air sacs in the fowl are known by a variety of names. Numerous diverticula, including the axillary, are reported by Sturkie (1954) to arise from the interclavicular air sac of certain species. McLeod and Wagers (1939) describe a large pouch leading from the main sac behind the coracoid bone into the axillary region in the chicken. According to Bradley (1951), the axillary sac in the chicken is a prolongation of the clavicular air sac. Rigdon et al. (1958) found that in the turkey there is a large cavity in the axilla that does not communicate with either the respiratory tract . . .

Three nitrile silicone polymers have been evaluated as liquid phases for gas chromatographic separation of the geometric isomers of methyl oleate, methyl linoleate, and methyl linolenate on capillary columns. A polymer of β-cyanoethylmethylsiloxane proved the most effective. This liquid phase separated oleate from elaidate, resolved the four geometric isomers of linoleate into three peaks, and divided the eight geometric isomers of linolenate into six peaks. Two other copolymers of dimethylsiloxane and β-cyanoethylmethylsiloxane gave poorer resolution ofcis-trans isomers, but showed different elution patterns for the geometric isomers of linoleate and linolenate.

Three biotin-dependent enzymes, pyruvate carboxylase (PC), propionyl CoA carboxylase (PCC), and beta-methylcrotonyl CoA carboxylase (beta MCC), were biochemically characterized in fibroblasts from two patients with neonatal multiple carboxylase deficiency. Genetic complementation analyses indicated that both cell lines, designated lines 1 and 2, were deficient in the various carboxylase activities and belonged to the bio complementation group. The activities of the three carboxylases became normal when line 2 cells were incubated in medium supplemented with biotin (1 mg/l) for 24 hrs, whereas 4-6 days were required to achieve maximum activities of PC, PCC, and beta MCC (57%, 46%, and 29% of mean normal enzyme activity, respectively) in line 1 cells incubated in medium containing up to 10 mg/1 biotin. Furthermore, PC activity in line 2 continued to increase under apparent gluconeogenic conditions in culture, but not in line 1. Thermostability studies suggested that biotin stabilizes PC and beta MCC in both cell lines. PC in line 1 cells incubated with or without biotin was less stable than that in normal or line 2 cells, and the less than normal increase of enzyme activities in line 1, especially that of PC, may represent incomplete biotination. These results indicate that there is biochemical heterogeneity within the bio complementation group. Immunotitration with antibodies prepared against purified pig heart PCC demonstrated normal quantities of cross-reacting material in both lines and no differences in the amount of this material after incubation with supplemental biotin, despite the seven- to 20-fold increase in PCC activity. Thus, the increase in carboxylase activity in both bio lines appears to represent activation of rpe-existing apocarboxylase rather than de novo enzyme synthesis. The primary defect in this form of multiple carboxylase deficiency may be in a common holocarboxylase synthetase or in biotin transport. If the defect is in the synthetase, the differences noted between the two bio lines could be explained by a difference in the enzyme's Km for biotin.

Prenatal diagnosis was carried out in 138 pregnancies at 1-in-4 risk for cystic fibrosis (CF) by using closely linked DNA markers, including XV-2c and KM-19. In fully informative families, 25 of 123 (20%) fetuses were predicted to be affected; 16 of these 25 pregnancies were terminated and 9 were continued. Postnatal sweat tests are completed in 42 cases; the diagnoses were confirmed in 4 of 4 infants predicted to be affected and in 37 of 38 infants predicted to be unaffected. One infant predicted to be a carrier had an abnormal sweat test after birth, but the mother also had an abnormal sweat test, and there was no evidence of an error in linkage analysis. The data indicate that prenatal diagnosis using linkage analysis is fully informative in most families and is highly reliable with either chorionic villus sampling or amniocentesis. Although outcome data are available on only 42 pregnancies, based on our experience, on general principles of linkage analysis, and on the tight linkage of the known DNA markers with CF, we recommend that DNA analysis replace microvillar intestinal enzyme analysis for 1-in-4 risk pregnancies when DNA is available from the propositus.

Summary1) Injections of 1-25 μg of DNP into chicken eggs, prior to incubation resulted in embryonic mortality after 3 days incubation, but did not produce lens changes. 2) Injections of 25-200 μg of DNP after 5 days incubation did not produce lenticular changes. 3) Cataracts were produced in embryos and hatched chicks from eggs injected with 200-500 μg of DNP after 8 days incubation. The size of cataract was roughly the same in hatched chicks as in embryos from the 500 μg group. 4) Injection of 750 μg of DNP after 15 days incubation produced an opacity evident after 20 hours. However, the opacity apparently regressed since the eyes of chicks hatched from this group appeared to be normal.

The first report of a cataract developing in man after the ingestion of dinitrophenol was in 1935.1Six years later Horner2collected from the literature 177 cases. The cataracts occurred approximately 15 months after the drug was received.3Horner,4in 1942, stated, "all attempts to produce experimental cataracts in laboratory animals by various and repeated doses of dinitrophenol have been unsuccessful." Bellows,5in 1944, also commented on the fact that ordinary laboratory animals do not develop cataracts when dinitrophenol is given. Krause6attributed this failure to a difference in the metabolism of the species. Robbins,7,8in 1944, experimentally induced cataracts for the first time in ducklings and chickens by the oral administration of dinitrophenol. He observed a fine gray opacity that progressed to involve most of the anterior portion of the lens. Upon continued feeding the changes in the anterior portion of the