Universal newborn screening: A roadmap for action.

Newborn screening (NBS) prevents morbidity and mortality by screening babies for selected disorders in the first days of life so that early diagnosis and treatment can be initiated. Congenital disorders impact an estimated 8 million or 6% of annual births worldwide, and of the top five that contribute 25% to the global burden of these disorders, three can be identified and managed by NBS. There are determined pockets of activity in Latin America, Sub-Saharan Africa, and the Asia Pacific region, where partnerships among government, non-governmental organizations, academia, the private sector and civil society are developing novel NBS programs that are both saving lives and preventing disability in those who survive.

A dosage sensitive locus at chromosome Xp21 is involved in male to female sex reversal.

Male to female sex reversal has been observed in individuals with duplications of the short arm of the X chromosome. Here we demonstrate that sex reversal results from the presence of two active copies of an Xp locus rather than from its rearrangement and that alterations at this locus constitute one of the causes of sex reversal in individuals with a normal 46,XY karyotype. We have named this locus DSS (Dosage Sensitive Sex reversal) and localized it to a 160 kilobase region of chromosome Xp21, adjacent to the adrenal hypoplasia congenita locus. The identification of male individuals deleted for DSS suggests that this locus is not required for testis differentiation. We propose that DSS has a role in ovarian development and/or functions as a link between ovary and testis formation.

ynergy of ethanol and a natural soporific--gamma hydroxybutyrate.

gamma-Hydroxybutyrate and ethanol, as well as gamma-butyrolactone and ethanol are potentiative with respect to duration of loss of the righting reflex (sleep time). The concentration of ethanol in the liver decreases from 30 to 90 minutes after rats are injected with ethanol, but there is no change when ethanol is injected with gamma-butyrolactone. In view of the fact that gamma-hydroxybutyrate is a natural intermediate in brain, the effects of ethanol on the central nervous system may be mediated through its interaction with gamma-butyrolactone.

Perinatal hypophosphatasia: tissue levels of vitamin B6 are unremarkable despite markedly increased circulating concentrations of pyridoxal-5'-phosphate. Evidence for an ectoenzyme role for tissue-nonspecific alkaline phosphatase.

"Perinatal" hypophosphatasia is the most severe form of this inborn error of metabolism, which is characterized by deficient activity of the tissue-nonspecific (liver/bone/kidney) isoenzyme of alkaline phosphatase (ALP) (TNSALP). We report that autopsy tissue from three affected subjects, which was profoundly low in ALP activity, had essentially unremarkable levels of pyridoxal-5'-phosphate (PLP), pyridoxal, and total vitamin B6 content despite markedly elevated plasma PLP levels (5,800, 14,500, and 98,500 nM; adult norm, 5-109 nM). Our findings help to explain the general absence of symptoms of vitamin B6 excess or deficiency in hypophosphatasia, and provide evidence that TNSALP acts as an ectoenzyme to regulate extracellular rather than intracellular concentrations of PLP (the cofactor form of vitamin B6) and perhaps other phosphate compounds.

Complementary DNA probes for the Duchenne muscular dystrophy locus demonstrate a previously undetectable deletion in a patient with dystrophic myopathy, glycerol kinase deficiency, and congenital adrenal hypoplasia.

Genomic DNA from a patient with dystrophic myopathy, glycerol kinase deficiency, and congenital adrenal hypoplasia was investigated using cDNA probes for the Duchenne muscular dystrophy (DMD) locus. Genomic probes had not detected a deletion in this patient. Southern analysis of Hind III-digested genomic DNA from this patient identified a deletion when the three distal Hinc II DMD cDNA fragments were used as probes. The deletion began in the genomic region corresponding to the 1.05-kb Hinc II cDNA fragment and extended through the 3' end of the DMD gene. This represents a centromeric breakpoint that corresponds to a position approximately 10.2-10.6 kb from the 5' end of the 14-kb DMD cDNA. These investigations demonstrate the value of the DMD cDNA probes for improved diagnoses in patients with molecular lesions involving the DMD locus. Furthermore, this novel deletion involving the coding portion of the 3' end of the DMD gene assists in the ordering of exons in this region and will provide insight into the functional role of the carboxy terminus of the DMD gene product, dystrophin.

Zebrafish dax1 is required for development of the interrenal organ, the adrenal cortex equivalent.

Mutations in the human nuclear receptor, DAX1, cause X-linked adrenal hypoplasia congenita (AHC). We report the isolation and characterization of a DAX1 homolog, dax1, in zebrafish. The dax1 cDNA encodes a protein of 264 amino acids, including the conserved carboxy-terminal ligand binding-like motif; but the amino-terminal region lacks the unusual repeats of the DNA binding-like domain in mammals. Genomic sequence analysis indicates that the dax1 gene structure is conserved also. Whole-mount in situ hybridization revealed the onset of dax1 expression in the developing hypothalamus at approximately 26 h post fertilization (hpf). Later, at about 28 hpf, a novel expression domain for dax1 appeared in the trunk. This bilateral dax1-expressing structure was located immediately above the yolk sac, between the otic vesicle and the pronephros. Interestingly, weak and transient expression of dax1 was observed in the interrenal glands (adrenal cortical equivalents) at approximately 31 hpf. This gene was also expressed in the liver after 3 dpf in the zebrafish larvae. Disruption of dax1 function by morpholino oligonucleotides (MO) down-regulated expression of steroidogenic genes, cyp11a and star, and led to severe phenotypes similar to ff1b (SF1) MO-injected embryos. Injection of dax1 MO did not affect ff1b expression, whereas ff1b MO abolished dax1 expression in the interrenal organ. Based on these results, we propose that dax1 is the mammalian DAX1 ortholog, functions downstream of ff1b in the regulatory cascades, and is required for normal development and function of the zebrafish interrenal organ.

Developmental expression of hexokinase 1 in the rat.

Hexokinase 1 (HK1) activity varies in a developmental stage- and tissue-specific manner and is a key step in glucose homeostasis and energy metabolism. We conducted studies to determine if HK1 expression is regulated at the transcriptional level. Expression of HK1 was examined in selected pre- and postnatal rat tissues using Northern blot analyses and RNAase protection assays. We found that brain and kidney exhibited significantly higher expression than heart, lung, spleen and skeletal muscle. Brain HK1 expression was constant prenatally, peaked at 7 days of age and reached a constant level after weaning. In kidney, HK1 expression was essentially constant or perhaps gradually decreased after weaning. HK1 transcription in heart, skeletal muscle and liver was higher during fetal stages than postnatally. Lung expression was essentially constant except in the adult. HK1 mRNA levels were compared to phosphoglycerate kinase (PGK) mRNA. PGK steady state mRNA levels were relatively constant in all tissues and developmental stages, except in skeletal muscle where there was a postnatal rise. The developmental profiles of HK1 and PGK mRNA expression are not consistent with enzyme activity measurements in all the tissues examined. We conclude that regulation of HK1 expression involves both transcriptional and posttranscriptional mechanisms that are tissue and stage specific.

Mutations in NR0B1 (DAX1) and NR5A1 (SF1) responsible for adrenal hypoplasia congenita.

Adrenal hypoplasia congenita (AHC) causes primary adrenal insufficiency due to the failure of development of the adrenal cortex. Clinical and pedigree data indicate that the condition is genetically heterogeneous. The predominant adrenal hypoplasia congenita locus, however, is the NR0B1 gene, at Xp21, encoding the protein DAX1. In this article, we present a compendium of published NR0B1 mutations and polymorphisms, and discuss them in the contexts of known biology and clinical applicability. The recent descriptions of patients with primary adrenal insufficiency due to mutations of NR5A1, which encodes SF1, are also discussed.

Nine novel mutations in NR0B1 (DAX1) causing adrenal hypoplasia congenita.

X-linked adrenal hypoplasia congenita (AHC) is caused by mutations in the NR0B1 gene. This gene encodes an orphan member of the nuclear receptor superfamily, DAX1. Ongoing efforts in our laboratory have identified nine novel NR0B1 mutations in X-linked AHC patients (Y81X, 343delG, 457delT, 629delG, L295P, 926-927delTG, 1130delA, 1141-1155del15, and E428X). Two additional families segregate previously identified NR0B1 mutations (501delA and R425T). Sequence analysis of the mitochondrial D-loop indicates that the 501delA family is unrelated through matrilineal descent to our previously analyzed 501delA family.

Genomic sequence of the DAX1 gene: an orphan nuclear receptor responsible for X-linked adrenal hypoplasia congenita and hypogonadotropic hypogonadism.

The gene responsible for X-linked adrenal hypoplasia congenita, DAX1, encodes a member of the nuclear hormone receptor superfamily. We sequenced 8851 bp that contained the DAX1 genomic region. The DAX gene was composed of two exons and one 3.4-kilobase intron. Putative TATA and GC boxes and a putative steroidogenic factor 1 response element were present in the 5'-flanking region. Two potentially polymorphic short tandem repeats were identified. The first exon encoded two putative novel zinc finger motifs within a putative DNA binding domain and part of the ligand binding domain, and the second exon encoded the remainder of the ligand binding domain. Although the putative DNA binding domain of DAX1 does not contain substantial sequence similarity to other nuclear hormone receptor superfamily members, the putative ligand binding domain had remarkable similarity to other family members. Single-strand conformational polymorphism analysis permitted identification of three new mutations in DAX1. In conclusion, single-strand conformational polymorphism analysis facilitates identification of mutations in the DAX1 gene, and the short tandem repeats may permit linkage analysis in families in which mutations are not yet identified. We speculate that DAX1 may be the most primitive member of the nuclear hormone receptor superfamily identified in mammals.

IMAGe, a new clinical association of intrauterine growth retardation, metaphyseal dysplasia, adrenal hypoplasia congenita, and genital anomalies.

We report three boys with adrenal hypoplasia congenita (AHC) and additional findings that represent a new syndrome, IMAGe: Intrauterine growth retardation, Metaphyseal dysplasia, AHC, and Genital anomalies. Each presented shortly after birth with growth retardation and severe adrenal insufficiency. Each of the three patients had mild dysmorphic features, bilateral cryptorchidism, a small penis, and hypogonadotropic hypogonadism. Skeletal surveys revealed metaphyseal dysplasia in all three and epiphyseal dysplasia in two. The patients had documented or suspected hypercalciuria and/or hypercalcemia, resulting in nephrocalcinosis in one and in prenatal liver and spleen calcifications in another. AHC presents most often either as an isolated abnormality, caused by mutations in the DAX1 gene, or as part of an Xp21 contiguous gene syndrome, caused by a deletion of the Duchenne muscular dystrophy, glycerol kinase, and DAX1 genes. All three patients with the IMAGe association had normal creatine kinase levels and no evidence of glycerol kinase deficiency. Sequence analysis of DNA from these patients revealed no mutation in the DAX1- or steroidogenic factor-1-coding sequences, nor was a deletion of DAX1 detected. Identification of the molecular basis of the IMAGe association will give new insight into the pathogenesis of this syndromic relationship involving bone, adrenal cortical, and pituitary development.

Ahch, the mouse homologue of DAX1: cloning, characterization and synteny with GyK, the glycerol kinase locus.

We cloned the murine full-length cDNA encoding Ahch, the mouse homologue of DAX1 (DSS-AHC Region on Human X Chromosome, Gene1) which is the gene responsible for human X-linked adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HH). Sequence analysis revealed that the murine and human cDNAs have 65% aa identity and 75% aa similarity overall. The cysteine residues in the putative DNA binding domain, which may interact with Zn2+ ions to form zinc fingers, are 100% conserved between the two species, indicating that the novel zinc-finger structures in DAX1 may be functional. In addition, mouse interspecific backcrosses show that the Ahch gene is closely linked to the glycerol kinase locus, GyK, on the mouse X chromosome, indicating that the order of the loci is conserved in this syntenic region between mouse and human.

Nr0b1 and its network partners are expressed early in murine embryos prior to steroidogenic axis organogenesis.

Ahch is an orphan nuclear receptor encoded by Nr0b1 on the murine X chromosome and is the ortholog of human DAX1. Nr0b1/NR0B1 expression at appropriate dosages is required for normal steroidogenic axis development: mutation of the human ortholog, NR0B1, results in adrenal hypoplasia congenita and hypogonadotropic hypogonadism; and duplication or transgenic overexpression in humans or mice, respectively, results in XY phenotypic females, a phenotype known as dosage sensitive sex-reversal. Complete loss of Nr0b1 by targeted deletion has been hypothesized to be lethal in embryonic stem (ES) cells and preliminary evidence suggested that ES cells might express Nr0b1. These investigations examined Nr0b1 expression and its network partners in both cultured ES cells and preimplantation embryos. We cultured ES cells in the absence or presence of differentiation agents and analyzed expression of Nr0b1 and associated network partners by northern blot hybridization and reverse transcriptase-polymerase chain reaction. Nrob1 was highly expressed by totipotent ES cells with reduced expression following induction toward individual germ layer fates. Nr5a1/Sf1, Wt1 and other genes that encode proteins known to interact with Nr0b1 were also expressed. Immunohistochemical analysis of preimplantation embryos for Ahch and key partners confirmed in vivo expression of network components. These findings are consistent with the existence of a potentially functional network of transcription factors, including Ahch, very early in embryonic development. These results validate ES cells as a developmentally dynamic model for mechanistic investigations into this regulatory network early in embryogenesis preceding organogenesis.

Glutaric acidemia: a metabolic disorder causing progressive choreoathetosis.

A boy with glutaric acidemia had psychomotor retardation first noted at age 6 months, recurrent metabolic acidosis, and a progressive quadriparesis with choreoathetosis. He died at age 3 1/2 years. Cultured skin fibroblasts lacked glutaryl-CoA dehydrogenase activity. There was a biochemical, but not a clinical, response to dietary restriction of lysine and tryptophan. The caudate and putamen of the brain showed severe loss of nerve cells and fibers with proliferation of astrocytes, as well as markedly reduced gamma-aminobutyric acid and glutamate decarboxylase activity.

Identification of new members of a carbohydrate kinase-encoding gene family.

In a sequence database search using the human glycerol kinase-encoding sequence (HUMGLYKINB) as a query, we identified six previously unidentified carbohydrate kinase sequences. Five of the six newly identified sequences appear to be known types of carbohydrate kinases, four are glycerol kinases and one is a gluconokinase. The sixth newly identified sequence, the Caenorhabditis elegans gene, CER08D7.7-CEF59B2.1, shows similarity to the family of carbohydrate kinases including other glycerol kinases, xylulokinases, gluconokinases, ribulokinases, rhamnulokinases, and fucokinases. A phylogenetic comparison of this newly identified Caenorhabditis elegans gene with the other members of the carbohydrate kinase family demonstrated that this sequence cannot be assigned to one of the known classes of carbohydrate kinases.

Amplification of bacterial DNA using highly conserved sequences: automated analysis and potential for molecular triage of sepsis.

OBJECTIVE: The clinical diagnosis of sepsis remains difficult, particularly in the young child, and would be improved by a rapid and reliable method for identification of bacteria in blood and other body fluids. Polymerase chain reaction (PCR) amplification of highly conserved DNA sequences found in all bacteria would permit fast and sensitive determination of the presence of bacteria in clinical specimens. METHODOLOGY: A primer pair for highly conserved regions of bacterial DNA encoding 16S ribosomal RNA (rDNA) was utilized for PCR amplification. PCR products were analyzed by gel electrophoresis, and, after modification of the primers, by an automated 96-well plate reader. RESULTS: rDNA was amplified from 12 different species of bacteria, including Gram-negative and -positive organisms. No signal was observed when total human DNA was used as template. Colorimetric analysis of amplified sequences using a 96-well format was also successful. CONCLUSIONS: We conclude that a single primer pair designed to anneal to a highly conserved region of bacterial DNA can amplify DNA specimens from a variety of different bacteria, while not amplifying human DNA. Such a molecular genetics approach can be fully automated with existing robotic technology. Because of speed, sensitivity, and cost, molecular triage of patients with signs and symptoms of possible bacterial infection will decrease morbidity and mortality among those with unrecognized bacteremia who are managed as outpatients, and will dramatically reduce hospital expenses from individuals who are admitted and are not bacteremic.

Newborn screening for phenylketonuria: predictive validity as a function of age.

Data from questionnaires were assembled for 109 infants with phenylketonuria (PKU) and 114 control infants to assess the predictive validity of newborn screening for PKU as a function of age. Patients with PKU had values of less than 4 mg/dL in cord blood and in samples from days 1, 2, and 4 through 7. The proportion of patients with PKU expected to fall below screening cutoffs of 2, 4, and 6 mg/dL was predicted for each age range. Using a cutoff of 4 mg/dL, approximately one third of patients with PKU would be missed by a sample taken from the neonate in the first 12 hours of life, and nearly 10% would be missed with a sample from the second 12 hours of life. This study shows that not all patients with PKU will be detected by newborn screening, and that the phenomenon of early nursery discharges must be considered in developing appropriate screening strategies.

Weaver syndrome: the changing phenotype in an adult.

We report on a 25-year-old woman who was diagnosed with Weaver syndrome after reevaluation because of the family's concern regarding recurrence risk for mental retardation in offspring of the woman's brother. The diagnosis was suggested on the basis of postnatal growth excess, camptodactyly, and developmental delay, but with a somewhat atypical facial appearance. When childhood photographs were reviewed, her facial characteristics were more consistent with those of Weaver syndrome in early childhood, but became less obvious with age. This is the second adult reported with Weaver syndrome and provides documentation of the adult phenotype. The diagnosis may be more difficult to make in adolescents and adults if one uses criteria developed for facial manifestations in young children.

San Luis Valley recombinant chromosome 8 and tetralogy of Fallot: a review of chromosome 8 anomalies and congenital heart disease.

Tetralogy of Fallot, the most common cyanotic heart defect, has not been closely associated with a specific chromosome defect. The San Luis Valley Recombinant Chromosome 8 [SLV Rec(8)] syndrome is strongly associated with congenital heart disease, particularly tetralogy of Fallot. This article reviews SLV Rec(8) syndrome and other chromosome 8 aberrations to suggest locations for cardiogenic genes. SLV Rec(8) [rec(8),dup q,inv(8)(p23q22)] syndrome has been found in Hispanic families in the southwestern United States. Congenital heart disease is found in 93.3% of SLV Rec(8) individuals (n = 45), with tetralogy of Fallot constituting 40.5% of all lesions and conotruncal defects, 55.6%. These frequencies exceed the incidence of tetralogy of Fallot (10%) and conotruncal defects (20%) among all children with heart defects (P less than 0.003 for both). Review of patients with deletion 8p (n = 13) showed heart defects in 84.6% with 27.3% being conotruncal defects. Among duplication 8q patients (n = 20), 45% had heart defects with conotruncal defects constituting 44%. Neither group differed significantly from expected in its incidence of conotruncal defects. Among patients with mosaic trisomy 8 (n = 47), 12 had heart abnormalities including one conotruncal defect. Among 3 patients with other rec(8) chromosomes, one had a ventricular septal defect. The cause of heart defects in SLV Rec(8) cannot be assigned to either the deletion of 8p or the duplication of 8q. The lack of an association between other chromosome 8 abnormalities and tetralogy of Fallot suggests that genes at the SLV Rec(8) breakpoints or an interaction between genes on both arms of chromosome 8 are important.