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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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The orphan nuclear receptor NR0B1 encodes the DAX1 protein, which stands for the dosage sensitive sex-reversal (DSS), adrenal hypoplasia congenita (AHC) critical region on the X-chromosome, gene 1. DAX1 was initially identified as part of a contiguous gene syndrome and is known to function in the proper formation of the adult adrenal gland. It has been hypothesized that DAX1 is responsible for the establishment and maintenance of the steroidogenic axis of development. Recent insight from the murine ortholog Dax1 along with reports of an alternatively spliced variant in humans suggests that Dax1 has additional functional roles beyond those previously understood. Here, we review DAX1/Dax1 known functional roles and the recently hypothesized function in the development of the embryo and in the maintenance of embryonic stem cell pluripotency.

Transcriptional network analysis in steroidogenic axis cell lines requires an understanding of cellular network composition and complexity. Previous studies have shown that absence of transcriptional network components in a cell line compromises that cell line's functional capacity for transcriptional regulation. Our goal was to analyze qualitatively steroidogenic axis-derived cell lines' expression of a putative transcriptional network involved in human and mouse development. To pursue this analysis we used Northern blots and a high density-multiplexed reverse transcription-polymerase chain reaction (HD-MRT-PCR) approach. Our results revealed that, while some members of this putative network were universally expressed, only a minority of the non-constitutive targeted transcripts were present in any single line. Based on our data and previously published results for contextual expression of these transcription factors, a model was constructed possessing the topology suggestive of a scale-free network: certain network members were highly connected nodes and would represent critical sites of vulnerability. The importance of these highly connected nodes for network function is supported by the severe phenotypes exhibited by human patients and animal models when these genes are mutated. We conclude that knowledge of network composition in specific cell lines is essential for their use as models to investigate functional interactions within selected subnetworks.

Arx is a homeobox-containing gene with a high degree of sequence similarity between mouse and zebrafish. Arx is expressed in the forebrain and floor plate of the developing central nervous systems of these vertebrates and in the presumptive cortex of fetal mice. Our goal was to identify genes in Xp22.1-p21.3 involved in human neuronal development. Our in silico search for candidate genes noted that annotation of a human Xp22 PAC (RPCI1-258N20) sequence (GenBank Accession No. AC002504) identified putative exons consistent with an Arx homologue in Xp22. Northern blot analysis showed that a 3.3kb human ARX transcript was expressed at high levels in fetal brain. A 5.9kb transcript was expressed in adult heart, skeletal muscle, and liver with very faint expression in other adult tissues, including brain. In situ hybridization of ARX in human fetal brain sections at various developmental stages showed the highest expression in neuronal precursors in the germinal matrix of the ganglionic eminence and in the ventricular zone of the telencephalon. Expression was also observed in the hippocampus, cingulate, subventricular zone, cortical plate, caudate nucleus, and putamen. The expression pattern suggests that ARX is involved in the differentiation and maintenance of specific neuronal cell types in the human central nervous system. We also mapped the murine Arx gene to the mouse genome using a mouse/hamster radiation hybrid panel and showed that Arx and ARX are orthologues. Therefore, investigations in model vertebrates may provide insight into the role of ARX in development. The recent identification of ARX mutations in patients with various forms of mental retardation make such studies in model organisms even more compelling.

Molecular genetics is rapidly moving from simple identification of a gene of interest to characterization of gene products as components in complex networks. Critical tools for gene product analysis require a rapid method for evaluation of contextual expression. Here, we describe a robust, high primer density, single-tube, multiplex reverse transcription (HD-MRT) technique. This approach is capable of analyzing for the presence of numerous transcripts when polymerase chain reaction (PCR) is subsequently employed for individual gene-specific sequence amplification (HD-MRT-PCR). This assay substantially increases the total number of different cDNAs for amplification beyond previously published techniques. Our approach simultaneously eliminates RNA quality control issues for samples run in parallel while improving efficiency in the use of time and materials. This assay is designed for broad applicability and accessibility, employs modifications of commercially available components, and allows more than 25 independently selected gene-specific primers to be used simultaneously. Our protocol allows multiplexed primers to behave similarly to uniplex RT reactions, while avoiding potential interference between gene-specific and/or nonspecific primers during annealing and reverse transcription. Expression of putatively networked homologous transcripts was analyzed in multiple cell lines and tissues from mouse and human to validate the technique., (Copyright 2001 Elsevier Science.)

Adrenal gland development is complex and poorly understood at the molecular level. Only a subset of patients with adrenal hypoplasia congenita (AHC) carry mutations in DAX1, a member of the nuclear hormone receptor superfamily. Therefore we set out to identify other candidate genes responsible for AHC by characterizing genes involved in fetal adrenal development. To identify these genes, we studied the differential expression of genes in fetal rat adrenals comparing tissues at 14 and 15 days postcoitum (dpc) since this period encompasses major morphological change in rat adrenal development. Fetal rat adrenals were dissected, cDNAs were prepared, and suppressive subtractive hybridization was performed. We isolated 126 clones of putatively differentially expressed clones and approximately 250 bp of each of the clones was sequenced. The most interesting putative developmental genes were examined. One member of the extracellular PTN/MDK (pleiotrophin/midkine) heparin-binding protein family involved in regulation of growth and differentiation was selected for initial study. We obtained full-length transcript by 3' rapid amplification of cDNA ends and performed Northern analysis on rat adrenal RNA from fetuses at 13, 14, 15, 17, and 19 dpc and newborns. Results from those analyses demonstrated the highest Mdk expression at days 13 and 14 followed by a moderate decrease of expression during the fetal stages thereafter. In the newborn, Mdk expression is nearly undetectable. Our results indicate that Mdk has a very specific pattern of fetal expression in the adrenals. We conclude that Mdk is involved early in fetal development of the rat adrenal. Therefore, MDK is a candidate gene for AHC not due to DAX1 mutations., (Copyright 2000 Academic Press.)

Fructose-1,6-diphosphatase (FDPase) deficiency is characterized by episodes of lactic acidemia, hypoglycemia, and ketonuria. Liver biopsy and subsequent enzyme analysis most reliably make the diagnosis. Review of the literature reveals 85 cases. Glycerol intolerance syndrome (GIS) is less well defined. There are only a handful of cases reported. We describe a patient with FDPase deficiency and significant glyceroluria and propose that GIS may be caused by partial deficiency of FDPase.

Because the initial step in the metabolism of glucose involves phosphorylation by hexokinase (HK), we tested the hypothesis that the expression of the isozymes, hexokinase type 1 (HK1) and hexokinase type 2 (HK2), would be different in rat mammary tissue during pregnancy and lactation. RNA was extracted from mammary tissue dissected from timed pregnant rats (from gestional days 10 to 21) and nursing rat mothers (up to postnatal day 5) for mRNA examination by reverse transcriptase and polymerase chain reaction (RT-PCR) using isozyme specific oligonucleotide primers to the HK1 and HK2 cDNAs. The HK1 mRNA was expressed in both the nonlactating and lactating mammary gland tissue, but HK2 mRNA was found only during lactation. We speculate that the pattern of HK expression might affect human milk production and quality., (Copyright 1999 Academic Press.)

The diagnosis of bacterial infections can be difficult and time consuming. Rapid and reliable molecular triage of potentially infected patients, particularly the young and the elderly, would prevent unnecessary hospitalizations, reduce associated medical costs, and improve the quality of care. Polymerase chain reaction (PCR) amplification utilizing a universal bacterial primer pair, followed by hybridization with species-specific probes, would allow rapid identification of the presence or absence of bacterial DNA, along with an identification of the bacterial species present. Molecular microbiological analyses will require access to bacterial strain standards that can be catalogued and distributed to clinical laboratories. We amplified template DNA in filter paper spots containing boiled bacteria from 14 clinical isolates using a universal primer pair for the 16S ribosomal RNA (rDNA) coding sequence. Species-specific probes were hybridized to the amplification products for bacterial species identification. We conclude that template DNA can be identified with species-specific probes after universal bacterial amplification with a single primer pair. We also demonstrate a rapid and efficient method for the long-term storage and cataloguing of bacterial DNA for use in quality control at clinical laboratories adopting molecular diagnostic methodologies. We speculate that PCR amplification combined with species-specific probe hybridization not only will represent an improvement over culture-based methods in terms of speed, sensitivity, and cost, but will also allow for the identification of unculturable bacteria and emerging or reemerging pathogenic organisms., (Copyright 1999 Academic Press.)

In mammals, sex is determined by the Y chromosome, which encodes a testis-determining factor (TDF). This factor causes the undifferentiated embryonic gonads to develop as testes rather than ovaries. The testes subsequently produce the male sex hormones that are responsible for all male sexual characteristics. In 1990, the sex-determining gene, TDF, was identified and termed SRY in humans (Sry in mice). It encodes a protein containing a high mobility group (HMG) motif, which confers the ability to bind and to bend DNA. Genetic evidence supporting SRY as TDF came from the observation of a male phenotype in XX mice transgenic for a small genomic fragment containing Sry, and from the study of XY sex-reversed individuals who harbor de novo mutations in the SRY coding sequence. Other non-Y-linked genes involved in sex determination were subsequently found by genetic analysis of XY sex-reversed patients not explained by mutations in SRY. These genes are WT1, SF1, DAX1, and SOX9. A regulatory cascade hypothesis for mammalian sex determination, proposing that SRY represses a negative regulator of male development, was recently supported by observation of mice that expressed a DAX1 transgene and developed as XY sex-reversed females. The role of some sex-determining genes, such as DAX1 and SF1, in the development of the entire reproductive axis, a functionally integrated endocrine axis, leads to a new concept. Normal sexual development may result from the functional and developmental integration of a number of different genes that play roles in sex determination, sexual differentiation, and sexual behavior., (Copyright 1998 Academic Press.)