Pro239Ser: a novel recessive mutation of the Pit-1 gene in seven Middle Eastern children with growth hormone, prolactin, and thyrotropin deficiency.

Pit-1, a member of the POU-homeo domain protein family, is one of the transcription factors responsible for anterior pituitary development and pituitary-specific gene expression. Here, we describe seven children with GH, PRL, and TSH deficiency from three, reportedly unrelated, Middle Eastern families, harboring a newly recognized Pro- > Ser recessive mutation in codon 239 of the Pit-1 gene. The mutated residue is located at the beginning of the second alpha-helix of the POU-homeodomain and is strictly conserved among all POU proteins. The Pro239Ser mutant binds DNA normally but is unable to stimulate transcription.

Novel mutations and polymorphisms in the Fanconi anemia group C gene.

Fanconi anemia (FA) is an autosomal recessive disorder associated with hypersensitivity to DNA cross-linking agents and bone marrow failure. At least four complementation groups have been defined, and the FA group C gene (FAC) has been cloned. We have screened 76 unrelated FA patients of diverse ethnic and geographic origins and from unknown complementation groups for mutations in the FAC gene either by chemical cleavage mismatch analysis or by single-strand conformational polymorphism (SSCP). Five mutations were detected in four patients (5.3%), including two novel mutations (W22X and L496R). Nine polymorphisms were detected, seven of which have not been described previously (663A-->G, L190F, IVS6 + 30C-->T, I312V, V449M, Q465R, and 1974G-->A). Six of the nine polymorphisms occurred in patients or controls from the Tswana or Sotho chiefdoms of South Africa and were not found in 50 unrelated European controls. Restriction site assays were established for all 8 pathogenic mutations identified in the FAC gene to date and used to screen a total of 94 unrelated FA patients. This identified only one other group C patient, who was homozygons for the mutation IVS4 + 4A-->T. This study indicates that the proportion of FA patients from complementation group C is generally likely to be less than 10%. Guidelines for the selection of FA patients for FAC mutation screening are proposed.

Genetic mapping of the FACC gene and linkage analysis in Fanconi anaemia families.

Fanconi anaemia is an autosomal recessive disorder associated with increased chromosome breakage and progressive bone marrow failure. The gene for complementation group C (FACC) has been cloned and mapped to chromosome 9q22.3, but neither its genetic location nor the proportion of patients belonging to group C is known. We have used a polymorphism within the FACC gene to localise it within a 5 cM interval on chromosome 9q, bounded by D9S196/D9S197 and D9S176. The genes for Gorlin's syndrome and multiple self-healing squamous epitheliomata have also been mapped to this interval. Linkage analysis with the three highly informative microsatellite polymorphisms flanking FACC in 36 Fanconi anaemia families showed that only 8% of families were linked to this locus. This indicates that the genes for the other Fanconi anaemia complementation groups must map to one or more different chromosomal locations. The markers also allowed rapid exclusion of 56% of the families in our panel from complementation group C, thus substantially reducing the number of patients who need to be screened for FACC mutations.

Familial growth hormone deficiency: a model of dominant and recessive mutations affecting a monomeric protein.

Two families with familial isolated GH deficiency (IGHD) were studied, type II (autosomal dominant) and type I (autosomal recessive), whose GH1 genes exhibit cosegregation with IGHD. DNA sequencing of the GH1 genes of the first family (IGHD II) demonstrated heterozygosity for a T-->C transition in the sixth base of the donor splice site of intron III. The GH1 gene mutation in the second family (IGHD I) was found, in a previous study, to be a G-->C transversion altering the first base of the donor splice site of intron IV. Interestingly, analysis of the transcripts derived from the mutant IGHD II allele revealed that the sequences corresponding to exon III were absent due to an exon skip that causes the loss of amino acids 32-71 from the mature GH protein. In contrast, the IGHD I mutation activates a cryptic donor splice site 73 bases upstream of the normal exon IV donor splice site causing loss of amino acids 103-126 of exon IV followed by a reading frameshift and synthesis of 94 novel amino acids before chain termination 88 nucleotides downstream of the normal GH stop codon. It is hypothesized that, because of the loss of protein sequences derived from exons IV and V, the IGHD I mutation products are not transported to secretory granules and thus cannot perturb secretion of the normal monomeric GH protein. In contrast the T-->C IGHD II mutant allele product retains these sequences and is transported to secretory granules where it can interact with the normal allele product producing a dominant-negative effect at the protein level.

Receptor mutations and haplotypes in growth hormone receptor deficiency: a global survey and identification of the Ecuadorean E180splice mutation in an oriental Jewish patient.

Eight different mutations were detected in the growth hormone (GH) receptor gene of patients with inherited GH receptor deficiency (GHRD; Laron syndrome) from five continents. All the mutations are located in the extracellular domain of the receptor and are predicted to cause gross structural abnormalities and non-functional receptor molecules. They include three nucleotide changes in the coding region causing translational stop signals, including the newly identified E183X mutation; two nucleotide changes in introns that affect splice junctions; two dinucleotide deletions that result in stop codons downstream; and one single nucleotide change that activates a donor splice site within an exon and results in a transcript missing 24 nucleotides. This latter mutation (E180splice) was first identified in a cohort of patients with GHRD from southern Ecuador. Based on the fact that the E180splice mutation generates a new cleavage site for the restriction enzyme MnlI, a simple diagnostic test has been developed that can be carried out on dried blood spots collected on filter paper. A total of 55 affected individuals from Ecuador has been found to be homozygous for this mutation. Asymptomatic carriers can also be detected, and 104 of 150 individuals screened were found to be carriers. Using this test, the E180splice mutation has recently been detected in one of two oriental Jewish patients from Israel.

Heterogeneous growth hormone (GH) gene mutations in familial GH deficiency.

The GH1 genes of probands of two families with familial isolated GH deficiency (IGHD) were sequenced. Double stranded sequencing of the polymerase chain reaction (PCR) amplification products from genomic DNA of two affected cousins in a consanguineous Turkish family revealed a G-->A transition in the 20th codon of the GH1 signal peptide. This substitution converts a TGG (Trp) to a TAG (stop) codon and generates a new AluI recognition site. PCR amplification of the GH1 alleles of family members, followed by AluI digestion, revealed that the G-->A transition segregated with the IGHD phenotype. In a Saudi Arabian family, a G-->C transversion was found that alters the first base of the donor splice site of intron IV. This substitution should perturb mRNA splicing, resulting in an altered protein product which should be unstable or bioinactive. This transversion also destroys an HphI site, which was used to assay samples from relatives. Digestion of PCR amplification products with HphI demonstrated cosegregation of the G-->C transversion with IGHD. These results demonstrate that familial IGHD is a heterogeneous disease that perturbs different steps in the expression of the GH1 gene.

A new autosomal recessive anomaly mimicking Fanconi's anaemia phenotype.

A family in which three siblings born to related parents all manifested clinical abnormalities characteristic of Fanconi's anaemia (microcephaly, short stature, slow growth, beak nose, micrognathia, skin dyspigmentation and forearm and thumb dysplasia in 2/3) is reported. All five family members had normal spontaneous chromosome breakage, a normal response to diepoxybutane and mitomycin C, and were fully informative for linkage with four DNA markers from chromosome 20q12-13.3 with no evidence for linkage. It is concluded that abnormalities typical for Fanconi's anaemia are inherited as an autosomal recessive without the defect responsible for increased chromosomal fragility and independently from the genes so far identified as being responsible for Fanconi's anaemia.

Genetic linkage studies of X-linked hypophosphataemic rickets in a Saudi Arabian family.

UNLABELLED: OBJECTIVE, PATIENTS AND DESIGN: X-linked hypophosphataemic rickets (HYP) is the most common inherited form of rickets and the gene causing this disorder has been localized to Xp22.3-p21.3 by linkage studies of affected families of Northern European origin. In addition, the locus order Xpter-(DXS207-DXS43,DXS197)-HYP-DXS41-X cen has been established and the flanking markers are useful for the presymptomatic diagnosis of HYP. However, a recent study indicates locus heterogeneity and this may hinder the use of the flanking markers for presymptomatic diagnosis in additional families and in particular those from different populations. We have therefore investigated one Saudi-Arabian family (13 affected and six unaffected members) with hypophosphataemic rickets for linkage to these and other X-linked markers. A total of 17 cloned human X chromosome sequences identifying restriction fragment length polymorphisms were used to localize the mutant gene causing this disorder in the Saudi Arabian family. RESULTS: Nine (four from Xp and five from Xq) of the 17 X-linked DNA probes proved informative and linkage was established between HYP and the DSX41 locus, peak LOD score = 4.22 (recombination fraction, theta = 0.00). A positive peak LOD score of 2.32 (theta = 0.05) was also obtained between HYP and the DXS207 locus. Thus, the HYP gene in this Saudi Arabian family is linked to two of the four flanking markers which demonstrated linkage in families of Northern European origin. CONCLUSION: We conclude that the X-linked hypophosphataemic rickets gene in a Saudi Arabian family is located in the Xp22.3-p21.3, a region where this gene has previously been mapped by linkage studies of families of Northern European origin. Our studies have not demonstrated locus heterogeneity, so the flanking markers for HYP previously established in the families of Northern-European origin will be useful in the genetic counselling and presymptomatic diagnosis of this disorder in the Saudi Arabian family.

Mortality, neoplasia, and Creutzfeldt-Jakob disease in patients treated with human pituitary growth hormone in the United Kingdom.

OBJECTIVE: To determine the cause of death and incidence of neoplasia in patients treated with human pituitary growth hormone. DESIGN: A long term cohort study established to receive details of death certification and tumour registrations through the Office of Population Censuses and Surveys and NHS central register. PATIENTS: All patients (1246 male, 662 female) treated for short stature with pituitary growth hormone under the Medical Research Council working party and health services human growth hormone committee. MAIN OUTCOME MEASURES: Death or development of neoplasia. RESULTS: 110 patients died (68 male, 42 female; aged 0.9-57 years) from 1972 to 1990. Fifty three death were from neoplasia responsible for growth hormone deficiency (27 craniopharyngioma, 24 other intracranial tumour, two leukaemia); two from histiocytosis X; and 13 from pituitary insufficiency. Six patients died of Creutzfeldt-Jakob disease, six of other neurological disorders, and eight of acute infection. Other deaths were apparently unrelated to growth hormone deficiency or its treatment. Seventeen tumours (in 16 patients) were identified during or after growth hormone treatment. Four were in patients with previous intracranial neoplasia and two were after cranial irradiation. Thirteen were intracranial, the others being Hodgkin's lymphoma, osteosarcoma, carcinoma of colon, and basal cell carcinoma. CONCLUSIONS: Recurrence or progression of intracranial tumours and potentially avoidable metabolic consequences of hypopituitarism were the main causes of death. Growth hormone treatment probably did not contribute to new tumour development. Creutzfeldt-Jakob disease after pituitary growth hormone treatment continues to occur in the United Kingdom. This cohort must remain under long term review.

A new syndrome of congenital hypoparathyroidism, severe growth failure, and dysmorphic features.

Twelve infants (six boys, six girls) with severe hypocalcaemic tetany or convulsions were seen over a three year period. Nine patients were symptomatic in the newborn period. Their hypocalcaemia was associated with hyperphosphataemia and very low concentrations of immunoreactive parathyroid hormone. None of the babies suffered from congenital cardiac disease. Cell mediated immunity, measured in five patients, was normal. There were no chromosomal abnormalities but all patients shared several dysmorphic features including deep set eyes, microcephaly, thin lips, beaked nose tip, external ear anomalies, micrognathia, and depressed nasal bridge. Mental retardation of varying degree was found in all patients. All had severe intrauterine and postnatal growth retardation. Four patients have died. The remaining eight patients are on treatments with vitamin D and calcium supplements with no change in their growth pattern. We believe that this association of congenital hypoparathyroidism with severe growth failure and dysmorphism represents a new syndrome.

Tissue and serum insulin-like growth factor I (IGF I) concentrations in rats subjected to temporary protein-energy malnutrition early in life.

Rats subjected to temporary protein-energy malnutrition and subsequent nutritional rehabilitation remain smaller than adequately fed animals, have a subnormal insulin secretion and persisting cellular hypoplasia in several tissues. This investigation studies whether impaired production of insulin-like growth factor I (IGF I) is another persisting consequence of malnutrition. Rats were subjected to severe protein-energy malnutrition between 3 and 6 weeks of age and subsequently fed adequate diet up to 12 weeks of age. Serum and tissue samples for analysis of IGF I were obtained at 12 weeks of age. IGF I concentrations were similar in serum, heart, liver and lung of previously malnourished and control rats. In the kidneys of previously-malnourished rats the IGF I concentration was twice that of control rats. Results suggest that during protein-energy malnutrition and subsequent nutritional rehabilitation IGF I tissue concentrations are primarily regulated by the prevailing plane of nutrition. It is speculated that the temporary protein-energy malnutrition blunts the cellular capacity for IGF I production and, except in the kidney, prevents increased IGF I tissue concentrations and associated compensatory growth.

Long-term effects on glucose tolerance and insulin secretory response to glucose following a limited period of severe protein or energy malnutrition in young rats.

The long-term effects on growth, glucose tolerance and insulin secretory response to glucose of temporary malnutrition early in life have been investigated. Rats were weaned onto either normal diet (18% protein), a protein-restricted diet (5% protein) or a diet adequate in protein but restricted in amount to equal the energy intake of protein-restricted rats ("energy restriction"). From 6 weeks of age and onwards all rats were fed normal diet. Body weight gain was inhibited by both protein and energy restriction but growth was resumed when rats were transferred to normal diet. Protein restriction impaired glucose tolerance and blunted insulin secretory response to glucose. Following refeeding glucose tolerance was normalized but insulin secretory response remained impaired at 12 weeks of age. Energy restriction did not initially affect glucose tolerance and insulin secretion. However, after refeeding male energy restricted rats developed a delayed and exaggerated insulin secretory response to glucose without concomitant deterioration of glucose tolerance. It is suggested that temporary protein restriction at a young age impairs pancreatic B-cell function and decreases peripheral sensitivity to insulin. By contrast, temporary energy restriction does not directly affect B-cell function but confers insulin resistance and compensatory increases of the insulin secretory response to glucose later in life. These models of malnutrition offer possibilities to further study long-term effects of early nutritional insults.

Protein-energy malnutrition induces changes in insulin sensitivity.

Early protein deprivation impairs glucose tolerance and insulin secretion, but the former generally recovers first. We have therefore investigated insulin sensitivity in vivo and in isolated muscle and cultured hepatocytes during protein-energy malnutrition and long-term follow-up in rats. Rats were weaned at 3 weeks onto normal rat chow (N rats) or onto 5% protein diet (LP rats). At 6 w of age LP rats were transferred to N chow. Insulin sensitivity was studied in the three systems at 3, 6, 12 and 24 w. At 6 w LP rats showed a greater and more prolonged fall in serum glucose in response to injected insulin than age--or weight--matched N, but by 12 w the difference was not significant. Similarly, soleus muscle from 6 w LP showed a higher basal rate of glucose transport and responded to a lower insulin dose than N, but there was no difference in sensitivity at 12 w. Hepatocytes from 6 w LP showed higher basal incorporation of glucose into glycogen than N, but insulin sensitivity was not different. Thymidine incorporation into hepatocyte DNA responded to lower insulin doses in 6 w LP than N. There was a decrease in insulin sensitivity with age in all experimental systems. Increased insulin sensitivity during malnutrition was seen in all tissues studied but differences between the two groups were not significant at 12 w and probably do not account for the normal glucose tolerance in the presence of low serum insulin levels previously reported.

Growth failure secondary to moyamoya syndrome.

We describe a boy who presented at the age of 7 years with short stature due to hypopituitarism. Six months after starting appropriate hormone replacement treatment at the age of 8 he suffered his first generalised convulsion. Further neuroradiological investigation led to the diagnosis of moyamoya syndrome.

Changes in leg length and height during treatment with somatotropin.

Knemometry has been used in a short term double blind placebo controlled trial in 13 patients with normal variant short stature receiving treatment with somatropin to assess the power of the change in lower leg velocity at one month to predict the increase in height velocity at six months. Used in this way the method has a positive predictive value and sensitivity of 90% and a negative predictive value and specificity of 50%. Although not a perfect discriminatory test, knemometry is a more reliable and less invasive way of analysing the likely value of a growth promoting treatment than metabolic assays in individual patients. Given the possible future rapid expansion of the use of somatropin in short stature of various aetiologies there is a need for a relatively simple and inexpensive means of evaluating response to treatment.

Long-term follow-up after early protein-calorie malnutrition in young rats: sex difference in glucose tolerance and serum insulin levels.

Acute protein-calorie malnutrition impairs both glucose tolerance and insulin secretion, and long-term pancreatic damage leading to malnutrition diabetes has been postulated. The present study has investigated this association in rats weaned onto 5% protein (LP) or 18% protein (normal, N) diet from age 3 weeks to 6 weeks. From 6 weeks both LP and N rats were fed N diet for the remainder of the experiment. LP rats did not grow while on the LP diet and remained significantly lighter for several weeks. Nose to tail tip length was identical for the two groups in both sexes at both 24 and 48 weeks, and mean body weight was not significantly less in LP than N after 18 weeks in either sex. Protein/DNA ratios in LP (an index of cell size) remained lower than N in heart, skeletal muscle, and lung at 24 and 48 weeks, but not in gut, liver, or kidney tissues. Thus, skeletal growth was apparently not impaired by the early malnutrition, but muscle tissue did not catch up. The similarity in final body weight implies greater adipose stores in older LP rats. At 12 weeks there was no difference in glucose tolerance tests (GTT) either between males and females within a dietary group or between N and LP, despite impaired insulin secretion in LP. Both fasting glucose levels and GTT deteriorated markedly between 12 and 48 weeks in all rats, but especially in LP males. Serum insulin levels following glucose injection were lower at 48 weeks than 12 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

Newborn splenic volumes vary under different malaria endemic conditions.

Ultrasound was used to measure newborn splenic dimensions and calculate the volumes in a malarious and a non-malarious region of Papua New Guinea. The median splenic volume of infants born in Madang, where malaria transmission is high throughout the year, was 5.2 cm3/kg, while that of infants born in Goroka, where malaria is not endemic, was 2.6 cm3/kg. The cause of this difference is unknown, but possible explanations include fetal exposure to malaria antigens in utero and the high incidence of inherited red cell disorders in the malarious regions of Papua New Guinea.