Consequences of JAG1 mutations.

BACKGROUND: Alagille syndrome (AGS) is a multi-system, autosomal dominant disorder with highly variable expressivity, caused by mutations within the Jagged1 (JAG1) gene. METHODS: We studied 53 mutation positive relatives of 34 AGS probands to ascertain the frequency of clinical findings in JAG1 mutation carriers. RESULTS: Eleven of 53 (21%) mutation positive relatives had clinical features that would have led to a diagnosis of AGS. Seventeen of the 53 (32%) relatives had mild features of AGS, revealed only after targeted evaluation following the diagnosis of a proband in their family. Twenty five of the 53 (47%) mutation positive relatives did not meet clinical criteria, and two of these individuals had no features consistent with AGS at all. The frequency of cardiac and liver disease was notably lower in the relatives than in the probands, characterising the milder end of the phenotypic spectrum. The characteristic facies of AGS was the feature with the highest penetrance, occurring almost universally in mutation positive probands and relatives. CONCLUSIONS: This study has implications for genetic counselling of families with AGS and JAG1 mutations.

Homozygosity for the V37I Connexin 26 mutation in three unrelated children with sensorineural hearing loss.

Mutations in the Connexin 26 (Cx26) gene have been found to account for approximately 20% of all childhood deafness. This number approaches 50% in documented recessive cases of hearing loss. Two mutations, 35delG and 167delT, account for the majority of reported mutations in this gene, but to date, more than 60 mutations have been described. No other single gene has yet been identified that contributes this significantly to the aetiology of hearing loss. Several mutations in this gene have been found to predominate in specific ethnic populations (167delT in Ashkenazi Jews and 235delC in Japanese individuals). While the majority of mutations found in Cx26 result in frame shifts and premature terminations, a number of missense mutations have also been identified. The V37I missense mutation has been reported as both a polymorphism and as a potentially disease-causing missense mutation. The present authors have identified three unrelated individuals with sensorineural hearing loss who are homozygous for this mutation. One individual is of Philippine ancestry, another is from a Chinese and Cambodian background, while the third is of Chinese ancestry, raising the possibility that this mutation may be more frequent among populations in eastern Asia.

Mutation analysis of Jagged1 (JAG1) in Alagille syndrome patients.

Alagille syndrome (AGS) is an autosomal dominant disorder caused by mutations in Jagged1 (JAG1), a ligand in the evolutionarily conserved Notch signaling pathway. Previous studies have demonstrated that a wide spectrum of JAG1 mutations result in AGS. These include total gene deletions, protein truncating, splicing and missense mutations which are distributed across the coding region of the gene. Here we present results of JAG1 mutation screening by SSCP and FISH in 105 patients with AGS. For these studies, new primers were designed for 12 exons. Mutations were identified in 63/105 patients (60%). The spectrum of the JAG1 mutations presented here is consistent with previously reported results. Eighty three percent (52/63) of the mutations were protein truncating, 11% (7/63) were missense, 2% (1/63) were splice site, and 5% (3/63) were total gene deletions demonstrable by FISH. Six of the missense mutations are novel. As has been reported previously, there is no apparent relationship between genotype and clinical phenotype.

Somatic and germ line mosaicism and mutation origin for a mutation in the L1 gene in a family with X-linked hydrocephalus.

X-linked hydrocephalus is caused by mutations in the gene for neural cell adhesion molecule L1 (L1CAM). In this report, we describe identification of a mutation in an isolated case of hydrocephalus with adducted thumbs. Tracing the origin of the mutation within the family showed a degree of somatic mosaicism in the asymptomatic maternal grandfather of the propositus. This report highlights the need to take mosaicism into account when counselling relatives of affected individuals.

Lateral meningocele syndrome: three new patients and review of the literature.

One female and two male patients with multiple lateral meningoceles are presented. They do not have neurofibromatosis or Marfan syndrome and share findings with the two previously described patients with multiple lateral meningoceles. The original report by Lehman et al. [1977: J Pediatr 90:49-54] was titled "familial osteosclerosis," because osteosclerosis was present in the proposita and her mother; the patient described by Philip et al. [1995: Clin Dysmorphol 4:347-351] also had increased bone density of the skull base and the sutures. Thickened calvaria were present in one of our patients; two had a prominent metopic suture. Other shared findings include multiple lateral meningoceles, Wormian bones, malar hypoplasia, downslanted palpebral fissures, a high narrow palate, and cryptorchidism in males. In addition, our patients showed ligamentous laxity, keloid formation, hypotonia, and developmental delay. A short umbilical cord was noted in two patients. One had a hypoplastic posterior arch of the atlas and an enlarged sella, as reported by Lehman et al. [1977]. Our patients appear to have the same syndrome as previously reported. We suggest it be called "lateral meningocele syndrome," because of this unique finding.

Production and characterization of monoclonal antibodies to dehydroepiandrosterone sulfate: application to direct enzyme-linked immunosorbent assays of dehydroepiandrosterone sulfate and androsterone/epiandrosterone sulfates in plasma.

Mice were immunized with 5-androstene-3 beta-ol-7,17-dione-7-CMO:bovine serum albumin (DHEA-7-O-CMO-BSA) or 5-androstene-3 beta-ol-17-one hemisuccinate-bovine serum albumin (DHEA-3HS-BSA) conjugates and monoclonal antibodies were produced, characterized, and selected for maximum DHEAS binding. Of these hybridomas, four clones from DHEA-3HS-BSA-immunized mice had acceptable criteria for the development of a competitive enzyme-linked immunosorbent assay (ELISA) for DHEAS in plasma. One hybridoma supernatant from DHEA-7-O-CMO-BSA-immunized mice showed 360% cross-reactivity to both androsterone sulfate and epiandrosterone sulfate. This allows the possibility of the direct determination of androsterone sulfate and epiandrosterone sulfate in plasma after correction for the DHEAS contribution. Both ELISAs employ a DHEA-3HS-thyroglobulin conjugate adsorbed to the wells of a standard 96-well microtiter plate. DHEAS in the standards or diluted plasma sample competes with immobilized DHEA-3HS-thyroglobulin for antibody-binding sites. Antibody is detected with anti-mouse-lg peroxidase by further washing, adding o-phenylenediamine substrate, and reading the absorbance at 492 nm. The ELISAs are simple, reproducible, and reliable and, to our knowledge, they are the first tests employing monoclonal antibodies to DHEAS.

Frontonasal malformation and cloacal exstrophy: a previously unreported association.

We report on a child with frontonasal malformation (FNM) and cloacal exstrophy, a combination of findings that have not been reported previously. In FNM and cloacal exstrophy, associated malformations are rare. FNM and cloacal exstrophy both represent abnormalities of the development of the midline field; this combination of anomalies in this patient suggests an impairment of caudal and cranial midline development during blastogenesis.

Autosomal dominant "Opitz" GBBB syndrome due to a 22q11.2 deletion.

We report on a family with autosomal dominant paternally inherited "Opitz" GBBB syndrome and an additional case with findings which have been reported in that syndrome. In each case the propositus presented with a vascular ring. Since a vascular ring may be a sign of a 22q11.2 deletion [Zacki et al., 1995], FISH (fluorescence in situ hybridization) studies were performed. These studies demonstrated a 22q11.2 deletion in the 3 affected individuals. Review of Opitz GBBB syndrome and the 22q11.2 microdeletion syndrome demonstrates significant overlap of manifestations including both facial characteristics and structural anomalies. Based on the phenotypic overlap and the presence of a 22q11.2 deletion in our patients with Opitz GBBB syndrome and the presence of a deletion in a patient with lung hypoplasia, absent pulmonary artery, and long segment tracheomalacia, we propose that, in some cases, the Opitz GBBB syndrome may be due to a 22q11.2 deletion. This enlarges the list of "syndromes" associated with the 22q11.2 deletion, which presently includes most patients with DiGeorge, velocardiofacial, and conotruncal anomaly face syndrome.

Short-term response of nonurea organic osmolytes in human kidney to a water load and water deprivation.

The cells of the inner medulla of the mammalian kidney accumulate high concentrations of nonurea organic osmolytes. The organic osmolytes found in the kidney include glycine betaine and sorbitol. This study was designed to measure changes in the urinary excretion of glycine betaine and sorbitol and the plasma concentration of glycine betaine in response to an acute water load (20 ml/kg) or acute water deprivation in young healthy males. In response to a water load the urinary excretion of glycine betaine and sorbitol increased parallel with or shortly after urinary urea excretion. The increase in urinary urea and sorbitol excretions preceded maximum minute volume, whereas peak glycine betaine excretion was closely related to maximum urine minute volume. Subsequently, urea, sorbitol, and glycine betaine excretion rates returned to baseline. In contrast, during water deprivation no change in glycine betaine, sorbitol, and urea urinary excretions occurred during the study period. Plasma glycine betaine concentration was stable during both diuresis and antidiuresis. We conclude that the organic osmolytes glycine betaine and sorbitol are components of a physiological and dynamic system in response to an acute water diuresis.

Abnormal glycine betaine content of the blood and urine of diabetic and renal patients.

In normal human plasma the concentrations of the renal osmolyte, glycine betaine, are usually between 20 and 70 mumol/l, in adult males (median 44 mumol/l) higher than in females (34 mumol/l). Concentrations are lower in renal disease (median 28 mumol/l) and normal in diabetes. Urinary excretion of glycine betaine shows no sex difference and is frequently elevated both in renal disease and in diabetes (medians: normal, 6.2, renal 12.3 and diabetes, 39.7 mmol/mol creatinine). The elevation in diabetes does not strongly correlate with known renal disease, nor with either urinary microalbumin or plasma creatinine. There is no correlation with glycated haemoglobin. The positive correlation with the excretions of another renal osmolyte, sorbitol, was highly significant in diabetic subjects. In the diabetic group there was also a significant negative correlation between plasma glycine betaine and urine microalbumin.

Glycine betaine and proline betaine in human blood and urine.

In healthy human subjects, glycine betaine concentrations in the blood plasma are normally between 20 and 60 mumol/l, adult males tending to have higher concentrations than females. Proline betaine concentrations are more variable, ranging from undetectable to about 50 mumol/l. Both betaines are present in urine. Whereas the urinary excretion of proline betaine reflects plasma concentrations, with high clearance rates, there is no correlation between plasma and urine glycine betaine concentrations. The apparent clearance rates are low (usually less than 5%). The proline betaine content of human kidney tissue is less than 0.1% of the glycine betaine content, and this is true also of rabbit tissue despite high concentrations of both betaines in rabbit circulation and urine. These data suggest that glycine betaine, but not proline betaine, is important in human and other mammalian biochemistry.

Reporter epitopes: a novel approach to examine transmembrane topology of integral membrane proteins applied to the alpha 1 subunit of the nicotinic acetylcholine receptor.

The development of a novel immunological method called the "reporter epitope" technique to probe the transmembrane topology of integral membrane proteins is described. Using this method, synthetic oligonucleotides encoding epitopes (reporter epitopes) for well characterized monoclonal antibodies (reporter mAbs) were inserted at various locations within the human acetylcholine receptor (AChR) alpha 1 subunit cDNA. The engineered subunits were then expressed along with Torpedo beta 1, gamma, and delta subunits in Xenopus oocytes, and the transmembrane location of the site of insertion was determined by the binding of the 125I-labeled reporter mAbs to whole oocytes. Control reporter epitope insertions at alpha 347 exhibited the expected cytoplasmic location. Reporter epitopes inserted at alpha 429 are located on the extracellular surface. Reporter epitopes that are 16-48 amino acids long do not disrupt assembly or function of hybrid AChRs when inserted near the carboxy terminus (at alpha 429) or in the large cytoplasmic domain (at alpha 347). However, because two reporter epitopes inserted at alpha 157 obliterated subunit assembly and a third reporter epitope when tolerated at this position was inaccessible from the extracellular surface and only marginally accessible after detergent solubilization of the AChRs, a definitive transmembrane location for this region was not possible. Nonetheless, the use of this approach has been successfully demonstrated, and it may be generally applicable to the study of other integral membrane proteins.

Organic osmolytes in human and other mammalian kidneys.

Osmotically-active organic solutes, or osmolytes, have been found in high concentration in the renal inner medulla of a wide variety of mammalian species, but their existence in human kidneys has not yet been shown. The aim of this study was to demonstrate the presence of osmolytes in the human kidney. Human tissues were obtained from kidneys removed surgically for diseases which involved only one pole of the kidney; in most cases this was a tumor. Animal kidneys analyzed were from dogs, pigs and rabbits. Inner medulla and cortex tissue samples were analyzed and found to contain the organic osmolytes glycine betaine, myo-inositol, sorbitol and glycerophosphorylcholine. The levels were much higher in the medulla than in the cortex. Further dissection of the human kidneys showed that sorbitol, glycerophosphorylcholine and glycine betaine were maximally concentrated at the papillary tip, while myo-inositol was found in highest concentration at the papillary base. Osmolytes were in low concentrations or undetectable in rabbit skeletal muscle, ureter and bladder. The organic osmolytes detected are likely to be physiologically important in humans. Studies in other mammals can be used as models for the investigation of the osmolyte system in human kidney function.

Same-day batch measurement of glycine betaine, carnitine, and other betaines in biological material.

Glycine betaine, carnitine, carnitine esters, butyrobetaine, and proline betaine (stachydrine) concentrations in biological materials can be reliably measured in 100-microliters samples, with a detection limit below 1 mumol/liter. The procedure is suitable for batches of more than 30 specimens and it is possible to obtain a single result within 2 h. The betaines are extracted into an acetonitrile:methanol mixture, dried with anhydrous disodium hydrogen phosphate containing argentous oxide. The 4-bromophenacyl ester derivatives are formed using 4-bromophenacyl triflate as reagent, in the presence of solid magnesium oxide as base. The derivatives are separated by high-performance chromatography on a silica column, in a mixed partition and ion-exchange mode.