Linkage of Bardet-Biedl syndrome to chromosome 16q and evidence for non-allelic genetic heterogeneity.

Bardet-Biedl syndrome is an autosomal recessive disorder characterized by mental retardation, obesity, retinitis pigmentosa, polydactyly and hypogonadism. Other findings include hypertension, diabetes mellitus and renal and cardiovascular anomalies. We have performed a genome-wide search for linkage in a large inbred Bedouin family. Pairwise analysis established linkage with the locus D16S408 with no recombination and a lod score of 4.2. A multilocus lod score of 5.3 was observed. By demonstrating homozygosity, in all affected individuals, for the same allele of marker D16S408, further support for linkage is found, and the utility of homozygosity mapping using inbred families is demonstrated. In a second family, linkage was excluded at this locus, suggesting non-allelic genetic heterogeneity in this disorder.

MHC class 2 deficiency and X-linked agammaglobulinaemia in a consanguineous extended family.

Manifestations of immunodeficiency within the same family are presumed to be the same disease. We report a consanguineous extended family where four patients have immunodeficiency, three have X-linked agammaglobulinaemia and one has major histocompatibility complex class 2 deficiency. Within one family, two rare genetic diseases with similar clinical manifestations can occur.

A novel mutation causing complete thyroxine-binding globulin deficiency (TBG-CD-Negev) among the Bedouins in southern Israel.

T4-binding globulin (TBG) is the major thyroid hormone transport protein in human serum. Inherited TBG abnormalities do not usually alter the metabolic status and are transmitted in X-linked inheritance. A high prevalence of complete TBG deficiency (TBG-CD) has been reported among the Bedouin population in the Negev (southern Israel). In this study we report a novel single mutation causing complete TBG deficiency due to a deletion of the last base of codon 38 (exon 1), which led to a frame shift resulting in a premature stop at codon 51 and a presumed truncated peptide of 50 residues. This new variant of TBG (TBG-CD-Negev) was found among all of the patients studied. We conclude that a single mutation may account for TBG deficiency among the Bedouins in the Negev. This report is the first to describe a mutation in a population with an unusually high prevalence of TBG-CD.

Two new mutations in the glucose-6-phosphatase gene cause glycogen storage disease in Hungarian patients.

Glycogen storage disease type 1a (von Gierke disease, GSD-1A) is caused by the deficiency of microsomal glucose-6-phosphatase (G6Pase) activity which catalyzes the final common step of glycogenolysis and gluconeogenesis. The cloning of the G6Pase cDNA and characterization of the human G6Pase gene enabled the identification of the mutations causing GSD-1a. This, in turn, allows the development of non-invasive DNA-based diagnosis that provides reliable carrier testing and prenatal diagnosis. Here we report on two new mutations E110Q and D38V causing GSD-1a in two Hungarian patients. The analyses of these mutations by site-directed mutagenesis followed by transient expression assays demonstrated that E110Q retains 17% of G6Pase enzymatic activity while the D38V abolishes the enzymatic activity. The patient with the E110Q has G222R as his other mutation. G222R was also shown to preserve about 4% of the G6Pase enzymatic activity. Nevertheless, the patient presented with the classical severe symptomatology of the GSD-1a.

A single-base deletion in the 3'-coding region of glycogen-debranching enzyme is prevalent in glycogen storage disease type IIIA in a population of North African Jewish patients.

Glycogen storage disease type III (GSD III) is an autosomal recessive disease caused by the deficiency of glycogen-debranching enzyme (AGL). The overall incidence of the disease is about 1:100,000 life births in the USA; however, it is unusually frequent among North African Jews in Israel (prevalence 1:5,400, carrier prevalence 1:35). All North African Jewish GSD III patients examined have both liver and muscle involvement. While all patients showed the characteristic features related to the liver enzyme deficiency, the peripheral muscular impairment varied from minimal to severe, with neuromuscular involvement. A single mutation in the AGL gene, the deletion of T at position 4,455 (4,455delT) in homozygous form, was found in this patient population. The mutation 4,455delT results in the change of 17 amino acids at the carboxy terminus of the AGL protein (1,486-1,502) and truncation of the last 30 amino acids of the normal AGL 1,532 amino acids. The mutation appears to be ethnic specific as it was not seen in 18 patients of different ethnic origins. This is the first report of a mutation in the AGL gene affecting a considerable number of GSD III patients in a defined population.

Linkage localization of the thoraco-abdominal syndrome (TAS) gene to Xq25-26.

The thoraco-abdominal syndrome (TAS) presents a closure defect confined to the ventral midline, manifested as ventral hernia of various degrees in all affected individuals and antero-lateral diaphragmatic defect manifested almost exclusively in affected males. The syndrome is inherited as an X-linked dominant trait affecting blastogenesis (XLB mutation). We studied 27 members of the TAS family for linkage on the X chromosome. The best lod score of 5.5 at theta 0.04 was found for the HPRT locus on Xq26.1. A multilocus lod score of 12.4 was observed when the linkage analysis utilized additional markers in Xq25-26.

Clinical and genetic characterization of an autosomal dominant nephropathy.

Autosomal dominant familial nephropathies with adult onset, no macroscopic cysts, and progressive deterioration include medullary cystic disease (ADMCKD) as well as other less specific entities. We studied a kindred of Jewish ancestry in which 15 members (both male and female) have suffered from chronic renal failure. The first evidence of renal involvement was observed between 18 and 38 years. It included hypertension followed by progressive renal insufficiency. No polyuria, anemia, gout, hematuria, nor proteinuria were seen. An average of 4.5 years elapsed from diagnosis to end-stage renal disease. Renal pathology at early stages of the disease showed extensive tubulointerstitial fibrosis and global glomerulosclerosis. Linkage analysis was performed at the two known loci of ADMCKD, on Chromosomes 1 and 16. Linkage to the chromosome 16 locus was excluded. However, linkage to the chromosome 1q21 locus of ADMCKD was established with a maximum two-point LOD score of 3.82 to D1S394. The disease interval could be narrowed to about 9 cM/7.4 Mb between D1S1156 and D1S2635. Multiple-point linkage analysis revealed a maximum LOD of 4.21, with a broad peak from markers D1S2858 and D1S2624. This report establishes linkage between a familial nephropathy characterized by hypertension and progressive renal failure to the locus described for ADMCKD, a disease classically associated with macroscopic corticomedullary cysts, salt-losing tubulointerstitial nephropathy, and anemia. This finding broadens the clinical spectrum of ADMCKD positioned on chromosome 1q21 locus.

Deletion of 8.5 Mb, including the FMR1 gene, in a male with the fragile X syndrome phenotype and overgrowth.

A four-year-old boy with severe psychomotor retardation, facial appearance consistent with the fragile X syndrome, hypotonia, and overgrowth was found to have a deletion including the fragile X gene (FMR1). The breakpoints of the deletion were established between CDR1 and sWXD2905 (approximately 200 kb apart) at Xq27.1 (centromeric) and between DXS8318 (612-1078L) and DXS7847 (576-291L) (approximately 250 kb apart) at Xq28, about 500 kb telomeric to the FMR1 gene. The total length of the deletion is approximately 8.5 Mb. The propositus's mother, who was found to be a carrier of the deletion, showed very mild mental impairment. Except for mental retardation, which is a common finding in all cases reported with similar deletions of chromosome Xq, this patient had generalized overgrowth, exceeding the 97th centile for height and weight. Obesity and increased growth parameters have been reported in other patients with deletions either overlapping or within a distance of 0.5 Mb from the deletion in the present patient. Thus, it is suggested that a deletion of the 8-Mb fragment centromeric to the FMR1 gene might have an effect on growth.

Glycogen storage disease type 1a in Israel: biochemical, clinical, and mutational studies.

Glycogen storage disease type 1a (von Gierke disease, GSD 1a) is caused by the deficiency of microsomal glucose-6-phosphatase (G6Pase) activity which catalyzes the final common step of glycogenolysis and gluconeogenesis. The recent cloning of the G6Pase cDNA and characterization of the human G6Pase gene enabled the characterization of the mutations causing GSD 1a. This, in turn, allows the introduction of a noninvasive DNA-based diagnosis that provides reliable carrier testing and prenatal diagnosis. In this study, we report the biochemical and clinical characteristics as well as mutational analyses of 12 Israeli GSD 1a patients of different families, who represent most GSD 1a patients in Israel. The mutations, G6Pase activity, and glycogen content of 7 of these patients were reported previously. The biochemical data and clinical findings of all patients were similar and compatible with those described in other reports. All 9 Jewish patients, as well as one Muslim Arab patient, presented the R83C mutation. Two Muslim Arab patients had the V166G mutation which was not found in other patients' populations. The V166G mutation, which was introduced into the G6Pase cDNA by site-directed mutagenesis following transient expression in COS-1 cells, was shown to cause complete inactivation of the G6Pase. The characterization of all GSD 1a mutations in the Israeli population lends itself to carrier testing in these families as well as to prenatal diagnosis, which was carried out in 2 families. Since all Ashkenzai Jewish patients harbor the same mutation, our study suggests that DNA-based diagnosis may be used as an initial diagnostic step in Ashkenazi Jews suspected of having GSD 1a, thereby avoiding liver biopsy.

Congenital insensitivity to pain with anhidrosis (CIPA) in Israeli-Bedouins: genetic heterogeneity, novel mutations in the TRKA/NGF receptor gene, clinical findings, and results of nerve conduction studies.

Congenital insensitivity to pain with anhidrosis (CIPA), a rare and severe disorder, comprises absence of sensation to noxious stimuli, inability to sweat, and recurrent episodes of hyperthermia. It has a relatively high prevalence in the consanguineous Israeli-Bedouins. Clinical studies of 28 patients are reported here. Using the linkage analysis approach, we linked the disease in 9 of 10 unrelated Israeli-Bedouin families with CIPA to the TrkA gene, which encodes the receptor for nerve growth factor. In one family, linkage was excluded, implying that another gene, yet unidentified, is involved. Two new mutations in the tyrosine kinase domain of the TrkA gene were identified in our CIPA patients: a 1926-ins-T in most of the southern Israeli-Negev CIPA patients, and a Pro- 689-Leu mutation in a different isolate of Bedouins in northern Israel. Eight prenatal diagnoses were made in the southern Israeli-Negev Bedouins, two by linkage analysis and six by checking directly for the 1926-ins-T mutation. Three polymorphisms in the TrkA protein kinase encoding domain were also observed.

Short stature in carriers of recessive mutation causing familial isolated growth hormone deficiency.

Isolated growth hormone deficiency (IGHD) IB is an autosomal recessive disorder characterized by a good response to exogenous growth hormone (GH) treatment without development of anti-GH antibodies. Patients with IGHD IB were found to be compound heterozygotes for deletion and frameshift mutations as well as homozygotes for splicing mutations in the GH-1 gene. Recently, a novel splicing mutation in the GH-1 gene was identified in an extended, consanguineous Arab-Bedouin family from Israel with IGHD IB. Prior to the identification of this mutation, a considerable number of children with short stature in this family were found normal on pharmacological stimulation for GH release. This observation prompted a genotype/phenotype correlation of potential heterozygotes in the family. Carriers of the mutant GH-1 allele were found as a group to have a significantly shorter stature than normal homozygote (mean standard deviation scores, 1.67 and -0.40, respectively, P<0.05). Moreover, 11 of 33 (33%) heterozygotes, but only 1 of 17 (5.9%) normal homozygotes, had their height at 2 or more SD below the mean. Overall, 48.5% of studied heterozygotes were found to be of appreciably short stature with height at or lower than the 5th centile (> or = -1.7 SD), whereas only 5.9% of the normal homozygotes did (P<0.004). This phenomenon of heterozygotes for a recessive mutation in the GH-1 gene manifesting short stature, might imply that some such mutations may account for non-GH deficiency reduced height in the general population.

Ataxia with isolated vitamin E deficiency in four siblings.

We describe 4 siblings of a consanguineous Bedouin family with Friedreich ataxia phenotype in whom low serum vitamin E levels without other indicators of fat malabsorption were detected. Although age of onset and some of the clinical features were alike in all 4 patients, the electrophysiological parameters were markedly abnormal in 2, but normal in the other 2. Erythrocytes revealed both membranous and intracellular evidence of oxidative damage. The mutations described in other families with ataxia with isolated vitamin E deficiency were not detectable, nor was an abnormal single-stranded conformation polymorphism pattern apparent in the three exons at the 3' region of the gene. Vitamin E administration in pharmacological doses improved the neurological condition in 2 patients and also corrected some of the patients' erythrocyte cell abnormalities. The finding of vitamin E deficiency in other cases of Friedreich ataxia phenotype may allow treatment at an early stage of the disease, when large dose Vitamin E therapy may reverse the neurological lesions.

Congenital insensitivity to pain. Orthopaedic manifestations.

We reviewed 13 patients with congenital insensitivity to pain. A quantitative sweat test was carried out in five and an intradermal histamine test in ten. DNA examination showed specific mutations in four patients. There were three clinical presentations: type A, in which multiple infections occurred (five patients); type B, with fractures, growth disturbances and avascular necrosis (three patients); and type C, with Charcot arthropathies and joint dislocations, as well as fractures and infections (five patients, four with mental retardation). Patient education, shoeware and periods of non-weight-bearing are important in the prevention and early treatment of decubitus ulcers. The differentiation between fractures and infections should be based on aspiration and cultures to prevent unnecessary surgery. Established infections should be treated by wide surgical debridement. Deformities can be managed by corrective osteotomies, and shortening by shoe raises or epiphysiodesis. Joint dislocations are best treated conservatively.

The 3849 + 10 kB C-->T mutation in a 21-year-old patient with cystic fibrosis.

Cystic fibrosis (CF) is the most common lethal inherited disease in the white population. It is characterized by exocrine gland epithelia dysfunction, which leads to pulmonary and pancreatic insufficiency. Since the cloning of the CF gene in 1989 and the identification of the most common CF mutation (delta F508), more than 400 different mutations have been described. These mutations appear to contribute to the heterogeneity of the CF phenotype and several reports have speculated on the relationship between the most common CF mutations and the patient's clinical status. We report the case of a 21-year-old woman with longstanding chronic pansinusitis, nasal polyposis, chronic cough and severe nasal crusting. During a period of five years she had been followed by her otolaryngologist and pediatric pulmonologist. Sweat tests performed at the age of 17 and 18 were within normal limits and she underwent repeated conventional sinonasal procedures, with no improvement in her clinical status. On her present admission, sweat tests showed a 70 meq/l chloride concentration. The diagnosis of CF was then confirmed by DNA analysis and the patient was found to carry the 3849 + 10 kB C-->T mutation. The early detection of this newly recognized form of CF in adults as well as in children presenting with sinonasal symptoms is critical for life expectancy and quality.

The PREPL A protein, a new member of the prolyl oligopeptidase family, lacking catalytic activity.

The PREPL (previously called KIAA0436) gene encodes a putative serine peptidase from the prolyl oligopeptidase family. A chromosomal deletion involving the PREPL gene leads to a severe syndrome with multiple symptoms. Homology with oligopeptidase B suggested that the enzyme cleaves after an arginine or lysine residue. Several PREPL splice variants have been identified, and a 638-residue variant (PREPL A) was expressed in Escherichia coli and purified. Its secondary structure was similar to that of oligopeptidase B, but differential-scanning calorimetry indicated a higher conformational stability. Dimerization may account for the enhanced stability. Unexpectedly, the PREPL A protein did not cleave peptide substrates containing a P1 basic residue, but did slowly hydrolyse an activated ester substrate, and reacted with diisopropyl fluorophosphate. These results indicated that the catalytic serine is a reactive residue. However, the negligible hydrolytic activity suggests that the function of PREPL A is different from that of the other members of the prolyl oligopeptidase family.

A microfluorometric assay for cholinesterases, suitable for multiple kinetic determinations of picomoles of released thiocholine.

A highly sensitive microfluorometric assay for cholinesterases has been developed. Enzymatic activity is measured by monitoring the thiocholine produced by specific hydrolysis of acetylthiocholine. This is carried out by reacting the thiocholine formed with the fluorogenic compound N-(4(7 diethylamino-4-methylcoumarin-3-yl)phenyl)maleimide to yield an intensely fluorescent product. The assay is linear over a range extending from a few picomoles to nanomoles of thiocholine. The specificity and accuracy of this microfluorometric assay were examined using microgram quantities of rat brain tissue as a source for cholinesterases. The specific activities and the Km values determined by this new method for both cholinesterase activities present in the brain (acetylcholine hydrolase, EC 3.1.1.7, and "nonspecific" cholinesterase-acylcholine acylhydrolase, EC 3.1.1.8) were identical to those reported earlier using the less sensitive spectrophotometric and radiometric methods. The background emission caused by nonenzymatic hydrolysis of the substrate is relatively low, and does not exceed background values encountered in other methods. The assay may be used for monitoring the kinetics of enzymatic activities in microscale reaction mixtures, providing a linear determination of the thiocholine produced over a period of at least 30 h at room temperature. The method can also be adapted for use in other enzymatic assays where reagents containing thiol groups can be produced or consumed.

Biosynthesis and secretion of catalytically active acetylcholinesterase in Xenopus oocytes microinjected with mRNA from rat brain and from Torpedo electric organ.

A novel technique was developed for monitoring the level of the mRNA species that direct the synthesis of acetylcholinesterase (AcChoEase; acetylcholine acetylhydrolase, EC 3.1.1.7), using microinjected Xenopus oocytes as a translation system. When injected with poly(A)-containing RNA from whole rat brain or rat cerebellum and from electric organ of Torpedo ocellata, Xenopus oocytes synthesize and secrete catalytically active cholinesterase. The newly synthesized enzyme, which is mostly secreted into the oocytes incubation medium, appears to be primarily AcChoEase because it is inhibited by the specific inhibitor BW 284C51. The new enzymatic activity can be detected after injection of as little as 12.5 ng of poly(A)-containing RNA per oocyte, and there is a linear dependence of the oocytes' ability to form AcChoEase on the amount of injected RNA. The AcChoEase mRNA displays a tau 1/2 of about 10 +/- 3 hr in injected oocytes. The abundance of AcChoEase mRNA in the total nonfractionated mRNA injected was calculated to be ca. 1 x 10(-5), a value similar to the level of AcChoEase protein determined in rat brain. The combination of the high turnover number of AcChoEase, the efficiency of the oocyte system, and the sensitivity of the assay used thus permit the accurate monitoring of the scarce mRNA species that direct the synthesis of this enzyme.