Non-type I cystinuria caused by mutations in SLC7A9, encoding a subunit (bo,+AT) of rBAT.

Cystinuria (MIM 220100) is a common recessive disorder of renal reabsorption of cystine and dibasic amino acids. Mutations in SLC3A1, encoding rBAT, cause cystinuria type I (ref. 1), but not other types of cystinuria (ref. 2). A gene whose mutation causes non-type I cystinuria has been mapped by linkage analysis to 19q12-13.1 (Refs 3,4). We have identified a new transcript, encoding a protein (bo, +AT, for bo,+ amino acid transporter) belonging to a family of light subunits of amino acid transporters, expressed in kidney, liver, small intestine and placenta, and localized its gene (SLC7A9) to the non-type I cystinuria 19q locus. Co-transfection of bo,+AT and rBAT brings the latter to the plasma membrane, and results in the uptake of L-arginine in COS cells. We have found SLC7A9 mutations in Libyan-Jews, North American, Italian and Spanish non-type I cystinuria patients. The Libyan Jewish patients are homozygous for a founder missense mutation (V170M) that abolishes b o,+AT amino-acid uptake activity when co-transfected with rBAT in COS cells. We identified four missense mutations (G105R, A182T, G195R and G295R) and two frameshift (520insT and 596delTG) mutations in other patients. Our data establish that mutations in SLC7A9 cause non-type I cystinuria, and suggest that bo,+AT is the light subunit of rBAT.

Immunologic studies of water-soluble human amyloid fibrils. Comparative studies of eight amyloid preparations.

Eight preparations of soluble amyloid and degraded amyloid (DAM) were compared immunologically. Unlike amyloid fibrils, six of eight preparations of DAM proved to be relatively strong immunogens. Antisera to DAM reacted weakly or not at all with normal human serum or extracts of normal tissues, but were specifically reactive with amyloid fibrils or DAM. Comparative studies of DAM'S from eight different subjects showed some degree of cross-reactivity among them, yet demonstrated that they were not identical. Similar conclusions were obtained by quantitative precipitin and complement fixation analyses. Comparison of the amyloid fibrils with the homologous DAM by complement fixation and absorption studies demonstrated the existence in DAM of antigenic determinants that were lacking or inaccessible in the native fibrils. A search for amyloid precursors and antibodies to amyloid in the sera of 12 patients proved unsuccessful.

Immunologic studies of the major nonimmunoglobulin protein of amyloid. I. Identification and partial characterization of a related serum component.

Antisera have been prepared against the major nonimmunoglobulin component of secondary and familial Mediterranean fever (FMF) associated amyloid which has been called A component or acid soluble fraction (ASF). The antisera were shown to be monospecific for ASF by precipitation of (125)I-labeled antigen and gave a reaction of identity with four different ASF preparations. The antisera were able to detect a circulating component in human serum that migrated in the alpha1-globulin region. This circulating component gave a line of identity with degraded ASF by double immunodiffusion. 57 normal sera and 89 sera from patients with diseases known to be frequently associated with amyloidosis were tested by immunodiffusion for the circulating ASF component. 7% of normal sera and 50-80% of the pathologic sera had elevated amounts of this component. Absorption studies showed that all normal sera probably have small amounts of this component while cord sera do not have detectable amounts. This component was partially purified and was shown to be slightly larger than albumin. The relation of the circulating component to the acid soluble fraction of amyloid is discussed.

A variant of prealbumin from amyloid fibrils in familial polyneuropathy of Jewish origin.

Amyloid fibrils were isolated from spleen and thyroid obtained at autopsy from one patient (S.K.O.) of Jewish origin with familial amyloidotic polyneuropathy. Gel filtration on Sephadex G100 after solubilization in 5 M guanidine HCl yielded three major components with 14,000, 9,000, and 5,000 mol wt, respectively. The two larger components shared antigenic determinants with human prealbumin. Amino acid analysis and amino terminal sequence studies revealed the 14,000-mol wt protein to be an intact prealbumin subunit. The 9,000-mol wt fragment obtained in highest yield encompassed the region from position 49-127 and the 5,000 mol wt fraction encompassed the amino terminal of prealbumin (position 1-48). An amino acid substitution (Gly/Thr) was detected at position 49, where enzymatic cleavage occurred. Thus, several prealbumin-derived fragments, predominantly the carboxyl end, constitute the amyloid fibrils in a heredofamilial amyloidosis syndrome of dominant inheritance.

Physical, chemical, and ultrastructural studies of water-soluble human amyloid fibrils. Comparative analyses of nine amyloid preparations.

Amyloid fibrils were isolated from the tissues of nine patients with amyloidosis in a state of high purity by homogenization of the tissue followed by extraction with distilled water. Physical, chemical, and ultrastructural studies suggest that amyloid fibrils from different individuals resemble each other, but are not identical. In tissue sections as well as by negative staining of isolated fibrils, morphologic variations were observed. Among the isolated fibrils at least three types were noted. The majority resembled those described previously. However, one subject had two types of fibrils which differed in size and appearance. Most of the preparations sedimented as a single component with a sedimentation coefficient of 45-50S or as a larger polymer. However, two of the preparations had sedimentation coefficients of 8-9S, and a third one had a major 95S component and a minor 9S fraction. While the preparations of amyloid were not sufficiently pure for amino acid analyses, peptide maps demonstrated differences among amyloid preparations from different subjects. The amyloid fibrils in their native state proved to be remarkably resistant to digestion by a number of proteolytic enzymes. Several chemical methods were tried to produce smaller subunits. Of these, the most successful one was the use of 0.1 M NaOH which yielded a smaller, soluble fraction with sedimentation coefficients ranging from 1.1 to 2.8S. Accompanying this degradation, there was little loss of peptides or carbohydrates. Based on the results of the chemical analyses, it is estimated that the subunit produced by sodium hydroxide had a molecular weight of approximately 35,000-40,000.

The amino acid sequence of a major nonimmunoglobulin component of some amyloid fibrils.

The complete amino acid sequence of a protein, acid soluble fraction, (ASF) which constitutes up to 50% of amyloid fibrils from a patient with familial Mediterranean fever has been obtained. Partial amino acid sequences of three other proteins from patients with secondary amyloidosis were identical in the regions studied except for an alanine-valine interchange in one. The ASF contains no cysteine, does not resemble any known immunoglobulin, and has not been detected as yet in myeloma-associated amyloid.

The characterization of soluble amyloid prepared in water.

Amyloid was extracted from the spleen of a patient with primary amyloidosis by homogenizing it at high speed with water after preliminary treatments, first to remove proteins soluble in saline, and then to remove salts. The extracts containing amyloid appeared to be clear at concentrations up to 6 mg/ml of protein. The material gave little sediment on being centrifuged up to 20,000 g for 1 hr, but the protein was sedimented at 100,000 g in 1 hr. The amyloid could be precipitated from the extracts by addition of NaCl to 0.0075 mole/liter or of CaCl(2) to 0.0025 mole/liter. The protein-bound Congo red formed a red precipitate and this property was used to estimate recovery and purity of amyloid during extraction. On electronmicroscopy the isolated amyloid proved to be morphologically pure. It existed either as single filaments measuring 60-80 A in diameter or as large aggregates of these filaments.Freshly isolated amyloid in water sedimented as a single homogeneous peak with an s degrees (20,[unk]) of about 45-50S. On standing, the solution became cloudy and more rapidly sedimenting components appeared. On electrophoresis the material migrated as a homogeneous peak towards the anode. The protein had an amino acid composition different from that of all known serum proteins. It was rich in acidic amino acids and had little cysteine and methionine and no hydroxyproline. The total content of carbohydrate was less than 2%.

Systemic senile amyloidosis. Identification of a new prealbumin (transthyretin) variant in cardiac tissue: immunologic and biochemical similarity to one form of familial amyloidotic polyneuropathy.

Isolated amyloid fibrils from three cases of systemic senile amyloidosis (SSA) contained subunit proteins with molecular masses of 14 (10-20%), 10-12 (60-80%), and 5-6 kD (5-10%) when fractionated under reducing and dissociating conditions. This grouping was identical to that seen in SKO, a case of familial amyloidotic polyneuropathy (FAP) studied earlier. Amino acid sequencing confirmed that SSA subunit proteins were in fact prealbumin (transthyretin). Complete sequence analysis of one SSA preparation revealed the presence of a new variant Pa (TTr) molecule with a single amino acid substitution of isoleucine for valine at position 122. Further studies used an antiserum specific for SKO IV, a subunit protein of SKO previously shown to correspond to carboxy-terminal 78 residues (positions 49-127) of (TTr). Anti-SKO IV reacted with SSA in tissue at equivalent dilutions to anti-Pa (TTr) and with the 10-12-kD fraction of SSA on Western blots; reactivity was blocked by SKO IV, but not by Pa (TTr). SSA is a form of systemic amyloidosis caused by tissue deposition of Pa (TTr) and its fragments, with shared conformational or subunit antigenicity to at least one form of FAP. Identification of a new variant Pa (TTr) molecule in one case suggests further that SSA may be a genetically determined disease expressed late in life.

An adult form of juvenile rheumatoid arthritis.

The conditions of five adults were eventually diagnosed as juvenile rheumatoid arthritis (Still's disease). Prolonged hospitalization, repetitive roentogenographic examinations, biopsies, laparotomies, and therapeutic trials with toxic agents preceded the establishment of the final diagnosis. Common early findings in all cases were prolonged "septic fever," polyarthralgia, and an elevated ESR. Three patients had a rash, four had splenomegaly, three had a vague history of a similar disease, and four had leukocytosis. Some of the patients were older than others described in the literature. Two received immunosuppressive agents and did relatively well. In view of our experience and the few reports in the literature, we concluded that juvenile rheumatoid arthritis in adults has to be seriously considered in the presence of prolonged septic fever, polyarthralgia, rash, and splenomegaly, before harmful drugs are given or risky procedures are performed.

A single mutated MEFV allele in Israeli patients suffering from familial Mediterranean fever and Behçet's disease (FMF-BD).

Although familial Mediterranean fever (FMF) is an autosomal recessive disorder, preliminary partial mutation analysis suggested that about 60% of FMF patients, who also suffer from Behçet's disease (FMF-BD), have only a single mutated FMF gene (MEFV). In this study, the possibility that patients with FMF-BD may indeed be carriers of a single mutated MEFV is further analysed. The presence of mutations in the coding region of MEFV of eight patients with FMF-BD, representing six families with 47 members, was determined by sequencing. A possible role for the non-carrier chromosome and for BD in the expression of FMF in patients with a single mutated MEFV allele was determined by analysing the association between these variables and the presence of FMF in heterozygous kin. Sequence analysis revealed that all eight patients had indeed only one mutation in the coding region of MEFV. The patients' non-carrier chromosomes converged into three different MEFV haplotypes and were shared by heterozygous unaffected kin in five of six families. BD was found in 10 of 11 carriers with FMF vs one of 16 carriers without FMF (P < 0.001). These results suggest that FMF may be expressed in individuals harbouring only one coding mutation in MEFV. The findings argue against a role for the non-carrier chromosome in the induction of FMF, and suggest that the FMF phenotype in this cohort was associated with the simultaneous presence of BD. These findings may mirror a more generalised rule, that FMF may be precipitated in carriers of a single mutated FMF gene by factors unrelated to the other MEFV allele.

Tumor necrosis factor in familial Mediterranean fever.

PURPOSE: The pleiotropic inflammatory effects of tumor necrosis factor (TNF) prompted a study of this cytokine in familial Mediterranean fever (FMF), a recurrent polyserositis of unknown etiology. PATIENTS AND METHODS: Thirty-six asymptomatic and 24 patients with acute FMF were studied and compared with 20 matched healthy subjects. TNF levels were measured by bioassay in the plasma and in supernatants of peripheral blood mononuclear cells (PBMC) incubated alone or with an inducer (lipopolysaccharide, phytohemagglutinin [PHA], or Sendai virus). Cytotoxicity could be abolished in all cases by preincubation with monoclonal anti-TNF-alpha antibodies. RESULTS: No TNF was found in plasma and non-induced PBMC supernatants. Induced TNF production was markedly decreased in patients with acute FMF and increased in asymptomatic FMF patients to levels over those of control subjects (p less than 0.05). Thus, PHA-induced TNF levels were 4 U/mL in patients with acute FMF, 25 U/mL in asymptomatic patients, and 14 U/mL in healthy control subjects (median values), and the other inducers gave similar results. Retesting of patients first studied during an acute episode when their disease was quiescent also revealed a fivefold increase in TNF production. These results were independent of the use of colchicine, which also had no effect on TNF levels when taken by volunteers (1 mg/day) or when added to the PBMC cultures (10(-7) M). CONCLUSIONS: Since TNF has a very short half-life in plasma, the capacity of PBMC to respond to TNF inducers may more accurately reflect its synthesis. A marked decrease in this response in acute FMF suggests "exhaustion" of cells that are already highly activated to produce TNF and the possible participation of TNF in the pathogenesis of FMF.

Characterization of amyloid deposits and P component from a patient with factor X deficiency reveals proteins derived from a lambda VI light chain.

Amyloid fibrils were isolated from a spleen obtained at surgery from a 58-year-old white man with primary amyloidosis presenting with factor X deficiency and responding dramatically to splenectomy. Gel filtration on Ultragel ACA 54 in 5 M guanidine 1 M acetic acid yielded components with molecular weights between 17,000 and 13,000. Two of them (17K and 15K) were studied in detail. Antigenic and amino acid sequence analysis showed that these proteins were related to lambda VI immunoglobulin light chain. The predominant protein subunits of the amyloid fibril of the deposits (17K) was processed at the carboxy terminus in the same section of the constant region as the only other lambda VI amyloid protein previously reported. Amino terminal sequence of the 15K protein revealed not only degradation at the C terminal, but also minor degradation at the amino terminal (three residues difference from the 17K species). P component was also isolated from the spleen and characterized. This represents the first antigenic and sequence analysis of tissue amyloid proteins and P component from a patient presenting with factor X deficiency and another example of amyloid proteins derived from the newly discovered amyloidogenic lambda VI light chain subgroup.

Amyloidosis associated with renal cell carcinoma of the AA type.

Amyloid fibrils were found at postmortem examination in a 70 year old woman with generalized amyloidosis associated with renal carcinoma (hypernephroma). Clinically, her amyloid disease presented as nephrotic syndrome. It was demonstrated by electrophoretic and amino acid sequence analysis studies that the amyloid fibrils contained AA protein identical to that found in amyloidosis associated with chronic inflammatory and infectious diseases as well as in the genetic form of familial Mediterranean fever.

AA protein in a case of "primary" or "idiopathic" amyloidosis.

Amyloidosis constitutes a group of diseases in which extracellular fibrils with a characteristic appearance are deposited in a variety of tissues. Several different proteins have been identified as the major subunits of the fibrils. In the primary and myeloma-associated type, the amyloid fibrils consist of immunoglobulin light chain fragments, whereas in the secondary type and the amyloid associated with familial Mediterranean fever the major component is the AA protein. In this report a 21 year old man of Yemenite extraction with no underlying disease and no family history of amyloidosis was found to have amyloid deposits composed of AA protein. Although clinically this might be classified as primary amyloidosis, the absence of light chain fragments makes that diagnosis unlikely. Therefore, it is suggested that whenever possible the clinical classification be supplemented by a description of the biochemical nature of the fibrils.

Familial Mediterranean fever in the colchicine era: the fate of one family.

In order to demonstrate the effect of prophylactic colchicine treatment on the natural history of familial Mediterranean fever (FMF), a family is presented with 6 out of 9 siblings affected by FMF. Each patient represents a different stage of the amyloidotic kidney disease of FMF and the effect of continuous colchicine treatment on its course. Considered together, the members of this family present an almost complete clinical, genetic, and behavioral picture of the disease.

Dominant inheritance in two families with familial Mediterranean fever (FMF).

Familial Mediterranean fever (FMF) is an autosomal-recessive disease which affects almost exclusively people of Mediterranean and Middle Eastern origin. We examined the possibility of a dominant inheritance of FMF among our 3,000 patients in Israel. Two hundred forty FMF patients were members of 77 families in which the disease affected more than one generation. In 75 of these families the occurrence of FMF in more than one generation was found to be consistent with a recessive mode of inheritance due to a high gene frequency (q) and consanguinity among parents of the patients. In 2 families, one of Ashkenazi and the other of Georgian Iraqi origin, in which FMF occurred in 4 consecutive generations, the mode of inheritance could be explained only by autosomal-dominant inheritance.

Twin studies in familial Mediterranean fever.

Familial Mediterranean fever (FMF) is a genetic disease characterized by recurrent short episodes of fever, accompanied by peritonitis, pleuritis, or arthritis. The disease is almost completely ethnically restricted to patients of Mediterranean descent--Sephardic Jews, Armenians, Anatolian Turks, and Arabs. Although many family studies have been performed, no twin study has been reported as yet. We studied 21 di- and monozygotic twin sets, identified among the 1,943 FMF patients in our registry. Full concordance was observed in all the 10 monozygotic twin sets. In the 11 dizygotic twins, concordance for FMF disease was found in only 3 pairs. Variability in the clinical manifestations and degree of severity have been noted within twins. These findings provide definitive evidence for the genetic cause of FMF. They also support the single gene autosomal recessive model, and provide support for the contention that the lower observed than expected incidence found in FMF is due to genetically affected but clinically undiagnosed patients.

Familial Mediterranean fever in two Bedouin families: mutation analysis and disease severity.

Familial Mediterranean fever (FMF) is an autosomal recessive disease prevalent among non-Ashkenazi Jews, Armenians, Arabs, and Turks. The Bedouin are nomad Arab tribes residing in desert margins of the Middle East and Arabia. FMF is quite rare in Bedouins, and here we report on two Bedouin families from southern Israel suffering from this disorder. The MEFV mutations found in the Bedouin patients M694I, V726A, and E148Q are consistent with their Arab origin. The disease severity score showed a mild to moderate severity disease in six patients. The Bedouins, leading a unique nomadic life, may prove instrumental in unraveling the role of environmental factors in the course and severity of FMF.

Clinical differences between North African and Iraqi Jews with familial Mediterranean fever.

Familial Mediterranean fever (FMF) is an autosomal recessive disease causing attacks of fever and serositis. The gene causing this disease, designated MEFV, was mapped to the short arm of chromosome 16, but has not yet been cloned. North African and Iraqi Jews constitute the two largest population groups suffering from the disease in Israel. In this report we compared the severity of the disease between these two populations. North African Jews were found to have a more severe disease manifested by an earlier age of onset, an increase in frequency and severity of joint involvement, a higher incidence of erysipelas-like erythema, and a higher dose of colchicine required to control symptoms. The involvement of additional genes, environmental factors, and different mutations in MEFV, may explain the clinical variation in disease severity between these two population groups.

Biochemical and clinical studies in Libyan Jewish cystinuria patients and their relatives.

Cystinuria is a hereditary disorder manifested by the development of kidney stones. Three subtypes of the disease have been described, based on urinary excretion of cystine and the dibasic amino acids in heterozygotes, and oral loading tests in homozygotes. Cystinuria is very common among Libyan Jews living in Israel. Recently, we mapped the disease-causing gene in Libyan Jews to 19q, and have shown a very strong founder effect. In this report we present the results of biochemical and clinical studies performed on Libyan Jewish cystinuria patients and members of their families. High levels of cystine and the dibasic amino acids in heterozygotes support previous data that cystinuria in Libyan Jews is a non-type I disease. Oral loading tests performed with lysine showed some degree of intestinal absorption, but less than in normal controls. Previous criteria for determining the disease type, based solely on urinary amino acid levels, proved useless due to a very wide range of cystine and the dibasic amino acids excreted by the heterozygotes. Urinary cystine levels were useful in distinguishing between unaffected relatives and heterozygotes, but were unhelpful in differentiating between heterozygotes and homozygotes. Urinary levels of ornithine or arginine, and the sum of urinary cystine and the dibasic amino acids, could distinguish between the last two groups. Among stone formers, 90% were homozygotes and 10% were heterozygotes; 15% of the homozygotes were asymptomatic.