Danish type gelsolin related amyloidosis: 654G-T mutation is associated with a disease pathogenetically and clinically similar to that caused by the 654G-A mutation (familial amyloidosis of the Finnish type)

BACKGROUND: Familial amyloidosis of the Finnish type (FAF, Finnish hereditary amyloidosis) is caused by a 654G-A mutation in the gelsolin gene on chromosome 9 resulting in the expression of mutant Asn-187 gelsolin which is abnormally proteolytically processed generating amyloidogenic fragments that polymerize into amyloid fibrils. We have recently shown that in a Danish and a Czech family with a clinical syndrome similar to FAF, including corneal lattice dystrophy, cranial neuropathy and skin changes, the disease is caused by another mutation at the same position, namely 654G-T predicting a Try-for-Asp substitution at 187 in secreted gelsolin. AIM: To undertake a closer examination of the Danish subtype of FAF and report immunohistochemical and biochemical findings. RESULTS: Immunostaining of plasma gelsolin isolated from heterozygous FAF of the Danish subtype revealed a pattern similar to that found in FAF-Asn 187. The > 60 kDa gelsolin species contain an epitope characteristic of the amyloid forming region as revealed by an amyloid specific antibody, whereas the approximately 50 kDa fragments are devoid of it. Compared with the wild-type gelsolin peptide (Asp-187), the corresponding mutant peptide (Tyr-187) showed dramatically increased fibrillogenicity as revealed by quantitative thioflavine-T based fluorimetry; ultrastructurally, amyloid-like fibrils were formed by the mutant peptide. Immunohistochemistry showed that antibodies directed against residues 231-242 of secreted gelsolin, representing the carboxy terminus of the sequence forming the amyloid protein (residues 173-243) laid down in the tissues in a fibrillar form in FAF, specifically labelled the amyloid deposited in rectum and skin in the Danish (654G-T) subtype. CONCLUSIONS: The 654G-T mutation in the gelsolin gene gives rise to an amyloid disease clinically and pathogenetically similar to that caused by the 654G-A mutation.     

Gelsolin gene mutation--at codon 187--in familial amyloidosis, Finnish: DNA-diagnostic assay.

Familial amyloidosis, Finnish (FAF), is an autosomal dominant form of systemic amyloidosis with lattice corneal dystrophy and progressive cranial neuropathy as principal clinical manifestations. We have shown that the novel amyloid fibril protein found in these patients is an internal degradation fragment of gelsolin, an actin-binding protein, and that it contains an amino acid substitution, asparagine for aspartic acid at position 15, that is due to a guanine-to-adenine transversion corresponding to codon 187 of human plasma gelsolin cDNA. To test that this mutation cosegregates with the disease high-molecular-weight genomic DNA was isolated from autopsied tissues or lymphocytes of 23 patients, 6 healthy relatives and 20 unrelated healthy control persons. Specific fragments were amplified with the polymerase chain reaction for oligonucleotide hybridization analysis using the slot-blot technique. The guanine-to-adenine transversion was found in all FAF patients tested, but in none of the control subjects. Our results show that the mutation (G to A) cosegregates with the disease phenotype, and that the slot-blot analysis can be used as a diagnostic assay, including prenatal evaluation.     

Gelsolin gene mutation—at codon 187—in familial amyloidosis,Finnish: DNA-diagnostic assay

Familial amyloidosis, Finnish (FAF), is an autosomal dominant form of systemic amyloidosis with lattice corneal dystrophy and progressive cranial neuropathy as principal clinical manifestations. We have shown that the novel amyloid fibril protein found in these patients is an internal degradation fragment of gelsolin, an actin-binding protein, and that it contains an amino acid substitution, asparagine for aspartic acid at position 15, that is due to a guanine-to-adenine transversion corresponding to codon 187 of human plasma gelsolin cDNA. To test that this mutation cosegregates with the disease high-molecular-weight genomic DNA was isolated from autopsied tissues or lymphocytes of 23 patients, 6 healthy relatives and 20 unrelated healthy control persons. Specific fragments were amplified with the polymerase chain reaction for oligonucleotide hybridization analysis using the slot-blot technique. The guanine-to-adenine transversion was found in all FAF patients tested, but in none of the control subjects. Our results show that the mutation (G to A) cosegregates with the disease phenotype, and that the slot-blot analysis can be used as a diagnostic assay, including prenatal evaluation.     

Gelsolin variant and beta-amyloid co-occur in a case of Alzheimer's with Lewy bodies.

Familial amyloidosis, Finnish type (FAF), is an autosomal dominant form of amyloidosis which is related to a point mutation in the gelsolin gene localized on chromosome 9. The mutation corresponds to codon 187 of the secreted form of gelsolin, and is expressed in the amyloid fibril at residue 15. Our original FAF patient was demented, and neuropathological analysis showed Alzheimer type brain lesions associated with both classical and cortical Lewy bodies. Furthermore, antiserum against the gelsolin-derived FAF amyloid reacted strongly with both classical and cortical Lewy bodies of this FAF patient. In preliminary experiments similar results were obtained in cases of Parkinson's disease and diffuse Lewy body disease. These observations may indicate a role for gelsolin in the pathogenesis of Parkinson's disease and related conditions.     

Obstructive sleep apnoea syndrome in hereditarygelsolin-related amyloidosis

Gelsolin-related amyloidosis (AGel amyloidosis) is a rare autosomal dominant disorder, reported worldwide in kindreds carrying a G654A or G654T gelsolin gene mutation. The main clinical signs are cutis laxa, cranial and peripheral neuropathy, and corneal lattice dystrophy but heavy intermittent snoring also occurs. To evaluate whether sleep apnoea is present we performed nocturnal sleep recordings, cephalometric and spirometric analyses and multiple sleep latency tests (MSLT) in five snoring patients with a G654A gelsolin gene mutation. Four patients had obstructive sleep apnoea syndrome (OSAS) with redundant oropharyngeal and hypopharyngeal soft tissues, macroglossia and cranial neuromuscular dysfunction. The fifth patient had hypersomnia without obstructive sleep apnoea. Nasal continuous positive airway pressure (CPAP) was an effective treatment. This study presents the first evidence in favour of an association between AGel amyloidosis and OSAS, but further studies are needed to define the prevalence of OSAS and the pathogenetic roles of amyloid and variant gelsolin in its evolution.     

In vitro expression analysis shows that the secretory form of gelsolin is the sole source of amyloid in gelsolin-related amyloidosis

Amyloidoses are a group of diseases where abnormal fibrillar protein deposits accumulate in patients' tissues. In familial amyloidosis of the Finnish type (FAF), or gelsolin-related amyloidosis, the amyloid subunit protein consists of gelsolin peptides of amino acids 173-243 with the disease causing substitution at Asp187. Gelsolin is an actin- modulating protein and exists in both secretory and intracellular forms both encoded by a single gene in chromosome 9. We have previously shown that the FAF-associated forms of secretory gelsolin carrying the Asp187Asn or Asp187Tyr mutations are abnormally processed in cells, resulting in the secretion of an aberrant 68 kDa carboxyterminal fragment. Here we demonstrate by N-terminal sequencing that the amino terminus of this abnormal fragment is the amino acid 173 and thus represents the N-terminus of the FAF amyloid. We also provide evidence that the same truncated gelsolin can be found among the aberrant gelsolin fragments detected in patients' CSF. Finally, we also expressed the FAF-associated forms of intracellular gelsolin in COS-1 cells, and found no abnormality in their processing opposite to secretory form. Our results provide strong evidence that the secretory gelsolin is solely responsible for the amyloid formation in FAF.      

Lewy bodies are immunoreactive with antibodies raised to gelsolin related amyloid-Finnish type.

Lewy bodies (LB) are intraneuronal, cytoplasmic inclusion bodies. They are invariably present in Parkinson's and diffuse LB diseases. Their composition by direct biochemical methods is unknown, although LBs are immunoreactive with a number of antibodies, including anti-ubiquitin and anti-neurofilament antibodies. Familial amyloidosis, Finnish type (FAF), is an autosomal-dominant form of systemic amyloidosis. The authors have isolated and partially sequenced the amyloid. The protein has significant sequence identity with gelsolin, an actin-modulating protein. Rabbit polyclonal antibodies raised to the FAF amyloid not only immunostain the amyloid but also LBs in the cortex and substantia nigra of Parkinson's and diffuse LB disease brains. Immunoreactivity is absorbed by the purified amyloid but is unaffected by ubiquitin. This provides a link between the LB and one of the human amyloidoses, FAF.     

Amyloid in familial amyloidosis, Finnish type, is antigenically and structurally related to gelsolin.

Immunohistochemical studies of six patients with familial amyloidosis, Finnish type, showed that their amyloid deposits did not react with polyclonal antibodies against the amyloid proteins of other, established forms of systemic or cerebral amyloidosis. However, strong immunoreactivity was observed with rabbit antiserum raised against a low molecular weight purified amyloid subunit isolated from one of the patients. This immunoreactivity was abolished by absorption with the low molecular weight amyloid fraction. The amino terminal sequence of the amyloid protein subunit was homologous to gelsolin, an actin-modulating protein, and the amyloid deposits in tissues reacted with a monoclonal antibody against gelsolin. These studies show that the amyloid protein in familial amyloidosis, Finnish type, is not related to previously identified forms of amyloid, including prealbumin (transthyretin) variants, but represents a novel amyloidogenic protein related to gelsolin, a plasma and cytoplasmic protein.     

Founder effect in spinal and bulbar muscular atrophy (SBMA) in Scandinavia

We haplotyped 13 Finnish, 10 Swedish, 12 Danish and 2 Norwegian SBMA (spinal and bulbar muscular atrophy, Kennedy disease) families with a total of 45 patients and 7 carriers for 17 microsatellite markers spanning a 25.2 cM region around the androgen receptor gene on chromosome Xq11-q12 in search of a genetic founder effect. In addition, the haplotypes of 50 Finnish, 20 Danish and 22 Swedish control males were examined. All the Scandinavian SBMA families shared the same 18 repeat allele for the intragenic GGC repeat, which was present in only 24% of the controls. Linkage disequilibrium was also seen for the closest microsatellite markers. In addition, extended haplotypes of the Finnish, Swedish and Danish SBMA families revealed country-specific common founder haplotypes, which over time became gradually shortened by recombinations. No common haplotype was found among the controls. The data suggest that the SBMA mutation was introduced into western Finland 20 generations ago. Haplotype analysis implies a common ancestor for the majority of Scandinavian SBMA patients.     

Toward understanding the pathogenic mechanisms in gelsolin-related amyloidosis: in vitro expression reveals an abnormal gelsolin fragment

Gelsolln-related amyloldosls, also called familial amyloldosls,Finnish type (FAF) Is an autosomal dominantly Inherited disordercharacterized by progressive polyneuropathy and corneal latticedystrophy. All the analyzed patients are found to carry a nucleotldesubsltutlon of A or T for G₆₅₄ In their gelsolln gene, whichat the protein level results In the conversion of the 187 aminoacid residue, aspartic acid, to asparaglne or tyrosine, respectively.In this study, we transfected mammalian mesenchymal COS-1 cellswith a derivative of the expression vector pCD-X containingcDNA coding for the wild-type (D₁₈₇) and mutant forms(N¹⁸⁷ andY¹⁸⁷) of plasma gelsolln. Both disease-associated mutant formsof gelsolln were found to be abnormally processed, which ledto the secretion of an aberrant 68 kDa gelsolin fragment Intothe culture media. This fragment most probably represents acarboxy-termlnal part of the protein and contains the suggestedamyloid-forming sequence. Initial data were also obtained forInvolvement of a metalloendoprotease in the pathologic processing.This aberrant proteolysis is likely to represent a crucial Initiatorstep in the cascade resulting in amyloid accumulation in patients'tissues.     

Detection of the founder effect in Finnish CADASIL families

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited cerebrovascular disease characterized by brain infarcts, cognitive decline and dementia. The disease is caused by at least 91 missense mutations, four deletions and one splice site mutation in the NOTCH3 gene, which maps to 19p13.1. In 18 out of the 21 Finnish CADASIL families so far identified, the causative mutation is an arginine to cysteine substitution in position 133 (R133C). Most of the families carrying this mutation originate from the western coast of Finland, thus suggesting a founder effect. No previous reports of a founder effect in CADASIL have been published. We haplotyped 60 patients from these 18 families for 10 microsatellite markers in order to determine whether the families descend from a common ancestor. We found a similar haplotype linked to the mutation in all 18 pedigrees, which indicates a single common ancestor for all the Finnish R133C families. The age analysis of the founder mutation places the introduction of the mutation in the late 1600s or early 1700s.     

Haplotype analysis in Icelandic and Finnish BRCA2 999del5 breast cancer families.

The 999del5 mutation is the single, strong BRCA2 founder mutation in Iceland and the most common BRCA1/2 founder mutation in Finland. To evaluate the origin and time since spreading of the 999del5 mutation in Iceland and in Finland, we constructed haplotypes with polymorphic markers within and flanking the BRCA2 gene in a set of 18 Icelandic and 10 Finnish 999del5 breast cancer families. All Icelandic families analysed shared a common core haplotype of about 1.7 cM. The common ancestors for the Icelandic families studied were estimated to trace back to 340-1000 years, not excluding the possibility that the mutation was brought to Iceland during the settlement of the country. Analysis of the Finnish families revealed two distinct haplotypes. A rare one, found in three families in the old settlement region in southwestern Finland, shared a four-marker (0.5 cM) core haplotype with the Icelandic 999del5 haplotype. A distinct approximately 6 cM haplotype was shared by seven 999del5 Finnish families estimated to have a common ancestry 140-300 years ago. These families cluster in two geographical regions in Finland, in the very same area as those with the rare haplotype and also in the most eastern, late settlement region of Finland. The results may indicate a common ancient origin for the 999del5 mutation in Iceland and in Finland, but distinct mutational events cannot be ruled out. The surprising finding of the same mutation in two completely different haplotypes in a sparsely populated area in Finland may suggest gene conversion.     

Identification of the Finnish founder mutation for diastrophic dysplasia (DTD).

Diastrophic dysplasia (DTD) is especially prevalent in Finland and the existence of a founder mutation has been previously inferred from the fact that 95% of Finnish DTD chromosomes have a rare ancestral haplotype found in only 4% of Finnish control chromosomes. Here we report the identification of the Finnish founder mutation as a GT-> GC transition (c.-26 + 2T > C) in the splice donor site of a previously undescribed 5'-untranslated exon of the diastrophic dysplasia sulfate transporter gene (DTDST); the mutation acts by severely reducing mRNA levels. Among 84 DTD families in Finland, patients carried two copies of the mutation in 69 families, one copy in 14 families, and no copies in one family. Roughly 90% of Finnish DTD chromosomes thus carry the splice-site mutation, which we have designated DTDST(Fin). Unexpectedly, we found that nine of the DTD chromosomes having the apparently ancestral haplotype did not carry DTDST(Fin), but rather two other mutations. Eight such chromosomes had an R279W mutation and one had a V340del deletion. We consider the possible implications of presence of multiple DTD mutations on this rare haplotype.     

Haplotype-phenotype correlation in Fukuyama congenital muscular dystrophy

In typical Fukuyama congenital muscular dystrophy (FCMD), peak motor function is usually only unassisted sitting or sliding on the buttocks, though a few patients are able to walk at some point. However, a few patients have a severe phenotype and never acquire head control. In addition, it is clinically difficult to differentiate this severe FCMD from Walker-Warburg syndrome (WWS) or from muscle-eye-brain disease (MEBD). In order to establish a genotype-phenotype correlation, we performed haplotype analysis using microsatellite markers closest to the FCMD gene (FCMD) in 56 Japanese FCMD families, including 35 families whose children were diagnosed as FCMD with the typical phenotype, 12 families with a mild phenotype, and 9 families with a severe phenotype. Of the 12 propositi with the mild phenotype, 8 could walk and the other 4 could stand with support; 10 cases were homozygous for the ancestral founder (A-F) haplotype whereas the other 2 were heterozygous for the haplotype. In the 9 severe cases, who had never acquired head control or the ability to sit without support, 3 had progressive hydrocephalus, 2 required a shunt operation, and 7 had ophthalmological abnormalities. Haplotype analysis showed that 8 of the 9 cases of the severe phenotype are heterozygous for the A-F haplotype, and the other one homozygous for the haplotype. We confirmed that at least one chromosome in each of the 56 FCMD patients has the A-F haplotype. The rate of heterozygosity for the A-F haplotypes was significantly higher in severe cases than in typical or mild cases (P < 0.005). Severe FCMD patients appeared to be compound heterozygotes for the founder mutation and another mutation. Thus, the present study yielded molecular genetic evidence of a broad clinical spectrum in FCMD.     

A founder mutation of CANT1 common in Korean and Japanese Desbuquois dysplasia

Desbuquois dysplasia (DBQD) is a severe skeletal dysplasia of autosomal recessive inheritance. DBQD is classified into types 1 and 2 based on presence or absence of hand anomalies. In a previous study, we found a CANT1 (for calcium-activated nucleotidase 1) mutation, c.676G>A in five DBQD families. They were all East Asians (Japanese or Korean). The high prevalence of the same mutation among Japanese and Korean suggested that it is a common founder mutation in the two populations. To examine a possible common founder, we examined the region around CANT1 in chromosomes with c.676G>A mutation by genotyping polymorphic markers in the region for the families. We examined their haplotypes using the family data. We identified in all families a common haplotype containing the CANT1 mutation that ranged up to 550 kb. The two unrelated carriers of the mutation in general populations in Korea and Japan could also have the haplotype. We estimated the age of the founder mutation as ～ 1420 years (95% CI=880-1940 years). The c.676G>A mutation of CANT1 commonly seen in Japanese and Korean DBQD should be derived from a common founder.     

Tracing past population migrations: genealogy of steroid 21-hydroxylase (CYP21) gene mutations in Finland

The genealogic origin of steroid 21-hydroxylase gene (CYP21) mutations and associated haplotypes was determined in 74 unrelated Finnish families with CYP21 deficiency (congenital adrenal hyperplasia, CAH). These families account for two thirds (85/119) of all diagnosed patients of Finnish descent found in this country. We recently demonstrated that multiple founder mutations each associated with a particular haplotype can be found in Finland. Interestingly, some of the haplotypes were identical to those observed in various European populations, whereas others have not been described elsewhere, indicating a local and perhaps a more recent origin. In the present report we show that each of the major founder haplotypes originates from a particular geographic region of Finland. Thus many local genetic isolates are to be expected in Finland. Our finding is in a clear contrast to the genetic diseases known as the 'Finnish disease heritage', in which only one mutation usually predominates. Some of the CYP21 haplotypes proved very informative for analysis of the history of the Finnish population. For example, the origin of one frequent haplotype was shown to cluster in a region assumed by archaeological data to be a major site of immigration by settlers of either Scandinavian or Baltic origin during the first centuries AD. As this haplotype is frequent in many European patient populations, we provide independent genetic evidence of this Iron Age immigration. On the other hand, another frequent haplotype found solely in Finland reflects a more recent (post 15th century) settlement expansion. Consequently, well characterised and sufficiently frequent autosomal gene markers can provide useful information on migrations both between and within populations.     

Cathepsin K gene mutations and 1q21 haplotypes in at patients with pycnodysostosis in an outbred population

The molecular genetics of the autosomal recessive disorder pycnodysostosis was studied in five independent families from an outbred Caucasian population. We found two new mutations and one recently described mutation in the cathepsin K gene by sequencing DNA from eight patients with pycnodysostosis: a one base transition in exon8, c926T > C, causing a single amino acid substitution leucine-->proline, L309P; A 3' splice site mutation in intron 2, c121-1G > A, causing deletion of all exon 3, 41V-81Mdel; and the exon 3 missense mutation c236G > A leading to residue G79E. In three of the families patients were homozygous for 926T > C. In the remaining two families patients were heterozygous for 926T > C and 121-1G > A in one case, and for 926T > C and 236G > A in the other case. Assays using genomic DNA were developed for all three mutations. We tested 150 healthy control persons and observed the mutation frequencies: 0 to 300 for 121-1G > A and 236G > A and 1 to 150 for 926T > C. One patient from each family was haplotyped with eight microsatellite markers surrounding the cathepsin K gene on chromosome 1q21. A very rare, P = 1.8 x 10(-6) to P = 0.0004, and highly preserved area around the presumed disease locus was common to all the patients. This haplotype was found on seven chromosomes identical by state, IBS, out of the possible eight carrying the 926T > C mutation. Founder effect, locus homogeneity, and allele heterogeneity regarding pycnodysostosis within this population are discussed. Finally, the first pregnancy and delivery described in a patient with pycnodysostosis is reported.     

Haplotype analysis suggests that the two predominant mutations in Japanese patients with holocarboxylase synthetase deficiency are founder mutations

Holocarboxylase synthetase (HCS) deficiency is a rare autosomal recessive disorder of biotin metabolism. Including three new Japanese patients we diagnosed in this study, ten Japanese families have, so far, been accumulated. In these families, the mutations 237Leu > Pro (sevenalleles) and 1067delG (five alleles) were predominant; 508Arg > Trp and 550Val > Met mutations were identified in three families in the heterozygous form and in one patient in the homozygous form, respectively. To determine the origin of these mutations, we identified new polymorphic microsatellite markers in the HCS gene and analyzed the haplotypes of the patients. All the 237Leu > Pro and the 1067delG alleles were associated with haplotype 2-2. This finding is consistent with the notion that these mutations are founder mutations in the Japanese population. Three Japanese 508Arg > Trp alleles were associated with several haplotypes, including 2-3 and 1-4. The haplotype of a Taiwanese patient homozygous for the 508Arg > Trp mutation was 2-3/2-3. The haplotype of one Japanese patient homozygous for the 550Val > Met mutation was 1-4/1-4, whereas that of a Jewish patient with the same homozygous mutation was 2-3/2-3. Both mutations were associated with at least two haplotypes and were found in several ethnic groups. The changes 508Arg > Trp and 550Val > Met occurred at CpG dinucleotide. The data suggest that these two mutations represent a mutational hot-spot. Received: August 31, 2000 / Accepted: September 28, 2000     

Unique origin and low penetrance of the 946delGAG mutation in Valencian DYT1 families

Mutations in the DYT1 gene cause idiopathic torsion dystonia (ITD) transmitted in families as an autosomal dominant trait with incomplete penetrance. The most common mutation, 946delGAG, has been observed in populations with different ethnic and geographic origins. We have investigated 40 individuals from 22 unrelated families with ITD originating from the Land of Valencia, Spain, for the presence of this mutation and we found 5 patients and 6 unaffected subjects from 4 families who were carriers of the mutation. This finding indicates that 18% of families may be diagnosed as DYT1 and that penetrance is reduced. We detected two different geographic and linguistic origins of the Valencian families. However, by haplotype analysis using D9S1260, D9S1261, D9S63 and D9S1262 as flanking markers, we demonstrated that all affected and unaffected carriers shared a common chromosome confirming identical origin of the mutation in the four families. We postulate a unique origin for the 946delGAG mutation in the Land of Valencia and, based on linguistic criterion, we propose that the mutation might have occurred at the beginning of the second millennium. Genetic analysis of another family from Castilla-La Mancha showed a different haplotype segregating with the disease, suggesting that at least two distinct mutational events for the 946delGAG mutation have occurred in Spain.