Homozygosity for the transthyretin-met30-gene in two Swedish sibs with familial amyloidotic polyneuropathy

Familial amyloidotic polyneuropathy (FAP) is an autosomal dominant inherited disorder. Recent biochemical studies have revealed that amyloid protein in FAP of Japanese, Swedish and Portuguese origin mainly consists of a variant transthyretin (TTR) (formerly called prealbumin) with one amino acid substitution of methionine for valine at position 30. In a 56-year-old man with typical polyneuropathy, gastrointestinal problems and vitreous amyloid, we diagnosed homozygosity for the TTR-met30-gene using RFLP analysis. In a family study, a sister presented the same homozygous RFLP pattern; however, in a careful clinical investigation we were not able to demonstrate any of the typical symptoms of FAP, nor could we demonstrate amyloid deposits in a biopsy skin specimen. This is the first report of homozygosity for the TTR-met30-gene, and it shows that the mutation of the protein involved in amyloid formation may be necessary but is clearly not sufficient for the clinical symptoms.     

Hereditary amyloidosis: detection of variant prealbumin genes by restriction enzyme analysis of amplified genomic DNA sequences

The autosomal dominant prealbumin amyloidoses are late-onset disorders characterized by varying degrees of peripheral neuropathy, nephropathy and cardiomyopathy. To date, seven different single amino acid mutations in the plasma protein prealbumin (transthyretin) have been found to be associated with amyloidosis and each is the result of a single nucleotide change in the prealbumin gene. By virtue of the restriction endonuclease sites created by the point mutations which give rise to the protein variants, direct DNA tests using Southern analysis have already been developed for detection of the Met-30, Ile-33, Ala-60, Tyr-77 and Ser-84 prealbumin genes. As an alternative to Southern analysis, we have amplified discrete regions of the prealbumin gene using polymerase chain reaction (PCR) and used restriction enzyme analysis of the PCR products to detect the Met-30, Ala-60, Tyr-77 and Ser-84 prealbumin genes after agarose gel electrophoresis and staining with ethidium bromide. In comparison to Southern analysis these alternative tests yield results much more quickly and avoid the use and handling of radioactively labeled probes.     

Biochemical effect of liver transplantation in two Swedish patients with familial amyloidotic polyneuropathy (FAP-met30)

Familial amyloidotic polyneuropathy (FAP) is an autosomal dominant inherited disorder characterized by progressive peripheral and autonomic neuropathy, associated with neural and systemic amyloid deposits. The amyloid fibrils contain a variant transthyretin (TTR) molecule (TTR met30), over 90% of which is produced in the liver. After liver transplantation in two patients with severe symptomatic FAP, only normal TTR was detectable in circulation. The two patients are being monitored at regular intervals, and, although in one patient there was no evidence of reduction in the quantity of amyloid present at 6 months, there had been no further progression of the neuropathy.     

Late onset type I familial amyloidotic polyneuropathy: Presentation of three autopsy cases in comparison with 19 autopsy cases of the ordinary type

Clinicopathological features of three autopsy cases of extremely rare late onset type I familial amyloldotic polyneuropathy were presented and compared with 19 autopsy cases of the ordinary type. In the late onset cases, the ages at onset and at death were 27.5 and 24.5 years older, respectively, compared with the ordinary type. Also, duration of the total clinical course form onset to death was 3.7 years less than in the late onset cases. The degree of amyloid deposition was more marked in the heart of the late onset cases, causing prominent cardiac hypertrophy. It was also marked In the kidneys or thyroid of two cases, but slight to moderate in the peripheral or autonomic nervous tissues in all cases. Immunohistochemical Investigation demonstrated the presence of transthyretin (TTR) as an amyloid precursor protein and of serum amyloid P-component in amyloid deposits in various organs and tissues of the late onset type. These findings, as well as serum levels of variant TTR, were similar to those of the ordinary type. These results suggest that there are some factors other than the amyloid precursor protein that effect the degree of amyloid deposition.     

Late-onset familial amyloid polyneuropathy with the TTR Met 30 mutation in France

Four unrelated French cases of familial amyloid polyneuropathy are reported. Clinical onset ranged from the sixth to the ninth decade. Sensory signs were predominant initially in the lower limbs; motor changes, and in one case autonomic involvement, appeared later. Amyloid disease was clinically limited to the peripheral nervous system. In two cases, there was no evidence of familial disease. DNA analysis was performed in these four patients and in two children of Patient 1. Restriction analysis of amplification products of exon 2 of the transthyretin gene was positive for the valine 30 to methionine mutation. These four unrelated patients live in different areas of France. Further studies are needed to determine whether these mutations have a common origin and whether they are related to the Portuguese mutation.     

Mitochondrial haplogroup is associated with the phenotype of familial amyloidosis with polyneuropathy in Swedish and French patients

Familial amyloidotic polyneuropathy (FAP) is a monogenic disease caused by mutations in the transthyretin ( TTR ) gene. The phenotype of the most common TTR mutation, V30M, varies within and between populations. Oxidative stress and protein misfolding are cellular processes involved in the development of FAP. Because the mitochondria are important for both these processes, we investigated if mitochondrial haplogroups are related to age at onset of the disease in Swedish and French FAP patients. Mitochondrial haplogroup analysis was performed on 25 early-onset (below 40 years) and 29 late-onset (above 51 years) Swedish FAP patients. DNA from 249 Swedish individuals served as controls. In addition, 6 early-onset and 17 late-onset French FAP patients were examined with 25 French controls. The haplogroup distribution among late-onset Swedish and French cases was similar to that found in the general populations, whereas among early-onset cases a different haplogroup distribution was seen. The relatively rare haplogroup K was significantly more common among early-onset cases. Our findings substantiate the suggestion that a genetic component, still to be found, affecting mitochondrial function has an impact on the amyloid generating process in transthyretin amyloidosis.     

Chain reaction of amyloid fibril formation with induction of basement membrane in familial amyloidotic polyneuropathy

Familial amyloidotic polyneuropathy (FAP) is characterized by extracellular deposition of amyloid fibrils caused by a point mutation in the transthyretin (TTR) gene. Despite data from a number of in vitro studies of TTR amyloidogenesis, many questions, including where and how these fibrils form in vivo and what is the impact of amyloid deposition on tissues, remain unanswered. Here, we analysed the relationship between amyloid fibril formation and micro‐environmental changes by using autopsy cardiac tissues from 11 patients with FAP and a smooth muscle cell line. Ultrastructural studies of cardiomyocytes and vascular smooth muscle cells showed that amyloid fibrils formed first at the basement membrane and that amorphous non‐fibrillar TTR deposits and premature fibrils predominated during the early stage of amyloid deposition. Immunohistochemical analyses revealed that expression of major components of the basement membrane, such as collagen IV, laminin, and fibronectin, increased in parallel with the accumulation of TTR amyloid fibrils. In vitro studies with a vascular smooth muscle cell line revealed that synthetic TTR aggregates increased expression of these basement membrane components. Serum levels of collagen IV in FAP patients were significantly higher than those in healthy controls. Our data thus indicate that TTR amyloid fibrils formed first at the basement membrane and that expression of basement membrane components that was induced by amyloid deposition contributed to further amyloid deposition. This chain reaction may have important implications for FAP pathogenesis. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Haplotype analysis of classical and mild phenotype of phenylketonuria in the German Democratic Republic

The restriction fragment length polymorphism (RFLP)-haplotypes have been analysed in 16 families from the northern part of the GDR at risk for classical and mild phenylketonuria (PKU). Ten different RFLP haplotypes associated with the normal and mutant phenylalanine hydroxylase (PAH) alleles were identified. Of the 32 mutant alleles analysed, 29 (90.6%) were associated with haplotypes 1, 2, 3 and 4; 53.1% of the mutant alleles were linked with haplotype 2. The distribution of RFLP haplotypes in 16 patients of clinical different PKU phenotypes (classical and mild) is reported.     

Detection and identification of dengue-1 virus in clinical samples by a nested-PCR followed by restriction enzyme digestion of amplicons

Dengue viruses cause a disease with clinical findings ranging from asymptomatic infections to severe manifestations, characterised by haemorrhage and shock and known as dengue haemorrhagic fever/dengue shock syndrome. Since this fever and syndrome usually results from sequential infections by distinct dengue serotypes, rapid detection and identification of dengue viruses circulating in endemic areas are important to implement control measures, and ultimately to avoid secondary infections that could result in dengue haemorrhagic fever/dengue shock syndrome. A nested-PCR was developed followed by restriction enzyme (Kpn I) digestion of the amplicons to differentiate dengue-1 from dengue-2. Seventy-five IgM-containing samples collected from 2 to 17 days after the beginning of the symptoms were examined. These samples were submitted to nested-PCR amplification, the amplicons were digested with Kpn I, and the results compared to virus isolation in C6/36 cells and to results obtained by the standard PCR. Out of 75 tested samples, virus was isolated from 2 (2.6%), 17 (22.7%) were positive by the regular PCR protocol, and 58 (77.3%) were positive by nested-PCR. All of the amplicons digested by Kpn I identified dengue-1 virus as the infecting strain. These results indicate that the nested-PCR provided a high yield of dengue genome amplification even in the presence of IgM antibodies, and restriction enzyme digestion defined rapidly the circulating serotype. Therefore, the combination of these techniques may be useful to rapidly identify dengue viruses in countries where dengue-1 and dengue-2 circulates, and this approach can also be applied to the other two serotypes.     

Evidence of the presence of amyloid substance in the blood of familial amyloidotic polyneuropathy patients with ATTR Val30Met mutation

Transthyretin (TTR) is a major amyloid fibril protein found in patients with familial amyloidotic polynuropathy (FAP) and senile systemic amyloidosis (SSA). Mainly synthesized in the live, TTR is transferred in the form of tetramer bound with thyroxine, retinol-binding protein (RBP) and lipoprotein in the blood. The aim of this study was to demonstrate the presence of amyloid substances in the blood by investigated the hemocoelom amyloid in different tissue sections from autopsies such as brain, kidney, heart and aorta arch tissue. Congo red staining was employed following by application of polarized light examination, to verify the presence of amyloid deposition in the tissues. Immunohistochemical staining was then performed to identify the specific type of amyloid deposition. Matrix-assisted laser desorption-ionization/time of flight mass spectrometry (MALDI-TOF/MS) was also used to analyze TTR mutation in FAP patients. All subjects were FAP ATTR Val30Met patients. In FAP patients, TTR amyloid deposition was found mainly in the tunica intima of the aortic arch. Interestingly, amyloid substance was found in the blood of FAP patient. Our results suggest that amyloid substance was present in the blood of FAP ATTR Val30Met patients.     

DNA fingerprinting of yeast strains by restriction enzyme analysis

Restriction endonuclease analysis was used as a new method to obtain genomic DNA fingerprints in yeast. Fifteen yeast strains belonging to the genera Saccharomyces and Zyggosaccharomyces were examined. Restriction fragments obtained with Apal or Kspl endonucleases were separated by SDS-PAGE and silver-stained. Analysis of the fingerprints showed that restriction enzyme digestion of genomic DNA can be successfully applied to yeast, enabling the differentiation between strains belonging to different or to the same species or genera.     

聚合酶链反应-单链构象多态性分析在家族性高胆固醇血症患者家系调查中的应用

目的　探讨聚合酶链反应 -单链构象多态性分析 (polymerase chain reaction- single strainconformation polymorphism,PCR- SSCP)在家族性高胆固醇血症 (familial hypercholesterolemiac,FH)患者家系分析中的应用价值。方法　对于经 PCR- SSCP筛查、DNA序列分析证实的 4例 FH患者 (1例纯合子 FH外显子 7发生点突变 ,1例杂合子点突变位于外显子 14,2例杂合子点突变位于外显子 4的 3′部分 ) ,用 PCR- SSCP分析各家系成员共 2 3例 ,并对基因型和表型进行比较。结果　各家系成员均从基因水平明确了诊断 ,2 3例个体中 ,除先证者外 ,还发现 1例纯合子 ,8例杂合子。结论　 PCR- SSCP可对 FH先证者家系进行分析 ,并对其家系成员早期诊断 ,以便提供咨询和指导。     

Wild-type transthyretin significantly contributes to the formation of amyloid fibrils in familial amyloid polyneuropathy patients with amyloidogenic transthyretin Val30Met

Wild-type transthyretin is inherently an amyloidogenic protein, but its contribution to the formation of amyloid fibrils remains unclear in familial amyloid polyneuropathy patients. Our aim in this study was to elucidate the ratio of wild-type transthyretin in amyloid deposits in familial amyloid polyneuropathy patients. Abdominal fat amyloid fibrils in 44 familial amyloid polyneuropathy patients with amyloidogenic transthyretin Val30Met who had not undergone liver transplantation were examined. The amyloid fibrils were extracted from abdominal fat tissues and the composition ratios of wild-type and variant transthyretin were analyzed with liquid chromatography tandem mass spectrometry. The amyloid fibrils in abdominal fat tissues were composed of not only variant but also wild-type transthyretin in most patients (mean ratio, 40.7% ± 27.5%). The composition ratios of wild-type transthyretin in patients older than 50 years were significantly higher than those in patients younger than 50 (50.7% ± 26.9% versus 30.7 ± 24.8%). Our results indicate that wild-type transthyretin significantly contributes to the formation of amyloid fibrils in familial amyloid polyneuropathy patients with amyloidogenic transthyretin Val30Met, and its contribution tends to increase in older patients, suggesting that aging may play an important role in wild-type transthyretin-derived amyloid fibril formation in familial amyloid polyneuropathy patients. This is the first report showing the relationship between wild-type transthyretin deposition and aging in familial amyloid polyneuropathy patients. In addition, wild-type transthyretin may be more strongly amyloidogenic than previously considered because it is detectable even in amyloid fibrils isolated from young familial amyloid polyneuropathy patients.     

两个家族性高胆固醇血症纯合子家系低密度脂蛋白受体基因分析

目的：分析家族性高胆固醇血症（familial hypercholesterolemia,FH)低密度脂蛋白受体（low density lipoprotein receptor,LDLR)的基因突变。方法：提取5个彼此无亲缘关系临床诊断为FH的纯合子患儿及其家系成员的基因组DNA,用聚合酶链反应－单链构象多态性分析方法,对LDLR基因的启动子和全部18个外显子进行突变检测,并对结果异常者进行DNA测序。结果：在两个家系分别发现A606T和C263R两种突变。结论：LDLR基因在以上两位点的突变可引起FH,中国FH患者的LDLR基因可能存在特有的突变位点。     

Five missense mutations at the adenosine deaminase locus (ADA) detected by altered restriction fragments and their frequency in ADA – patients with severe combined immunodeficiency (ADA – SCID)

Severe combined immunodeficiency (SCID) is a heterogeneous syndrome, due to X-linked and autosomal recessive defects. A significant proportion of the autosomal recessive forms of SCID are due to mutations at the adenosine deaminase (ADA) locus. Nine different mutations at the ADA locus, including 7 missense point mutations, have been reported in children with ADA – SCID. We could detect 5 of the 7 missense mutations associated with ADA – SCID by alterations in restriction fragments utilizing standard restriction digestion of genomic DNA and hybridization of radiolabelled ADA genomic probes to Southern transfers. We additionally developed more rapid nonradioactive methods employing digestion of genomic DNA amplified by PCR that also detected all 5 mutations. Using these methods, we have examined a sample of 45 ADA – SCID chromosomes and report that these 5 missense mutations account for one third of the ADA – chromosomes studied, with 2 mutations being relatively common.     

HLA Class II typing by digestion of PCR-amplified DNA with allele-specific restriction endonucleases will fail to unequivocally identify the genotypes of many homozygous and heterozygous individuals

Abstract: Recently, a new technique for HLA class II genotyping has been introduced, the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, claimed to be a practical alternative to conventional serological and cellular HLA class II typing (1-3). The PCR-RFLP technique is ingenious, relatively rapid and does not require hybridization with sequence-specific oligonucleotide probes. However, analysis of whether homozygous and heterozygous combinations of PCR-RFLP patterns for the various investigated HLA class If loci are unique or not unfortunately shows that only 19% of DRB homozygous and heterozygous combinations are unique. The figures for the DQA1 , DQB and DPB loci are 56%, 29% and 65%, respectively. As not all nucleotide sequences analyzed in the above-mentioned studies (1-3) gave rise to unique PCR-RFLPs and as more sequences now are known, for the DRB1, DRB3, DRB5 and DPB1 loci (4), the frequencies of unique PCR-RFLP patterns for the different HLA class II loci will be reduced even further. Thus, the present analysis demonstrates that the PCR-RFLP technique, performed as described in references 1-3, is not yet ready to be used for routine HLA class II genotyping. The resolution of the PCR-RFLP method can be improved by various modifications. However, the role of a modified PCR-RFLP technique in HLA class II typing still remains to be shown.     

Investigation of varicella-zoster virus DNA by the polymerase chain reaction in healthy children with varicella vaccination

Investigation of varicella-zoster virus (VZV) DNA in 20 healthy children with varicella vaccination was carried out by using the polymerase chain reaction (PCR) and nested double PCR. Samples of peripheral blood mononuclear cells (PBMC) and throat swabs were simultaneously collected 3 times (before, 1 week, and 4 weeks after vaccination) for PCR analysis. One sample of PBMC was also obtained from each of the 12 healthy children with varicella during the acute phase as a positive control. VZV DNA was not found by the first PCR in any samples except one PBMC of a control subject. The nested double PCR, which is a more sensitive method for VZV DNA detection, was applied to the same samples. The viral DNA was detected in every PBMC of the controls, and in 2 (16.7%) PBMC at 1 week and in 6 (50%) PBMC at 4 weeks after vaccination in the 12 vaccinees with seroconversion. In 3 of 4 vaccinees who were seropositive before vaccination, VZV DNA was detected in PBMC at 1 or 4 weeks after vaccination. The three vaccinees showed a booster immunization with a significant increase in antibody liters. In contrast, no VZV DNA could be detected in any throat swabs of all the vaccinees nor in PBMC of the vaccinees who did not Seroconvert. © 1994 Wiiey-Liss, Inc.     

Characterization of common and rare human papillomaviruses in Portuguese women by the polymerase chain reaction,restriction fragment length polymorphism and sequencing

The present study aimed to provide additional information on the prevalence of mucosal human papillomavirus (HPV) types in Portuguese women by using polymerase chain reaction, restriction fragment length polymorphism and sequencing. HPV was detected in 15.5% (15/97) of the control samples, 23.5% (12/51) of atypical squamous cells of undetermined significance, 52.8% (28/53) of low‐grade lesions, 82.4% (28/34) of high‐grade lesions, and 100% (44/44) of carcinomas. Overall, 28 HPV types were detected: 11 high‐risk (HPV 16, 18, 31, 33, 35, 39, 45, 52, 56, 58, and 59), 3 probable high‐risk (HPV 53, 66, and 73), 6 low‐risk (HPV 6, 11, 44, 61, 70, and 81), and 8 unknown‐risk types (HPV 34, 62, 67, 71, 83, 84, 102, and 108). The most prevalent type was HPV 16, detected in 33.8% of women infected with HPV, followed by HPV 58 (9.2%), HPV 33 (7.0%), HPV 18 (6.3%), HPV 53 (5.6%), HPV 31 and 56 (4.9% each), HPV 6 (3.5%), and HPV 66 and 81 (2.8% each). Of 44 cervical carcinoma samples, 71% were associated with HPV 16 (60%) and HPV 18 (11.1%), followed by the high‐risk types 33 (11.1%), 35 (4.4%), 45 (4.4%), and 56 (2.2%), the probable high‐risk type 53 (4.4%) and the unknown‐risk type 67 (2.2%). This study provides information on the most common HPV types in Portuguese women and suggests that the current prophylactic HPV 16/18 vaccine may be useful for the prevention of cervical cancer in this population. J. Med. Virol. 82:1024–1032, 2010. © 2010 Wiley‐Liss, Inc.     

A new DIG-PCR-EIA method for the detection of Chlamydia pneumoniae DNA in clinical samples

Chlamydia pneumoniae, a common respiratory pathogen, may also play a role in the pathogenesis of other chronic conditions. For accurate detection of infected persons and verification of results obtained by other PCR methods, a DIG-PCR-EIA method was evaluated. In the DIG-PCR-EIA, a 437 bp DNA sequence was amplified and hybridized with a newly synthesized 229 bp biotin-labeled probe. The end product was detected by an enzyme immunoassay. The sensitivity of DIG-PCR-EIA was compared with Southern blot hybridization and one-step HR/HL PCR, which was the routine method used. DNA was detected to the level of 20 elementary bodies of DIG-EIA-PCR compared to less than 2 by Southern blot, and 200 by HR/HL PCR. Thus a 100-fold increase in sensitivity could be expected by DIG-EIA-PCR compared to the routine method. Throat swabs and adenoid tissue from 22 children with otitis and middle ear secretions from 29 children, as well as throat swabs from 179 blood donors, were analyzed with DIG-EIA-PCR, HL/HR PCR and nested touchdown PCR. 32% of the ear secretions were positive by DIG-EIA-PCR as compared to 5% by the other two methods. Three adenoid tissue samples were positive by all methods applied. Among the child and adult throat samples, 18% and 32%, respectively, were positive by DIG-EIA-PCR and 5% and 10% by HR/HLPCR. The results indicate the suitability of DIG-PCR-EIA for verification of results of HR/HL PCR. DIG-PCR-EIA has a potential for increased sensitivity and adaptation for automation. It should be further evaluated using various types of tissue specimens and DNA extraction methods.     

血管紧张素原基因核心启动子区域突变与藏族原发性高血压的关联分析

目的 寻找血管紧张素原(angiotensinogen, AGT)基因核心启动子区域存在的突变,分析该突变在中国西藏人群中的分布以及与原发性高血压的关联.方法 以藏族103例原发性高血压患者和82名健康受试者为研究对象进行病例-对照研究.用聚合酶链反应-单链构象多态性(polymerase chain reaction/single strand conformation polymorphism, PCR/SSCP)分析和自动荧光测序方法,对AGT基因核心启动子区域DNA序列进行突变分析;用聚合酶链反应-限制性片段长度多态性(polymerase chain reaction/restriction fragment length polymorphism, PCR-RFLP)方法分析AGT基因(-6)位点多态性.结果 PCR/SSCP分析发现,AGT基因转录起始位点上游(-20)位存在A→C突变,统计分析显示,藏族正常人群与高血压人群中该位点A等位基因均有较高的发生频率(0.9175,0.9124),突变位点多态性分布无统计学差异(P＞0.8).AGT基因转录起始位点上游(-6)位点存在A→G突变,在藏族正常人群中等位基因A和G分布频率分别为0.780和0.220,原发性高血压群体中它们的分布频率分别为0.626和0.374,两者之间存在差异(P＜0.025).结论 (1)藏族群体中AGT基因(-20)A等位基因有较高的分布频率;(2)AGT基因(-6)G等位基因在藏族高血压患者群体中发生频率较高,可能是藏族原发性高血压的遗传易感因子.