The molecular defect in type IIB von Willebrand disease. Identification of four potential missense mutations within the putative GpIb binding domain.

Type IIB von Willebrand Disease (vWD) is characterized by the selective loss of large von Willebrand Factor (vWF) multimers from plasma, presumably due to their increased reactivity with platelets and subsequent clearance from the circulation. Using the PCR, one of a panel of four potential missense mutations was identified in each of the 14 patients studied from 11 unrelated families. None of these substitutions was encountered in a large panel of normal DNAs. These changes all represent C----T transitions at CpG dinucleotides, proposed "hot spots" for mutation in the human genome. The four resulting amino acid substitutions, Arg543----Trp, Arg545----Cys, Val553----Met, and Arg578----Gln, are all clustered within the GpIb binding domain of vWF. Disruption of this latter functional domain may explain the pathogenesis of Type IIB vWD. By sequence polymorphism analysis, the Arg543----Trp substitution was shown to have occurred as at least two independent mutational events. This latter observation, along with the identification of mutations in all 14 patients studied and their localization to the GpIb binding domain, all strongly suggest that these substitutions represent the authentic defects responsible for Type IIB vWD. This panel of mutations may provide a useful diagnostic tool for the majority of patients with Type IIB vWD.     

Two novel missense mutations causing adrenoleukodystrophy in Italian patients.

The authors present two new missense mutations in exon 1 of the adrenoleukodystrophy (ALD) gene. The first, a C813T transition, results in the substitution Pro143 Ser in the third putative transmembrane domain of the adrenoleukodystrophy protein (ALDP) in an adult onset case. The second, a de novo C709T transition, results in a substitution Ser 108 Leu between the second and the third putative transmembrane segments, in a childhood case.     

Large deletion of the peroxisomal acyl-CoA oxidase gene in pseudoneonatal adrenoleukodystrophy.

We have cloned the cDNA encoding human peroxisomal acyl-CoA oxidase, the first enzyme in the peroxisomal beta-oxidation of very long chain fatty acids. Its nucleotide sequence was found to be highly homologous (85%) to the rat cDNA counterpart. An 88% homology between rat and human was found in the COOH-terminal end of the cDNA which includes the Ser-Lys-Leu peroxisomal targeting signal common to many peroxisomal proteins. The gene spans approximately 30-40 kb and is poorly polymorphic. Southern blot analyses were performed in two previously reported siblings with an isolated peroxisomal acyl-CoA oxidase deficiency (pseudoneonatal adrenoleukodystrophy). A deletion of at least 17 kb, starting down-stream from exon 2 and extending beyond the 3' end of the gene, was observed in the two patients. These observations provide a molecular basis for the observed acyl-CoA oxidase deficiency in our family. In addition, our study will enable the characterization of the genetic defect in unrelated families with suspected acyl-CoA oxidase disorders.     

Identification of mutations in 23S rRNA gene of clarithromycin-resistant Mycobacterium intracellulare.

Clarithromycin is a potent macrolide that has been used for treating infections with nontuberculous mycobacteria. Pairs of susceptible and resistant Mycobacterium intracellulare strains were obtained from patients with chronic pulmonary M. intracellulare infections undergoing monotherapy with clarithromycin. Nucleotide sequence comparisons of the peptidyltransferase region in 23S rRNAs from parental and resistant strains revealed that in three of six resistant strains, for which the MIC was > 32 micrograms/ml, a single base was mutated (Escherichia coli equivalent, A-2058-->G, C, or U). As the modification of adenine 2058 by dimethylation is a frequent cause of macrolide resistance in a variety of different bacteria, we suggest that mutation of A-2058 confers acquired resistance to clarithromycin in M. intracellulare.     

Identification of novel mutations of the CFTR gene in a Japanese patient with cystic fibrosis.

Cystic fibrosis (CF) is an inheritable disorder characterized by defective epithelial chloride transport and progressive lung disease, caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The subject of this study was an 8-year old Japanese boy, who developed typical CF symptoms including meconium ileus, pancreatic insufficiency, an elevated sweat chloride concentration and pulmonary disease. Analysis of the CFTR gene of this patient revealed compound heterozygous mutations in exon 11 (1742 delAC) and intron 9 (1525-18 GtoA) of the CFTR gene.     

Point mutations in the steroid-binding domain of the androgen receptor gene of five Japanese patients with androgen insensitivity syndrome.

We analyzed the androgen receptor (AR) gene in five Japanese patients diagnosed with androgen insensitivity syndrome (AIS). All AR genes from the five patients had single-nucleotide substitutions, which introduced a premature termination codon in three patients (Gln640, Arg752, and Gln640 and Trp751), and a single amino acid substitution in two patients (Arg831 to Gln, and Leu812 to Phe). All the mutations occurred in the steroid-binding domain, comprising exons D through G. The three patients with the premature termination codon(s) and the one patient with Arg831Gln were clinically diagnosed as having complete AIS, while the patient with Leu812Phe had a partial form of AIS. Pubic skin fibroblasts from four of the five patients did not show detectable androgen binding. These data on mutations that have not been reported previously, provide valuable information for the further characterization of structural and functional relationships in the steroid-binding domain of the AR protein.     

Plasmapheresis in treatment of adrenoleukodystrophy.

Adrenoleukodystrophy (ALD) is an X-linked disorder of metabolism of very long chain fatty acids (VLCFAs) with a frequency of up to 1:20,000 in males. VLCFAs C24:0 and C26:0 accumulate in the cholesterol ester and ganglioside fraction in plasma and red cells. Symptoms of ALD are ataxia, loss of visual and auditory functions, and cerebral convulsions. Up to the present, no sure therapeutic approaches have been established. Efforts were reported by dietary regimens with VLCFA restriction and glyceroltrioleate and glyceroltrierucate intake. In the present trial, we report a 55-year-old Caucasian male suffering from progressive ALD with spastic paraparesis. He has had a positive family history since the eighteenth century. In this patient treated with maximum dietary therapy over a period of 60 months, no normalization of C24:0 and C26:0 was reached, and neurological disorders were progressive. As a result, plasmapheresis was applied during the period 1990-1994 and since then for more selective adsorption of VLCFAs, dextran sulfate adsorption (Liposorber, Kaneka, Japan). During this period (64 months), C24:0 and C26:0 levels were reduced by 54.5% and 51.8%, respectively (p = 0.0001). The patient experienced a significant improvement in performance and general well-being. There has been no further progression of neuronal disorders to document.     

Identification of mutations in the apolipoprotein B-100 gene and in the PCSK9 gene as the cause of hypocholesterolemia

BACKGROUND: Characterization of the normally occurring mutations as the cause of hypocholesterolemia may increase our understanding of the normal lipid metabolism. METHODS: DNA from 93 unrelated hypocholesterolemic subjects with a mean (+/-SD) value for total serum cholesterol of 3.3 (+/-0.5) mmol/l) were subjected to DNA sequencing of the individual exons of the apolipoprotein B-100 (apoB-100) gene and of the proprotein convertase subtilisin/kexin 9 (PCSK9) gene. The same analyses were also performed in 23 unrelated subjects with autosomal dominant hypercholesterolemia who had unusually low levels of total serum cholesterol. RESULTS: Of the 93 hypocholesterolemic subjects, 9 subjects (9.7%) were heterozygous for a truncating mutation in the apoB-100 gene and six subjects (6.5%) were heterozygous for a loss-of-function mutation in the PCSK9 gene. Of the 23 subjects with autosomal dominant hypercholesterolemia, four subjects (17.4%) were heterozygous for mutations in the apoB-100 gene. CONCLUSION: Truncating mutations in the apoB-100 gene are slightly more common as the cause of hypocholesterolemia compared to loss-of-function mutations in the PCSK9 gene. It appears that mutations in the apoB-100 gene may completely normalize the lipid profile in subjects with autosomal dominant hypercholesterolemia, whereas loss-of-function mutations in the PCSK9 gene do not have a sufficient cholesterol-lowering capacity.     

Identification of two mutations in human xanthine dehydrogenase gene responsible for classical type I xanthinuria.

Hereditary xanthinuria is classified into three categories. Classical xanthinuria type I lacks only xanthine dehydrogenase activity, while type II and molybdenum cofactor deficiency also lack one or two additional enzyme activities. In the present study, we examined four individuals with classical xanthinuria to discover the cause of the enzyme deficiency at the molecular level. One subject had a C to T base substitution at nucleotide 682 that should cause a CGA (Arg) to TGA (Ter) nonsense substitution at codon 228. The duodenal mucosa from the subject had no xanthine dehydrogenase protein while the mRNA level was not reduced. The two subjects who were siblings with type I xanthinuria were homozygous concerning this mutation, while another subject was found to contain the same mutation in a heterozygous state. The last subject who was also with type I xanthinuria had a deletion of C at nucleotide 2567 in cDNA that should generate a termination codon from nucleotide 2783. This subject was homozygous for the mutation and the level of mRNA in the duodenal mucosa from the subject was not reduced. Thus, in three subjects with type I xanthinuria, the primary genetic defects were confirmed to be in the xanthine dehydrogenase gene.     

Protective protein gene mutations in galactosialidosis.

Four different protective protein cDNA mutations, 146A-->G (Q49R), 193T-->C (W65R), 268-269TC-->CT (S90L), and 1184A-->G (Y395C), were identified in six Japanese galactosialidosis patients with various phenotypic manifestations, and another mutation, 746T-->A (Y249N), in a patient of French-German origin with an atypical clinical course. Y395C was a common mutation in four Japanese patients in infancy and childhood; two juvenile patients were compound heterozygotes of Y395C and another common mutation, SpDEx7, and the other two infants were compound heterozygotes of Y395C and mutant alleles other than SpDEx7. We confirmed these mutations in genomic DNA by direct-sequence analysis or restriction-site analysis. The mutant cDNA clones, transiently expressed in a transformed galactosialidosis cell line, did not restore the secondarily deficient beta-galactosidase or alpha-neuraminidase activity except for the Y249N mutation that expressed some carboxypeptidase activity and restored the two lysosomal enzyme activities. Pulse-chase analysis detected a small amount of the mature form, as well as the precursor, in the cells transfected with the Y249N cDNA. Only precursor proteins were detected, mature proteins not appearing for the other mutant cDNAs.     

新疆少数民族苯丙酮尿症患者的突变基因分析

目的 探讨新疆少数民族苯丙酮尿症(PKU)患者的基因突变特征,为有针对性的防治策略提供科学依据.方法 采用单链构象多态性分析技术和PCR产物直接测序方法 ,检测12例少数民族PKU患者的苯丙氨酸羟化酶(PAH)基因突变.结果 从12例维吾尔族、回族和哈萨克族PKU患者(24个PAH等位基因)中,检出13种基因突变,包括错义突变8种、无义突变1种、剪切位点突变3种,其中突变频率最高的是EX6-96A＞G和P281L,EX6-96A＞G常见于国内和亚洲地区,P281L多见于欧洲各地.突变频率较高的R243Q、R111X、R176X和F161S 4种突变中,其中R243Q是我国北方地区居首位的突变,R111X处于第3位.而R176X和F161S在世界范围内都极为少见,尤其F161S是具有中国特色的基因突变,是第2次在中国人中发现.结论 新疆少数民族中的PAH突变基因不仅与亚洲黄种人和欧洲及拉美人种表现出密切的联系,而且也存在着明显的差异,形成了自身独立的遗传规律和特点,是我国一个十分特殊的PAH突变基因分布区域.     

云南省几个民族葡萄糖—6—磷酸脱氢酶基因突变型分析

目的：分析云南籍汉族及少数民族葡萄糖－６－磷酸脱氢酶（Ｇ６ＰＤ）缺乏症患者的Ｇ６ＰＤ基因突变。方法：应用自然引物或“错配引物”介导的多聚酶链反应限制性酶切分析方法，分析２９例云南籍几个民族Ｇ６ＰＤ缺乏症患者Ｇ６ＰＤ基因突变型。结果：发现１３８８突变１９例，１３７６突变１例，未定型９例。２９例标本中有７例为少数民族，分别为彝族３例（１３８８突变）、白族２例（１３８８突变）、傣族１例（１３８８突变）、纳西族１例（１３８８突变）。除傣族外，在其余三个少数民族中检出Ｇ６ＰＤ基因１３８８突变在国内尚属首次。结论：这种突变的发生早于少数民族形成之前，并可推测云南省Ｇ６ＰＤ缺乏症患者中Ｇ６ＰＤ基因突变型分布比例，有助于了解分子进化进程。     

Dravet综合征SCNIA基因新突变及遗传咨询

摘要:目的对2 例癫痫伴精神运动发育迟缓的患儿进行遗传学病因分析,旨在为患儿治疗及其家庭遗传咨询、生育指导提供依据。方法提取2例患儿及 其父母外周血基因组DNA并采用全外显子测序技术进行检测,按照美国医学遗传学与基因组 学学会(ACMG,2015)标准进行致病性分析,通过Sanger测序验证致病变异。结果全外显子测序结果发现,2例患儿电压门控性钠离子通道a1亚单位基因(SCNIA)基因存在c.5354T>C(p.11785T)和c.4380T>A(p.Y1460* )新发de novo杂合突变,分 别评估变异为可疑致病、致病突变位点,Sanger测序验证了该突变并确认双方父母相应位点均为野生型。结论结 合临床和基因诊断信息,2例患儿均被诊断为常染色体显性遺传病Dravet综合征,明确患儿的致病原因对于合理治疗方案的制定及其 家庭的优生优育、遗传咨询具有重要的意义。     

Missense mutations of the tissue-nonspecific alkaline phosphatase gene in hypophosphatasia.

Hypophosphatasia is an inborn error of metabolism that is characterized clinically by defective bone mineralization and biochemically by deficient activity of the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP) in serum and in tissues. Clinical severity is extremely variable, ranging from death in utero to pathologic fractures first presenting in adulthood. Severe forms of the disease are inherited in an autosomal recessive fashion; the modes of transmission of mild forms are uncertain. Deficiency of TNSALP activity in this condition suggests that mutations in the TNSALP "candidate" gene are the primary defects. This hypothesis was supported in 1988 by the demonstration, in one inbred infant, that an identical missense mutation in both alleles of the gene encoding TNSALP caused lethal hypophosphatasia. Here we summarize the work leading to that discovery and discuss the recent identification of additional missense mutations in the TNSALP gene associated with the entire clinical spectrum of hypophosphatasia.     

Identification of mutations in the gene for glucose-6-phosphatase, the enzyme deficient in glycogen storage disease type 1a.

Glycogen storage disease (GSD) type 1a is an autosomal recessive inborn error of metabolism caused by a deficiency in microsomal glucose-6-phosphatase (G6Pase), the key enzyme in glucose homeostasis. Southern blot hybridization analysis using a panel of human-hamster hybrids showed that human G6Pase is a single-copy gene located on chromosome 17. To correlate specific defects with clinical manifestations of this disorder, we identified mutations in the G6Pase gene of GSD type 1a patients. In the G6Pase gene of a compound heterozygous patient (LLP), two mutations in exon 2 of one allele and exon 5 of the other allele were identified. The exon 2 mutation converts an arginine at codon 83 to a cysteine (R83C). This mutation, previously identified by us in another GSD type 1a patient, was shown to have no detectable phosphohydrolase activity. The exon 5 mutation in the G6Pase gene of LLP converts a glutamine codon at 347 to a stop (Q347SP). This Q347SP mutation was also detected in all exon 5 subclones (five for each patient) of two homozygous patients, KB and CB, siblings of the same parents. The predicted Q347SP mutant G6Pase is a truncated protein of 346 amino acids, 11 amino acids shorter than the wild type G6Pase of 357 residues. Site-directed mutagenesis and transient expression assays demonstrated that G6Pase-Q347SP was devoid of G6Pase activity. G6Pase is an endoplasmic reticulum (ER) membrane-associated protein containing an ER retention signal, two lysines (KK), located at residues 354 and 355. We showed that the G6Pase-K355SP mutant containing a lysine-355 to stop codon mutation is enzymatically active. Our data demonstrate that the ER protein retention signal in human G6Pase is not essential for activity. However, residues 347-354 may be required for optimal G6Pase catalysis.     

Novel mutations in the 5'-UTR of the FOLR1 gene.

We have previously reported two novel mutations in the 5'-untranslated region (UTR) of the gene for folate receptor-alpha (FOLR1). In our search for additional mutations, 92 patient samples with elevated levels of homocysteine were screened by single-strand conformation polymorphism (SSCP) between nt -425 and -782, and -712 and -1110. Between nt -425 and -782 we did not find any mutations. Between nt -712 and -1110 there were three novel mutations. One subject had two mutations very close to each other, c.-856C>T and c.-921T>C. Two subjects had a c.-1043G>A mutation. To get an idea of the prevalence of FOLR1 mutations in an unselected population, we also screened 692 healthy school children for mutations. In this cohort, between nt -188 and +272 we discovered one novel mutation, a single nucleotide substitution, c.-18C>T, in addition to five children with the 25-bp deletion mutation previously described by us. Thus, so far we have discovered six novel mutations in the 5'-UTR region of the gene for folate receptor-alpha. We genotyped all 17 subjects with a FOLR1 mutation for the methylenetetrahydrofolate reductase (MTHFR) 677C>T polymorphism, and developed new single-nucleotide polymorphism (SNP) genotyping protocols for MTHFR 1298A>C and 1793G>A utilising Pyrosequencing technology. None of the 17 subjects had the 677TT genotype, which ruled out this as a cause of elevated homocysteine levels, which was observed in some of the subjects. Further studies of mutations in the 5'-UTR of FOLR1, and in particular of their interplay with folate intake status, are warranted.     

Therapeutic plasma exchange in treatment of adrenoleukodystrophy.

Adrenoleukodystrophy (ALD) is an X-linked disorder of metabolism of very long-chain fatty acids (VLCFA) with a frequency of up to 1:20,000 in males. VLCFA C 24:0 and C 26:0 accumulate in the cholesterol ester and ganglioside fraction in plasma and red cells. Symptoms of ALD are ataxia, loss of visual and auditory functions, and cerebral convulsions. Presently, no sure therapeutic approaches are established. Efforts were reported by dietary regimens with VLCFA-restriction and glycerol trioleate and glycerol trierucate intake. In the present trial, we report on a 58-year-old white male suffering from progressive ALD with spastic paraparesis. He has a positive family history back to the 18th century. In this patient, although maximum dietary therapy was applied over a period of 60 months, no normalization of VLCFA C24:0 and C26:0 was reached, and neurological disorders were progressive. As a result, therapeutic plasma exchange (TPE) was applied from 1990 to 1994. Then, for more selective adsorption of VLCFA, dextran-sulfate adsorption (Liposorber, Kaneka, Osaka, Japan) until 1996, and after that, immunoadsorption (Therasorb, Baxter, Munchen, Germany) were used. During these periods (total, 101 months), VLCFA C 24:0 and C 26:0 levels were reduced by 55% and 50% (p < 0.001). The patient experienced a significant improvement in performance and general well-being. No further progression of neuronal disorders was documented. This anecdotal data suggest a very beneficial effect of TPE in treatment of progressive ALD.     

Molecular analysis of ABCD1 gene in Indian patients with X-linked adrenoleukodystrophy

BackgroundX-linked Adrenoleukodystrophy (X-ALD), with an incidence of 1:14,000 is the most frequent monogenic demyelinating disorder worldwide. The principal biochemical abnormality in X-ALD is the increased levels of saturated, unbranched very long chain fatty acids (VLCFA). It is caused by mutations in ABCD1 gene. No molecular data on X-ALD is available in India and mutational spectrum in Indian patients is not known.MethodsWe standardized conformation sensitive gel electrophoresis (CSGE) method to detect mutations in ABCD1 gene in twenty Indian patients with X-ALD. The results were confirmed by sequencing. Genotype–phenotype correlation was also attempted. Prenatal diagnosis (PND) in one family was done using chorionic villi (CV) sample at 12 weeks of gestation.ResultsOut of twenty, causative mutations could be identified in twelve patients (60%). Six reported and four novel mutations were identified. Three polymorphisms were also observed. No hot spot was found. No significant genotype–phenotype correlation could be established.ConclusionsThe study identified the mutation spectrum of Indian X-ALD patients, which enabled us to offer accurate genetic counseling, carrier detection and prenatal diagnosis where needed.