Spectrum of PEX6 mutations in Zellweger syndrome spectrum patients

The autosomal recessive Zellweger syndrome spectrum (ZSS) disorders comprise a main subgroup of the peroxisome biogenesis disorders. The ZSS disorders can be caused by mutations in any of 12 different currently identified PEX genes resulting in severe, often lethal, multi‐systemic disorders. Defects in the PEX6 gene are the second most common cause for ZSS disorders. The encoded protein PEX6 belongs to the AAA ATPase family and contains two AAA cassettes and an AAA protein family signature. The PEX6 gene consists of 17 exons and previously mutations in the PEX6 gene were found to be scattered over all exons. We developed a post‐PCR high‐resolution melting (HRM) curve assay to scan the PEX6 gene for potential sequence variations followed by selective sequencing to identify these. We analyzed the PEX6 genes of 75 patients assigned to the PEX6 complementation group. We identified a total of 77 different mutations of which 47 mutations have not been reported previously, and 14 polymorphic variants. © 2009 Wiley‐Liss, Inc.     

Mutation analysis of PEX7 in 60 probands with rhizomelic chondrodysplasia punctata and functional correlations of genotype with phenotype

PEX7 encodes the cytosolic receptor for the set of peroxisomal matrix enzymes targeted to the organelle by the peroxisome targeting signal 2 (PTS2). Mutations in PEX7 cause rhizomelic chondrodysplasia punctata (RCDP), a distinct peroxisome biogenesis disorder. In previous work we described three novel PEX7 mutant alleles, including one, L292X, with a high frequency due to a founder effect. We have now extended our analysis to 60 RCDP probands and identified a total of 24 PEX7 alleles, accounting for 95% of the mutant PEX7 genes in our sample. Of these, 50% are L292X, 13% are IVS9+1G>C, and the remainder are mostly private. IVS9+1G>C occurs on at least three different haplotypes and thus appears to result from recurrent mutation. The phenotypic spectrum of RCDP is broader than commonly recognized and includes minimally affected individuals at the mild end of the spectrum. To relate PEX7 genotype and phenotype, we evaluated the consequence of the disease mutation on PEX7 RNA by Northern analysis and RT/PCR. We evaluated the function of the encoded Pex7 protein (Pex7p) by expressing selected alleles in fibroblasts from RCDP patients and assaying their ability to restore import of a PTS2 marker protein. We find that residual activity of mutant Pex7p and reduced amounts of normal Pex7p are associated with milder and variant phenotypes.     

Genomic structure and identification of 11 novel mutations of the PEX6 (peroxisome assembly factor‐2) gene in patients with peroxisome biogenesis disorders

The PEX6 (peroxisome assembly factor‐2, PAF‐2) gene encodes a member of the AAA protein (ATPases associated with diverse cellular activities) family and restores peroxisome assembly in fibroblasts from peroxisome biogenesis disorder patients belonging to complementation group C (group 4 in the United States). We have now clarified the genomic DNA structure of human PEX6 and identified mutations in patients from various ethnic groups. The human PEX6 gene consists of 17 exons and 16 introns, spanning about 14kb. The largest exon, exon 1, has at least 952 bp nucleotides. Eleven novel mutations (18 alleles) were identified by direct sequencing of the PEX6 cDNA from 10 patients. All these mutations have been confirmed in the corresponding genomic DNA. There was no common mutation, but an exon skip was identified in two unrelated Japanese patients. Most of the mutations led to premature termination or large deletions of the PEX6 protein and resulted in the most severe peroxisome biogenesis disorder phenotype of Zellweger syndrome. A patient with an atypical Zellweger syndrome had a missense mutation that was shown to disrupt the cell's ability to form peroxisomes. This mutation analysis will aid in understanding the functions of the PEX6 protein in peroxisomal biogenesis. Hum Mutat 13:487–496, 1999. © 1999 Wiley‐Liss, Inc.     

Identification of a new complementation group of the peroxisome biogenesis disorders and PEX14 as the mutated gene

Peroxisome biogenesis disorders (PBD) are lethal hereditary diseases caused by abnormalities in the biogenesis of peroxisomes. At present, 12 different complementation groups have been identified and to date, all genes responsible for each of these complementation groups have been identified. The peroxisomal membrane protein PEX14 is a key component of the peroxisomal import machinery and may be the initial docking site for the two import receptors PEX5 and PEX7. Although PEX14 mutants have been identified in yeasts and CHO-cells, human PEX14 deficiency has apparently not been documented. We now report the identification of a new complementation group of the peroxisome biogenesis disorders with PEX14 as the defective gene. Indeed, human PEX14 rescues the import of a PTS1-dependent as well as a PTS2-dependent protein into the peroxisomes in fibroblasts from a patient with Zellweger syndrome belonging to the new complementation group. This patient was homozygous for a nonsense mutation in a putative coiled-coil region of PEX14, c.553C>T (p.Q185X). Furthermore, we showed that the patient's fibroblasts lacked PEX14 as determined by immunocytochemical analysis. These findings indicate that there are 13 genotypes in PBD and that the role of PEX14 is also essential in humans.     

The peroxin Pex6p gene is impaired in peroxisomal biogenesis disorders of complementation group 6

Human genetic peroxisomal biogenesis disorders (PBDs), such as Zellweger syndrome, comprise 13 different complementation groups (CGs). Eleven peroxin genes, termed PEXs, responsible for PBDs have been identified, whereas pathogenic genes for PBDs of 2 CGs, CG-A (the same CG as CG8 in the United States and Europe) and CG6, remained unidentified. We herein provide several lines of novel evidence indicating that PEX6, the pathogenic gene for CG4, is impaired in PBD of CG6. Expression of PEX6 restored peroxisome assembly in fibroblasts from a CG6 PBD patient. This patient was a compound heterozygote for PEX6 gene alleles. Accordingly, by merging CG6 with CG4, human PBDs are now classified into 12 CGs. Received: December 25, 2000 / Accepted: February 5, 2001     

Identification of novel mutations and sequence variation in the Zellweger syndrome spectrum of peroxisome biogenesis disorders

Peroxisome biogenesis disorders (PBD) are a heterogeneous group of autosomal recessive neurodegenerative disorders that affect multiple organ systems. Approximately 80% of PBD patients are classified in the Zellweger syndrome spectrum (PBD‐ZSS). Mutations in the PEX1, PEX6, PEX10, PEX12, or PEX26 genes are found in approximately 90% of PBD‐ZSS patients. Here, we sequenced the coding regions and splice junctions of these five genes in 58 PBD‐ZSS cases previously subjected to targeted sequencing of a limited number of PEX gene exons. In our cohort, 71 unique sequence variants were identified, including 18 novel mutations predicted to disrupt protein function and 2 novel silent variants. We identified 4 patients who had two deleterious mutations in one PEX gene and a third deleterious mutation in a second PEX gene. For two such patients, we conducted cell fusion complementation analyses to identify the defective gene responsible for aberrant peroxisome assembly. Overall, we provide empirical data to estimate the relative fraction of disease‐causing alleles that occur in the coding and splice junction sequences of these five PEX genes and the frequency of cases where mutations occur in multiple PEX genes. This information is beneficial for efforts aimed at establishing rapid and sensitive clinical diagnostics for PBD‐ZSS patients and interpreting the results from these genetic tests. © 2008 Wiley‐Liss, Inc.     

Novel PEX1 mutations and genotype-phenotype correlations in Australasian peroxisome biogenesis disorder patients

The peroxisome biogenesis disorders (PBDs) are a group of neuronal migration/neurodegenerative disorders that arise from defects in PEX genes. A major subgroup of the PBDs includes Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), and infantile Refsum disease (IRD). These three disorders represent a clinical continuum with Zellweger syndrome the most severe. Mutations in the PEX1 gene, which encodes a protein of the AAA ATPase family involved in peroxisome matrix protein import, account for the genetic defect in more than half of the patients in this PBD subgroup. We report here on the results of PEX1 mutation detection in an Australasian cohort of PEX1-deficient PBD patients. This screen has identified five novel mutations, including nonsense mutations in exons 14 and 19 and single nucleotide deletions in exons 5 and 18. Significantly, the allele carrying the exon 18 frameshift mutation is present at moderately high frequency (approx. 10%) in this patient cohort. The fifth mutation is a missense mutation (R798G) that attenuates, but does not abolish PEX1 function. We have evaluated the cellular impact of these novel mutations, along with that of the two most common PEX1 mutations (c.2097-2098insT and G843D), in PBD patients by determining the levels of PEX1 mRNA, PEX1 protein, and peroxisome protein import. The findings are consistent with a close correlation between cellular phenotype, disease severity, and PEX1 genotype.     

Serum soluble interleukin-6 receptor in MRL/lpr mice is elevated with age and mediates the interleukin-6 signal

The characteristics of soluble interleukin-6 receptor (sIL-6R) in murine sera were examined. To investigate a relationship between serum sIL-6R level and autoimmune diseases, quantitative analysis of serum sIL-6R in MRL/lpr mice was performed by an enzyme-linked immunosorbent assay. The serum sIL-6R level in MRL/lpr mice of both sexes was below the detection limit (< 1.0 ng/ml) at 8 weeks of age, but it increased in accordance with age and reached 42 ± 9.3 ng/ml in female and 31 ± 13 ng/ml in male mice at 30 weeks of age. In MRL/+ mice, although an age-associated increase in serum sIL-6R level was observed, it was much less extensive than that in MRL/lpr mice. Elevated serum sIL-6R level at the age of 30 weeks was observed in female and male (NZB × NZW)F1 mice (32 ± 10 ng/ml and 17 ± 5.0 ng/ml, respectively), and male BXSB/Mpj Yaa mice (42 ± 18 ng/ml), suggesting that elevated serum sIL-6R in aged mice is one of the characteristics of autoimmune-prone mice. Quantitative analysis of serum IL-6 in MRL/lpr revealed that the serum sIL-6R level correlated well with the serum IL-6 level. We also showed that sIL-6R in the sera from MRL/lpr mice could mediate the IL-6 functions through the IL-6 signal-transducing receptor component gpl30, suggesting that elevated production of sIL-6R may partly contribute to development of autoimmune disease in MRL/lpr mice.     

Recessively inherited,late onset spondylar dysplasia and peripheral corneal opacity with anomalies in urinary mucopolysaccharides: A possible error of chondroitin-6-sulfate synthesis

Two male and two female sibs with an unusual form of spondyloepiphyseal dysplasia were reported. The main clinical features were low stature, moderate shortness of trunk and neck, abnormal span: height ratio, low-normal UBS: LBS ratio, and peripheral corneal punctate opacities only seen by the slitlamp. Normal mental status was present. Typical metachromatic granules were not seen either in bone-marrow cells or in peripheral blood cells. The X-ray picture showed spondylar and pelvic dysplasias. Qualitative rather than quantitative anomalies were shown in the urinary mucopolysaccharides, mostly involving chondroitin-6-sulfate. The genetic data are consistent with autosomal recessive inheritance.     

IL-6 levels predict disease variant and extent of organ involvement in patients with mastocytosis

Mastocytosis is often associated with organ involvement and hematological disorders. Patients may also exhibit elevated levels of plasma IL-6. To gain insight into the relevance of this observation, we correlated plasma levels of IL-6 and soluble IL-6 receptor (sIL-6R) with multiple disease parameters in 29 patients with mastocytosis. Mean plasma IL-6 levels were elevated in patients compared to healthy controls (P < 0.0001). Disease category significantly correlated with plasma IL-6 levels, as did severity of bone marrow pathology, organomegaly, and extent of skin involvement. In plasma, there was a positive correlation of IL-6 to total tryptase, alkaline phosphatase, IgM, white blood cell count, prothrombin time, partial thromboplastin time, and neutrophil numbers. There was an inverse correlation to hemoglobin. sIL-6R levels were not elevated. These observations demonstrate that IL-6 is a useful surrogate marker of severity of hematologic disease and suggest that IL-6 contributes to pathology.     

黛力新联合维生素B6治疗伴负性情绪的功能性消化不良临床疗效研究

目的 探讨黛力新联合维生素B6片辅助治疗伴负性情绪的功能性消化不良的临床效果, 为功能性消化不良（FD）的临床治疗提供参考。方法 选择云浮市人民医院2016年6月~2018年1月收治的112例伴有抑郁焦虑等负性情绪的FD患者,采用随机数字表法分为观察组和对照组,每组56例,两组患者均给予常规基础治疗,观察组患者在常规治疗的基础上给予黛力新（氟哌噻吨美利曲辛片）、维生素B6片口服治疗,对两组患者治疗后的心理状态采用SDS及SAS量表进行评估,并对躯体症状、总体疗效进行比较和分析。结果 观察组患者SAS评分、SDS评分低于对照组（P＜0.05);观察组患者躯体症较前缓解（P＜0.05）,且治疗组各项评分低于对照组（P＜0.05）,观察组治疗总有效率为 92.72%,对照组治疗总有效率为68.52%,差异具有统计学意义（P＜0.05)。结论 对于伴情绪障碍的FD患者,在常规治疗基础上加用氟哌噻吨美利曲辛片联合维生素B6片治疗,可有效减轻患者精神和躯体症状,提高临床疗效。     

Interleukin-6 (IL-6)--373 A9T11 allele is associated with reduced susceptibility to chronic periodontitis in Japanese subjects and decreased serum IL-6 level

We investigated whether interleukin-6 (IL-6) gene polymorphisms could be associated with chronic periodontitis (CP) and serum IL-6 level. One hundred and twelve CP and 77 non-CP Japanese subjects were analyzed for IL-6 -597 (G/A), -572 (C/G), -373 (A(n)T(m)), -190 (C/T), and -174 (G/C) polymorphisms. We could only detect -572 and -373 polymorphisms and found that the frequency and carriage rate of the -373 A9T11 allele were significantly higher in non-CP subjects. Enzyme-linked immunosorbent assay confirmed that the -572 and -373 G[A9T11] haplotypes were associated with lower serum IL-6 level. These findings suggest that IL-6 -373 A9T11 allele could be associated with reduced susceptibility to CP among Japanese subjects and decreased serum IL-6 level.     

Loss-of-function variation in the DPP6 gene is associated with autosomal dominant microcephaly and mental retardation

The molecular basis of autosomal dominant microcephaly, a disorder associated with small head circumferences that results in variable mental retardation, is largely unknown. In the present study, we conducted a variation analysis of the DPP6 gene in patients with autosomal dominant microcephaly and variable mental retardation. The copy number variation analysis of DPP6 was performed on DNA samples from 22 patients with microcephaly using high-resolution, array-based genomic hybridization, and sequence analysis was performed to screen mutations in another 50 microcephalic patients. Two de novo deletions and one missense mutation in familial microcephalic patients were identified. The transfection of plasmids encoding green fluorescent protein-pLLU2G-shDPP6 fusion proteins in mouse brains revealed that the decreased expression of the DPP6 gene slightly reduced the weight of the mouse brains and resulted in mouse learning disabilities compared with their wild-type littermates. Our data indicate that the loss-of-function variations in DPP6 are associated with autosomal dominant microcephaly and mental retardation. DPP6 appears to play a major role in the regulation of proliferation and migration of neurons in neurogenesis, most likely by participating in neuronal electrical excitability, synaptic integration, and plasticity.     

Human herpesvirus 6 (HHV-6) is transmitted from parent to child in an integrated form and characterization of cases with chromosomally integrated HHV-6 DNA

We obtained 7,566 peripheral blood mononuclear cell (PBMC) samples from 2,332 individuals and screened them for human herpesvirus infection. We identified five individuals who persistently harbored high copy numbers of human herpesvirus 6 (HHV-6) DNA in their PBMCs. HHV-6 DNA was also detected in other somatic tissues of these individuals. Five additional cases were identified among their family members. For two of these families, chromosomally integrated HHV-6 DNA (CIHHV-6) was detected in the PBMCs by fluorescence in situ hybridization. The prevalence of CIHHV-6 among all the subjects was 0.21%. The HHV-6 DNA was variant B in four families and variant A in one family. Antibodies to immediate early antigen and glycoprotein B were detected in 57 and 14% of individuals with CIHHV-6 and in 0 and 60% of healthy volunteers without CIHHV-6, respectively. HHV-6 could not be isolated from PBMCs with CIHHV-6. These cases shared no clinical features, and included three healthy individuals. Our data suggest that CIHHV-6 is rare but detectable in the general population and that hereditary transmission is one of the routes of HHV-6 transmission.     

A novel truncating variant within exon 7 of KAT6B associated with features of both Say–Barber–Bieseker–Young–Simpson syndrome and genitopatellar syndrome: Further evidence of a continuum in the clinical spectrum of KAT6B‐related disorders

KAT6B sequence variants have been identified in both patients with the Say–Barber–Biesecker–Young–Simpson syndrome (SBBYSS) and in the genitopatellar syndrome (GPS). In SBBYSS, they were reported to affect mostly exons 16–18 of KAT6B, and the predicted mechanism of pathogenesis was haploinsufficiency or a partial loss of protein function. Truncating variants in KAT6B leading to GPS appear to cluster within the proximal portion of exon 18, associated with a dominant‐negative effect of the mutated protein, most likely. Although SBBYSS and GPS have been initially considered allelic disorders with distinctive genetic and clinical features, there is evidence that they represent two ends of a spectrum of conditions referable as KAT6B‐related disorders. We detected a de novo truncating variant within exon 7 of KAT6B in a 8‐year‐old female who presented with mild intellectual disability, facial dysmorphisms highly consistent with SBBYSS, and skeletal anomalies including exostosis, that are usually considered component manifestations of GPS. Following the clinical diagnosis driven by the striking facial phenotype, we analyzed the KAT6B gene by NGS techniques. The present report highlights the pivotal role of clinical genetics in avoiding clear‐cut genotype‐phenotype categories in syndromic forms of intellectual disability. In addition, it further supports the evidence that a continuum exists within the clinical spectrum of KAT6B‐associated disorders.

     

The neuroprotective enzyme CYP2D6 increases in the brain with age and is lower in Parkinson's disease patients

Cytochrome P450 2D6 (CYP2D6) is a drug-metabolizing enzyme expressed in the brain that also metabolizes endogenous neural compounds (e.g., catecholamines) and inactivates neurotoxins (e.g., 1-methyl-4-thenyl-1,2,3,6-tetrahydropyridine; MPTP). Genetically poor CYP2D6 metabolizers are at higher risk for developing Parkinson's disease (PD), a risk that increases with exposure to pesticides. As age is a risk factor for PD we measured the ontogenic expression of CYP2D6 in human brain, and compared brain CYP2D6 levels in PD cases with age-matched controls. CYP2D6 increased from fetal to 80 years of age (n = 76), exhibiting 3 distinct phases of change. Compared with PD controls, PD cases had approximately 40% lower CYP2D6 levels in the frontal cortex, cerebellum, and the hippocampus, even when controlling for CYP2D6 genotype. In contrast, CYP2D6 levels in cases were similar to controls in PD-affected brain areas, the substantia nigra, and caudate, consistent with higher astrocytic and cellular CYP2D6 staining observed in PD cases. In summary, the lower CYP2D6 levels in PD cases may have reduced their ability to inactivate PD-causing neurotoxins contributing to their disease risk.     

The interleukin-6 promoter polymorphism is associated with elevated leukocyte,lymphocyte, and monocyte counts and reduced physical fitness in young healthy smokers

Smoking and interleukin-6 are important factors in driving inflammation. This study assessed the relationship between smoking, interleukin-6 genotype, physical fitness, and peripheral blood count in healthy young men. For this interleukin-6 promoter polymorphism –174 genotype-phenotype association study 1,929 healthy German male aviators recruited at the central German Air Force Institute of Aviation Medicine were stratified by smoking habits. Cardiovascular fitness was expressed as maximal physical working capacity (PWCmax) in watts per kilogram body weight as assessed by maximal exercise testing by cycle ergometry up to physical exhaustion. Smokers had higher leukocyte and lymphocyte counts than nonsmokers and lower PWCmax. In the overall study population the C allele of the interleukin-6 polymorphism was weakly associated with elevated leukocytes and lymphocytes; in nonsmokers the interleukin-6 polymorphism was not associated with altered phenotypes, but in smokers the interleukin-6 C allele was associated with higher leukocytes, lymphocytes, and monocytes and with lower PWCmax. Smoking is thus associated with elevated leukocytes and lymphocytes and with reduced physical fitness. Gene carriers with the interleukin-6 C allele may suffer particularly from cigarette smoking.Abbreviations CRP C-reactive protein - IL Interleukin - PWCmax Maximal physical working capacity     

Deletion or triplication of the α3(VI) collagen gene in three patients with 2q37 chromosome aberrations and symptoms of collagen-related disorders

Two new cases of del(2)(q37.1) and one case of partial trp(2)(q37) are studied by FISH with cosmid probes from the COL6A3 and PAX3 genes mapped in 2q37.3 and 2q36, respectively. While the PAX3 gene dosage appeared unaffected, the COL6A3 gene was found to be deleted and triplicated, respectively. This finding could explain features of connective tissue disorders such as joint laxity and hypotonia or joint stiffness and epiphyseal dysplasia, particularly documented by congenital dislocation of the radial head. Karyotype-phenotype correlations witii reference to published cases are discussed.