Identification of a cytogenetic deletion and of four novel mutations (Q69X,I172F,G188V,G197R) affecting the gene for ornithine transcarbamylase (OTC) in Spanish patients with OTC deficiency

A deletion of at least 11.5 cM in the paternal X chromosome mapping between microsatellites DXS989 and DXS1003 and encompassing the genes for ornithine transcarbamylase (OTC), retinitis pigmentosa GTPase regulator (RPGR) and dystrophin, was associated with the loss of band Xp21 in a female patient with OTC deficiency. Another four female patients were heterozygous for point mutations in the OTC gene: the nonsense mutation Q69X or the missense mutations I172F, G188V and G197R. In the OTC amino acid sequence, I172 and G197 are proximate to residues involved in catalysis, and G188 is within a loop joining helix 5 and strand 6 in the core of the ornithine‐bindingdomain. Therefore, the mutations of these residues may cause structural changes affecting catalysis and/or the architecture of the ornithine domain. The mutation appeared “de novo” in the patients or, in one case, in the mother of the patient, in agreement with the predominance of “de novo” mutations in female patients of OTC deficiency. There was full agreement between the results of mutational analysis and of allopurinol testing in the patients and their female relatives, supporting the value of the allopurinol test in the detection of carriers of OTC deficiency. This deficiency is a genetically heterogeneous X‐linked condition. Hum Mutat 14:352–353, 1999. © 1999 Wiley‐Liss, Inc.     

Proportions of spontaneous mutations in males and females with ornithine transcarbamylase deficiency

We used specific mutation analysis to estimate the proportions of males and females with ornithine transcarbamylase (OTC) deficiency whose mutations occurred in the germ cells of one of the parents. The mutations were identified in the probands, and subsequently carrier testing was performed on their mothers and some of the grandmothers. Of 28 OTC deficient males, only 2 (7%) had sporadic mutations (95% CI, 0.6–18.5%), whereas of 15 OTC deficient females, 12 (80%) had sporadic mutations (95% CI, 63–99%) (P < 0.001). Based on these results we estimated the male/female mutation rate ratio (ν/μ) in the OTC gene to be approximately 52. Assuming a fitness for males with OTC deficiency of 0 and the proportion of new female mutants at 0.80, the estimated fitness of heterozygous females is 0.4. Because of the difference in mutation rates between male and female germ cells, we suggest that 9/10 or higher, rather than the conventional 2/3 proportion, be applied when estimating prior risk of carrier status in a mother of one affected male. The prior risk of a mother of an affected female is much lower, approximately 2/10. © 1995 Wiley-Liss, Inc.     

Ornithine carbamoyltransferase deficiency: molecular characterization of 29 families

Ornithine carbamoyltransferase deficiency is the most common inherited defect of the urea cycle. We examined 28 male and 9 female patients from 29 families and identified 25 distinct mutations in OTC, 14 of which were novel. Three novel missense mutations (p.Ala102Pro, p.Pro158Ser, p.Lys210Glu) and a novel deletion of the Leu43 are not directly involved either in the enzyme active site or in the intersubunit interactions; however, the mutations include conserved residues involved in intramolecular interaction network essential for the function of the enzyme. Three novel large deletions – a 444 kb deletion affecting RPGR, OTC and TSPAN7, a 10 kb‐deletion encompassing OTC exons 5 and 6 and a 24.5 kb‐deletion encompassing OTC exons 9 and 10 – have probably been initiated by double strand breaks at recombination‐promoting motifs with subsequent non‐homologous end joining repair. Finally, we present a manifesting heterozygote carrying a hypomorphic mutation p.Arg129His in combination with unfavorably skewed X‐inactivation in three peripheral tissues.     

一种新的鸟氨酸氨基甲酰转移酶基因突变E122G的鉴定

目的：测定1个迟发型鸟氨酸氨基甲酰转移酶（ornithine transcarbamylase,OTC)缺乏症的中国汉族家系的基因突变及患者OTC基因外显子序列。方法：用聚合酶链反应－单链构象多态性结合PCR产物直接测，鉴定其突变类型。结果：在该家系患者OTC基因中确认了1个新的错义突变E122G，位于第4外显子的保守残基（GAA→GGA）。结论：OTC基因中E122G突变引起迟发型OTC缺乏症，是1个尚未报道的突变。     

Regulatory region of the Aspergillus nidulans argB gene

Summary We have constructed a series of deletion plasmids which contain the Aspergillus nidulans argB gene for ornithine carbamoyltransferase (OTC). These deletions comprise the 5 upstream sequence of the argB gene. The pro^– arg^– strain of A. nidulans was transformed with the above plasmids. Several arg⁺ transformants of integration types I and II, obtained using each of the deletion plasmids, were studied, and their ability to de-repress OTC level by proline starvation was compared. It was concluded that nucleotides located between –150 and –50 by upstream of the argB gene are significant for its cross-pathway regulation. This regulatory region contains three copies of the TGACTC hexanucleotide which is a cis-acting regulatory sequence of general amino acid control in yeast.Abbreviations bp base pairs - kb 10³ base pairs - OTC ornithine carbamoyltransferase - ORF open reading frame     

High-frequency detection of deletions and variable rearrangements at the ornithine transcarbamylase (OTC) locus by oligonucleotide array CGH

Ornithine transcarbamylase (OTC) deficiency is an X-linked inborn error of metabolism characterized by impaired synthesis of citrulline from carbamylphosphate and ornithine. Previously reported data suggest that only approximately 80% of OTC deficiency (OTCD) patients have a mutation identified by OTC gene sequencing.To elucidate the molecular etiology in patients with clinical signs of OTCD and negative OTC sequencing, we subjected their DNA to array comparative genomic hybridization (aCGH) using a custom-designed targeted 44 k oligonucleotide array. Whenever possible, parental DNA was analyzed to determine the inheritance or to rule out copy number variants in the OTC locus.DNA samples from a total of 70 OTCD patients were analyzed. Forty-three patients (43/70 or 61.5%) were found to have disease-causing point mutations in the OTC gene. The remaining 27 patients (27/70 or 38.5%) showed normal sequencing results or failure to amplify all or part of the OTC gene. Among those patients, eleven (11/70 or 15.7%) were found to have deletions ranging from 4.5 kb to 10.6 Mb, all involving the OTC gene. Sixteen OTCD patients (16/70 or 22.8%) had normal sequencing and oligoarray results. Analysis of the deletions did not reveal shared breakpoints, suggesting that non-homologous end joining or a replication-based mechanism might be responsible for the formation of the observed rearrangements.In summary, we demonstrate that approximately half of the patients with negative OTC sequencing may have OTC gene deletions readily identifiable by the targeted oligonucleotide-based aCGH. Thus, the test should be considered in OTC sequencing-negative patients with classic symptoms of the disease.     

Next‐generation DNA sequencing of a Swedish malignant hyperthermia cohort

Malignant hyperthermia (MH)‐related mutations have been identified in the ryanodine receptor type 1 gene (RYR1) and in the dihydropyridine gene (CACNA1S), but about half of the patients do not have causative mutations in these genes. We wanted to study the contribution of other muscle genes to the RYR1 phenotypes. We designed a gene panel for sequence enrichment targeting 64 genes of proteins involved in the homeostasis of the striated muscle cell. Next‐generation sequencing (NGS) resulted in >50,000 sequence variants which were further analyzed by software filtering criteria to identify causative variants. In four of five patients we identified previously reported RYR1 mutations while the fifth patient did not show any candidate variant in any of the genes investigated. In two patients pathogenic variants were found in other genes known to cause a muscle disorders. All but one patient carried likely benign rare polymorphisms. The NGS technique proved convenient in identifying variants in the RYR1. However, with a clinically variable phenotype‐like MH, the pre‐selection of genes poses problems in variant interpretation.     

Mutations and polymorphisms in the human ornithine transcarbamylase (OTC) gene

Ornithine transcarbamylase (OTC) deficiency is the most common inherited disorder of the urea cycle and is transmitted as an X-linked trait. Defects in the OTC gene cause a block in ureagenesis resulting in hyperammonemia, which can lead to brain damage and death. Three previous mutation updates for the OTC gene have been published, in 1993, 1995, and 2002. The most recent comprehensive update, in 2002, contained 244 mutations including 13 nondisease-causing mutations and polymorphisms. This current update reports 341 mutations, of which 93 have not been previously reported, and an additional 29 nondisease-causing mutations and polymorphisms. Out of the 341 mutations, 149 were associated with neonatal onset of hyperammonemia (within the first week of life), 70 were seen in male patients with later onset of hyperammonemia, and 121 were found in heterozygous females (one unknown). Along with the reported mutations, residual enzyme activities and other pertinent clinical information are included whenever available. Most mutations in the OTC gene are specific to a particular family ("private" mutations). They are distributed throughout the gene, with a significant paucity of mutations in the 32 first codons encoding the "leader" peptide (exon 1 and the beginning of exon 2). Almost all mutations in consensus splice sites confer a neonatal onset phenotype. Using the current molecular screening methods, mutations are found in about 80% of the patients. The remaining patients may have mutations in regulatory domains or mutations deep in the introns, which constitute 98.5% of the genomic sequence. In addition, a phenocopy of OTC deficiency caused by mutations in another unknown gene cannot be excluded.     

Molecular analysis of two uncharacterized sequence variants of the VHL gene

Mutations in the VHL gene cause von Hippel–Lindau disease, a cancer predisposing syndrome characterized by a variety of benign and malignant neoplasms. We report the molecular characterization of two sequence variants of the VHL gene: a synonymous substitution c.462 A>C in exon 2 and a duplication of 11 bp in the promoter region (c.−65_−55dup11). The first variant is a pathogenic mutation because, although it does not change the sense of the affected codon, it causes skipping of exon 2 in the affected allele by altering the splicing consensus site at the 3′ end of exon 2. The 11 bp duplication represents a nonpathogenic variant. In fact, although it affects a critical region of the VHL promoter, it was found in healthy controls, and we show that carrier individuals express both VHL alleles at equimolar levels. Our data underline the importance of careful evaluation of the potential pathogenicity of sequence variants that may not belong to the obvious disease-causing mutation categories, or that affect relevant regulatory regions. mRNA analysis will be required to ultimately resolve this issue.Maddalena Martella and Leonardo Salviati contributed equally to this work.     

Germline mutation profile of the VHL gene in von Hippel-Lindau disease and in sporadic hemangioblastoma

Von Hippel-Lindau (VHL) disease is a dominantly inherited disorder predisposing those afflicted to hemangioblastomas of the central nervous system and the retina, renal cell carcinomas, pheochromocytomas, and pancreatic tumors. The disease has been associated with mutations of the VHL gene. The screening of 92 unrelated patients with VHL disease for point mutations in this gene revealed 61 DNA variants. In addition, a search for EcoR1 rearrangements revealed germline anomalies in 5 patients. The 61 variants could be subdivided in 20 mutations predicted to alter the open reading frame (8 nonsense mutations, 8 frame shift mutations, and 4 mutations in consensus splicing sites) and 43 DNA sequence variants of a priori unknown biological consequence (4 in-frame insertions or deletions, 36 missense mutations, and 3 apparently silent variations). The 3′ end of the coding sequence of the VHL gene, which encodes the Elongin binding domain was the site of 5 of 20 truncating mutations (25%) and of 18 of 41 DNA variants (44%) causing uncertain functional impairment. A similar screening in 18 patients with sporadic hemangioblastoma revealed 2 missense DNA variants. In order to corroborate this latter observation, a systematic screening for germline alteration of the VHL gene might be performed in a larger series of sporadic hemangioblastoma. If this preliminary result is confirmed, more than 10% of sporadic hemangioblastoma might be related to a mild VHL disease, thus a follow-up program similar to that recommended in cases of VHL disease should probably be discussed in the corresponding families. Hum Mutat 12:424–430, 1998. © 1998 Wiley-Liss, Inc.     

Mutations and polymorphisms in the human ornithine transcarbamylase gene

Deletions of variable size involving one or more exons, 29 different missense, nonsense, or frameshift mutations, and three polymorphisms have been found in patients with ornithine transcarbamylase (OTC) deficiency. Most of the deletions and mutations were found in patients with severe disease manifested clinically as acute neonatal hyperammonemia. A small number of mutations or somatic mosaicism for deletions were found in males with “late onset” disease and in heterozygous females who were symptomatic. Approximately 10–15% of all molecular alterations associated with OTC defi ciency are large deletions involving all or part of the OTC gene with or without contiguous genes on the short arm of the X chromosome. Approximately 10% of all point mutations involve the CpG dinucleotide of codon 141 with a CGA→CAA transition producing a deleterious Arg→Gln substitu tion in position 109 of the mature enzyme and causing the elimination of a TaqI recognition site. The majority of the remaining mutations in the OTC gene are unique to the affected family and are usually not found in unrelated patients. To date, two mutations have been described in the sequence of the “leader” peptide, 23 mutations have been found in the coding sequence of the “mature” enzyme, and four mutations have been discovered in splicing recognition sites. Approximately 20 single base polymorphisms have been postulated to exist by comparing two reported OTC gene sequences; six of these substitutions cause amino acid changes of which three have been confirmed in patients. Of the known point mutations, 27 are single base substitutions: 17 missense, 6 nonsense, 4 splice site, and the remaining 2 are single base deletions. © 1993 Wiley-Liss, Inc.     

Cloning and characterization of human ORNT2: a second mitochondrial ornithine transporter that can rescue a defective ORNT1 in patients with the hyperornithinemia-hyperammonemia-homocitrullinuria syndrome,a urea cycle disorder

We recently characterized the mitochondrial ornithine transporter (ORNT1), the gene defective in the hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome, a urea cycle disorder. Despite the apparent functional ablation of ORNT1 in 10 French-Canadian probands with the ORNT1-F188 Delta allele, these patients are mildly affected when compared to patients with other urea cycle disorders such as deficiency of ornithine transcarbamylase. Given that the inner mitochondrial membrane is impermeable to solutes, we hypothesize that other unidentified carriers have some degree of functional redundancy with ORNT1. Using conserved sequences of mammalian and fungal mitochondrial ornithine transporters, we screened the Expressed Sequence Tag database for additional transporters belonging to the ORNT subfamily. Here we identify a new intronless gene, ORNT2, located on chromosome 5. The gene product of ORNT2 is 88% identical to ORNT1, targets to the mitochondria and is expressed in human liver, pancreas, kidney, and cultured fibroblasts from control and HHH patients. When ORNT2 is overexpressed transiently in cultured fibroblasts from HHH patients, it rescues the deficient ornithine metabolism in these cells. Our results suggest that ORNT2 may in part be responsible for the milder phenotype in HHH patients secondary to a gene redundancy effect. We believe ORNT2 arose from a retrotransposition event. To our knowledge, this is the first report of a functional retroposon (ORNT2) that can rescue the disease phenotype of the gene it arose from, ORNT1. As such, ORNT2 may eventually become a candidate for pharmacological-based approaches to correct a urea cycle disorder.     

Adenosine A1 receptor and bipolar affective disorder: systematic screening of the gene and association studies

In the present study we sought to identify genetic variation in the adenosine A₁ receptor (A₁AR) gene on chromosome 1q31-32.1, which through alteration of protein function or level of expression might contribute to the genetic predisposition to bipolar affective disorder. We performed a systematic mutation scan of the whole coding sequence as well as 5′ and 3′ untranslated regions by means of single-strand conformation analysis. The region upstream to the coding sequence we investigated contains two functional promoters. Screening 42 patients with bipolar affective disorder, we detected 11 DNA sequence variants (48T/A, 267 + 275C/T, 805T/G, 1777C/A, 1827C/T, 1904C/T, 2126G/T, 2294insT, 2776C/T, 2777del36, 2819T/G). Determining the frequency of these variants in 42 anonymous blood donors, we observed a non-significant (P < 0.06) trend towards an underrepresentation of the 2126T variant in patients when compared to controls. On the other hand, the 2777del36 and the 2819G variant were not found among the controls. These findings were followed up in a large independent replication sample. However, we were not able to confirm the initial findings in the second sample. Our data suggest that genetically determined variation of the A₁AR and its two promoters do not play a major role in the development of bipolar affective disorder. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 81:18–23, 1998. © 1998 Wiley-Liss, Inc.     

鸟氨酸氨甲酰基转移酶缺陷症三例临床和基因突变分析

目的 分析鸟氨酸氨甲酰基转移酶缺陷症(ornithine carbamoyltransferase deficiency,OTCD)的临床表现、遗传学特点,从基因水平了解OTCD的致病因素,达到基因诊断和遗传咨询的目的.方法 应用聚合酶链反应扩增3例OTCD患者的鸟氨酸氨甲酰转移酶(ornithine carbamoyltransferase,OTC)基因各个外显子所在片段,并进行直接测序,以检测突变.结果 例1在生后6月以呕吐起病,为错义突变T262I,其母亲表型正常,为T262I杂合突变;例2在2岁时以烦躁不安起病,为错义突变R277W,例2父母未行基因检测;例3以嗜睡在新生儿期起病,为错义突变I172M,其母表型正常,为I172M杂合突变.结论 基因分析是诊断OTCD有效可行的方法.T262I突变及R277W突变是症状较轻的温和突变,I172M突变是起病年龄早及症状重的突变.对致病基因进行突变检测不仅可以诊断OTCD,而且可以发现无症状的基因携带者,为遗传咨询及产前诊断提供依据.

Abstract：

Objective To analyze the clinical and genetic characteristics of three children with ornithine carbamoyltransferase deficiency(OTCD), and to provide a practical method for gene diagnosis and genetic counseling of the disease. Methods All exons of the ornithine carbamoyltransferase (OTC) gene were screened by polymerase chain reaction-DNA direct sequencing in the three OTCD patients. Results One patient firstly presented as vomiting at 6 month of age. A missense mutation of T262I was detected. His mother had the same mutation without any clinical symptoms. The second patient presented as restlessness, and had a missense mutation of R277W. Gene analysis of his parents was not available. The third patient presented as neonatal lethargy, harbored a missense mutation of I172M. His mother had the same mutation without any clinical symptoms. Conclusion Gene mutation analysis is a feasible way for diagnosing OTCD. Patients with I172M mutation present symptom early, while those with T262I and R277W mutations manifest symptoms later. Gene mutation analysis will be important for asymptomatic and prenatal diagnosis and genetic counseling.     

Loss of interaction between plectin and type XVII collagen results in epidermolysis bullosa simplex

Plectin is a linker protein that interacts with intermediate filaments and β4 integrin in hemidesmosomes of the epidermal basement membrane zone (BMZ). Type XVII collagen (COL17) has been suggested as another candidate plectin binding partner in hemidesmosomes. Here, we demonstrate that plectin–COL17 binding helps to maintain epidermal BMZ organization. We identified an epidermolysis bullosa (EB) simplex patient as having markedly diminished expression of plectin and COL17 in skin. The patient is compound heterozygous for sequence variants in the plectin gene (PLEC); one is a truncation and the other is a small in‐frame deletion sequence variant. The in‐frame deletion is located in the putative COL17‐binding domain of plectin and abolishes the plectin–COL17 interaction in vitro. These results imply that disrupted interaction between plectin and COL17 is involved in the development of EB. Our study suggests that protein–protein binding defects may underlie EB in patients with unidentified disease‐causing sequence variants.     

Genetic variant spectrum in 265 Chinese patients with hemophagocytic lymphohistiocytosis: Molecular analyses of PRF1, UNC13D,STX11, STXBP2, SH2D1A,and XIAP

Hemophagocytic lymphohistiocytosis (HLH) is a rare life‐threatening hyperinflammatory disease. This study aimed to investigate the frequencies and distributions of inherited variants in PRF1, UNC13D, STX11, STXBP2, SH2D1A, and XIAP genes in Chinese patients with HLH. A total of 265 patients diagnosed with HLH from January, 2010 to December, 2016 were recruited and analyzed for the 6 genes. Genetic variants were observed in 87 (32.83%) patients. 36 (13.58%) exhibited variants in UNC13D, 18 (6.79%) exhibited PRF1 variants, 10 (3.77%) had variants in XIAP, 9 (3.40%) exhibited variants in STXBP2, 6 (2.26%) carried variants in SH2D1A, 1 (0.38%) had STX11 variant, and 7 (2.64%) exhibited digenic variants. Monoallelic variants were the most common, which accounted for 49.43% of all cases with variants. All variants were confirmed to be germline‐derived. The present study describes a distinct variant spectrum in Chinese patients with HLH, whereby UNC13D is the most frequently mutated gene with missense variants that are the most common molecular defects. The variant profile of Chinese HLH patients is quite different from that of Western cohorts but similar to that of Korean patients, yet showing its own uniqueness. This racial difference shows the role of genetic background in the occurrence of HLH.     

Screening the human bradykinin B2 receptor gene in patients with cardiovascular diseases: Identification of a functional mutation in the promoter and a new coding variant (T21M)

To elucidate if genetic variants in the bradykinin B₂ receptor (B₂) gene occur that could affect receptor expression and function, we screened for mutations in the promoter and in the coding region of the human B₂ gene. In our initial study we analyzed 92 consecutive, unrelated subjects (including 25 patients with hypertrophic cardiomyopathy, 18 patients with dilated cardiomyopathy (DCM), 25 patients with hypertension, 18 patients with coronary heart disease, and 6 patients with valvular heart disease) using nonradioactive polymerase chain reaction–single-strand conformation polymorphism analysis as mutation screening method. We detected eight as yet unknown polymorphic sites in the promoter region of the B₂ gene (−845 C/T, −704C/T, −649 insG, −640 T/C, −536 C/T, −412 C/G, −143 C/T and −78 C/T) with allele frequencies between 0.5 and 13%. One of them (−412 C/G) destroys a Sp1 binding site and abolishes protein binding to this Sp1 site in human umbilical vein endothelial cells and human vascular smooth muscle cells. In the protein-coding region one new coding variant (T21M) with the potential to create a truncated receptor isoform was detected. We determined the frequency of the promoter variant at position −412 (C → G) and the newly identified coding variant (T21M) in extended samples of 69 patients with HCM, 163 patients with DCM, 109 patients with hypertension, and 173 healthy anonymous blood donors. The promoter variant (−412C/G) was found in one blood donor and the T21M mutation was not found in the control population. Therefore, it appears that these mutations are rare events and the determination of clinical significance will be a demanding task in the future. Am. J. Med. Genet. 80:521–525, 1998. © 1998 Wiley-Liss, Inc.