Molecular characterization of erythropoietic protoporphyria in South Africa

Background  Erythropoietic protoporphyria (EPP) results from a partial deficiency of ferrochelatase (FECH). Clinical expression normally requires coinheritance of a common hypomorphic FECH allele (IVS3-48C) in trans to a deleterious (primary) FECH mutation.
Objectives  To characterize South African subjects with EPP, by identification and assessment of FECH sequence variations, including the IVS3-48C polymorphism.
Methods  Polymerase chain reaction amplification, single-strand conformational polymorphism analysis and restriction endonuclease analysis were employed to identify and determine the frequencies of FECH sequence variations, including the IVS3-48C polymorphism, in a study cohort of symptomatic and asymptomatic South African EPP family members, and a matched control cohort.
Results  We identified 29 patients from 18 families. With the exception of one family, who may represent a phenocopy of EPP, the presentation of EPP was typical. All were of European immigrant stock, and we have not identified EPP in other ethnic groups. Ten sequence variations were identified, including four apparent disease-causing mutations, the IVS3-48T/C polymorphism and five further polymorphisms. The molecular basis of EPP was established for 15 of the 17 families. A 5-bp deletion in exon 7 (757_761delAGAAG) was present in 12 of these families and haplotype studies in these families suggested a single mutational event and thus a local founder effect for this deletion. The other mutations were family specific and included two previously described splice-site mutations (IVS3+2T>G and IVS7+1G>A) and a novel 7-bp deletion in exon 4 (356_362delTTCAAGA).
Conclusions  The IVS3-48C allele appears to modulate the phenotypic expression of EPP in the South African EPP cohort as observed in other populations.     

Late presentation of erythropoietic protoporphyria: case report and genetic analysis of family members

Erythropoietic protoporphyria (EPP) is an inherited disorder of haem biosynthesis caused by decreased activity of the enzyme ferrochelatase (FECH), which catalyses the insertion of iron into protoporphyrin, the last step in haem biosynthesis. Development of clinically overt EPP usually requires inheritance of a severe FECH mutation trans to a low-expression FECH variant (FECH IVS3-48C), which is present in 13% of the U.K. population. Reduced FECH activity leads to accumulation of protoporphyrin in various tissues. An excess amount of free protoporphyrin in the skin causes photosensitivity. EPP usually presents in early childhood or infancy, with painful burning and pruritus within minutes of light exposure. Onset of symptoms in adults is rare and often associated with acquired somatic mutation of the FECH gene secondary to haematological malignancy. Here we describe a patient with EPP, in whom the presenting clinical symptom, night-time itch, did not appear until middle age and who had an asymptomatic sister with the same FECH genotype.     

红细胞生成性原卟啉病一家系亚铁螯合酶基因突变检测

目的 通过对一例红细胞生成性原卟啉病家系亚铁螯合酶基因突变检测,探讨红细胞生成性原卟啉病的遗传特征。方法 收集一例红细胞生成性原卟啉病家系中4例患者及3例表型正常者和50例无亲缘关系健康个体外周血标本,采用PCR扩增亚铁螯合酶基因全部11个外显子及侧翼序列并进行直接测序。结果 先证者及其母亲、姐姐、表兄、外祖父中检测到一个新的剪接突变位点间隔序列（IVS）3+1G→A,其外祖母和父亲未发现该突变;正常对照未发现此突变。先证者及其父亲、姐姐、表兄、外祖父两个单核苷酸多态性为IVS1-23T/C和IVS3-48C/T,而先证者的母亲与外祖母为IVS1-23C/C和IVS3-48T/T。结论 发现红细胞生成性原卟啉病家系一个新的基因突变位点,该突变可能与两个低表达等位基因IVS1-23T和IVS3-48C共同导致红细胞生成性原卟啉病的临床表型。     

A novel splicing mutation and haplotype analysis of the FECH gene in a Chinese family with erythropoietic protoporphyria

Background Erythropoietic protoporphyria (EPP) is a rare autosomal dominant disorder of haeme biosynthesis resulting from a partial decrease in ferrochelatase (FECH) activity leading to excessive accumulation of protoporphyrin. Clinical manifestation normally requires coinheritance of a common hypomorphic FECH allele and a deleterious FECH mutation. Objective The aim of this study was to characterize the inheritance of a Chinese family with EPP at molecular level, by identification and analysis of FECH sequence variation, including the IVS3‐48C polymorphisms. Methods Polymerase chain reaction amplification was employed to identify the FECH sequence variation, including the IVS3‐48C polymorphism, in a Chinese EPP family and a matched control cohort. Results A splicing mutation in IVS3 + 1G→A was identified in the proband as well as his symptomatic sister, cousin, his grandfather and his asymptomatic mother, but was absent in his father and grandmother. All the family members with overt photosensitivity carried the low‐expressed allele IVS3‐48C, which were absent in the asymptomatic EPP patients of this family. Conclusion We described a novel splicing FECH mutation in a Chinese EPP family and analysed the hypomorphic IVS3‐48C allele, which were believed to be responsible for generating the phenotypic symptoms in this family.     

Clinical, biochemical, and genetic study of 11 patients with erythropoietic protoporphyria including one with homozygous disease

OBJECTIVE: To study the mutations in the ferrochelatase gene (FECH) and the phenotypic expression of erythropoietic protoporphyria (EPP) in a group of Spanish patients. DESIGN: Case series. SETTING: University-based hospital. PATIENTS: Eleven unrelated patients with EPP and 19 asymptomatic relatives from 10 families. MAIN OUTCOMES MEASURES: Measurement of protoporphyrin concentration in red blood cells and feces by fluorometry and chromatography. Analysis of the mutations of the FECH gene by single-strand conformation analysis. Expression of mutations in Escherichia coli. RESULTS: FECH gene mutations were found in all 11 patients. Ten were heterozygous and carried the IVS3-48C low-expression allele. Three novel mutations were found: IVS4 + 1delG, 347-351delC, and 130_147dupl 18. One patient did not present the IVS3-48C polymorphism and was found to harbor a novel A185T missense mutation in both alleles. The familial study confirmed a recessive mode of inheritance of the disease. The A185T mutation showed a residual activity 4% of normal when expressed in E coli. This patient presented cutaneous photosensitivity similar to the heterozygous cases, but a higher protoporphyrin accumulation in erythrocytes, microcytic anemia, and early signs of liver engagement. FECH mutations were found in 10 healthy relatives, none of whom carried the low-expression allele. The frequency of the IVS3-48C allele among 180 nonporphyric Spanish individuals was 5.2%. CONCLUSIONS: These findings confirm, among a group of Spanish patients, that most cases of EPP result from the coinheritance of IVS3-48C and a mutation in the FECH gene, and also document the existence of patients with mutations in homozygosity that may present a more severe form of the disease.     

红细胞生成性原卟啉病一家系中亚铁螯合酶基因突变的检测

目的 对一红细胞生成性原卟啉病（EPP）家系进行基因突变研究,探讨基因突变与临床表现的关系,为进一步开展基因诊断和基因治疗奠定基础。方法 收集家系资料,抽取家系中患者、正常人及与该家系无关的50例正常人的外周血,提取外周血基因组DNA。应用PCR方法扩增外周血基因组DNA亚铁螯合酶（FECH）基因的第1至11外显子及其侧翼序列。PCR产物直接进行双向测序以检测突变。结果 根据临床表现和卟啉测定结果,患者明确诊断为红细胞生成性原卟啉病。PCR扩增得到预期DNA片段。PCR 产物直接测序结果：家系中先证者、其妹和其父FECH基因第1内含子供体剪接位点检测到一个杂合突变（IVS1 ＋ 1G→C）,该突变为国际首次报道。还在先证者、其妹和其母FECH基因第1内含子受体端检测到一个与低表达等位基因相关的多态性（IVS1-23C/T）。结论 报道一FECH基因第1内含子供体剪接位点的新突变,该突变可能引发FECH基因缺陷,是EPP家系中患者发病的分子基础。     

一例红细胞生成性原卟啉病临床分析及FECH基因检测

收集1例红细胞生成性原卟啉病患者的临床资料及外周血,提取患者DNA,对FECH基因进行Sanger测序,与100名正常人进行对比.患者皮损多位于暴露部位,皮损粗糙、增厚,面部皮损呈蜡样光泽,手背部可见密集肤色粟粒大丘疹.患者尿液、粪便和血清Wood灯下呈浅粉色;外周血涂片检查电光源下见大量红细胞,荧光相见大量红细胞的红...     

A homoallelic FECH mutation in a patient with both erythropoietic protoporphyria and palmar keratoderma

Background Erythropoietic protoporphyria (EPP) is a hereditary disorder caused by the deficiency of ferrochelatase (FECH) in the haem biosynthetic pathway. In the majority of families, EPP is transmitted as a pseudodominant trait. Autosomal recessive form of EPP is found in only about 3% of the families. Objectives In this study, we describe a 6‐year‐old boy who suffered from both EPP and palmar keratoderma. Methods and Results A novel homoallelic missense mutation (p.Ser318Tyr) was identified in the FECH gene. In addition, a region of homozygosity of approximately 6.8 Mb was observed in chromosome 18 of the patient by both microsatellite and SNP array. The parents of the patient, both of Palestinian (Jordanian) origin, were heterozygous for the S318Y mutation, although no history of consanguinity was known. Microsatellite genotyping identified a partial haplotype from each parent that corresponds to the region of homozygosity in the patient. Assuming S318Y is a founder mutation, the number of generations separating the two parents from their common ancestor from whom they inherited S318Y was estimated as 21.7 (95% CI 3.42–69.7). Conclusion EPP was therefore inherited as an autosomal recessive trait in the family. This study confirms the association between palmar keratoderma and autosomal recessive EPP.     

Gene dosage analysis identifies large deletions of the FECH gene in 10% of families with erythropoietic protoporphyria

Erythropoietic protoporphyria (EPP) is an inherited cutaneous porphyria characterized by partial deficiency of ferrochelatase (FECH), accumulation of protoporphyrin IX in erythrocytes, skin, and liver, and acute photosensitivity. Genetic counseling in EPP requires identification of FECH mutations, but current sequencing-based procedures fail to detect mutations in about one in six families. We have used gene dosage analysis by quantitative PCR to identify large deletions of the FECH gene in 19 (58%) of 33 unrelated UK patients with EPP in whom mutations could not be detected by sequencing. Seven deletions were identified, six of which were previously unreported. Breakpoints were identified for six deletions (c.1-7887-IVS1+2425insTTCA; c.1-9629-IVS1+2437; IVS2-1987-IVS4+352del; c.768-IVS7+244del; IVS7+2784-IVS9+108del; IVS6+2350-TGA+95del). Five breakpoints were in intronic repeat sequences (AluSc, AluSq, AluSx, L1MC4). The remaining deletion (Del Ex3-4) is likely to be a large insertion-deletion. Combining quantitative PCR with routine sequencing increased the sensitivity of mutation detection in 189 unrelated UK patients with EPP from 83% (95% CI: 76-87%) to 93% (CI: 88-96%) (P=0.003). Our findings show that large deletions of the FECH gene are an important cause of EPP. Gene dosage analysis should be incorporated into routine procedures for mutation detection in EPP.     

Incomplete erythropoietic protoporphyria caused by a splice site modulator homozygous IVS3‐48C polymorphism in the ferrochelatase gene

Erythropoietic protoporphyria (EPP) is an inherited cutaneous porphyria caused by both the partial deficiency of ferrochelatase (FECH) and the existence of cytosine at IVS3‐48 in trans to a mutated FECH allele. However, physicians occasionally encounter patients with EPP with a mild phenotype associated with a slight increase in the erythrocyte‐free protoporphyrin concentration and no FECH gene mutations. In this study, genetic analyses were performed on three patients with a mild phenotype of EPP, with photosensitivity, slightly increased erythrocyte‐free protoporphyrin concentrations and only a few fluorocytes in the peripheral blood. After obtaining the patients’ and their parents’ informed consent, a direct sequence analysis of the FECH gene and a restriction fragment length polymorphism analysis were performed on samples from the patients. The FECH gene mutation was not detected in the direct sequence analyses in any of the patients. However, all three patients had the homozygous IVS3‐48C polymorphism. These findings suggest that homozygous IVS3‐48C polymorphism of the FECH gene is associated with a slight elevation of the protoporphyrin level in erythrocytes, resulting in a mild EPP phenotype.     

Seasonal palmar keratoderma in erythropoietic protoporphyria indicates autosomal recessive inheritance

Erythropoietic protoporphyria (EPP) is an inherited disorder that results from partial deficiency of ferrochelatase (FECH). It is characterized clinically by acute photosensitivity and, in 2% of patients, liver disease. Inheritance is usually autosomal dominant with low penetrance but is recessive in about 4% of families. A cross-sectional study of 223 patients with EPP in the United Kingdom identified six individuals with palmar keratoderma. We now show that these and three additional patients, from six families, have an inherited subtype of EPP which is characterized by seasonal palmar keratoderma, relatively low erythrocyte protoporphyrin concentrations, and recessive inheritance. No patient had evidence of liver dysfunction; four patients had neurological abnormalities. Patients were hetero- or homoallelic for nine different FECH mutations; four of which were previously unreported. Prokaryotic expression predicted that FECH activities were 2.7-25% (mean 10.6%) of normal. Neither mutation type nor FECH activity provided an explanation for the unusual phenotype. Our findings show that palmar keratoderma is a clinical indicator of recessive EPP, identify a phenotype that occurs in 38% of reported families with recessive EPP that to our knowledge is previously unreported, and suggest that patients with this phenotype may carry a lower risk of liver disease than other patients with recessive EPP.     

Molecular epidemiology of erythropoietic protoporphyria in the U.K.

Summary Background Erythropoietic protoporphyria (EPP) is a cutaneous porphyria caused by mutations in the ferrochelatase (FECH) or, less frequently, the delta‐aminolaevulinate synthase 2 (ALAS2) gene. Predictive genetic counselling requires accurate molecular diagnosis and knowledge of patterns of inheritance. Objectives To investigate the molecular epidemiology of EPP in the U.K. Methods DNA samples from 191 unrelated patients resident in the U.K. were analysed for mutations in the FECH and ALAS2 genes and for the FECH IVS3‐48 dimorphism. Results Mutations were identified in 179 (94%) patients. Most (169; 94%) had a FECH mutation on one allele and were classified as having pseudodominant EPP (psdEPP); seven (4%) patients had FECH mutations on both alleles (autosomal recessive EPP) and three (2%) patients had ALAS2 mutations (X‐linked dominant protoporphyria). The FECH IVS3‐48C allele was strongly associated with psdEPP and with the absence of mutations at the FECH or ALAS2 loci. Fifty‐six FECH mutations were identified, 19 being previously unreported. Missense mutations were predominant in autosomal recessive EPP (82%) but not in psdEPP (32%). One mutation (c.314 + 2T>G) was present in 41 (24%) of EPP families, most of whom appeared to be descended from a common ancestor resident in the north of England. Conclusions These data define the prevalence and molecular epidemiology of each type of EPP in the U.K.     

Genetic analysis of the ferrochelatase gene in eight Japanese patients from seven families with erythropoietic protoporphyria

A decrease in the activity of ferrochelatase (FECH; EC 4.99.1.1), the terminal enzyme of the heme biosynthetic pathway, results in erythropoietic protoporphyria (EPP; MIM 177000). We analyzed the FECHgene in eight Japanese EPP patients from seven non-consanguineous families and found two distinct genomic DNA abnormalities. In six patients from five families, there was a G-to-A point-mutation at the first position of the intron 9 donor site; it resulted in aberrant splicing and skipping of exon 9 in FECH mRNA. In one patient, we found an A-to-G point-mutation 4 bases from the 3" terminus of intron 4 that led to the in-frame insertion of 3 bases in mRNA. No allelic anomalies, except for 3 single nucleotide polymorphisms were detected in another patient. We analyzed intron polymorphism at IVS3-48, known to be associated with the phenotypic expression of EPP, in these eight patients and 152 healthy Japanese volunteers. All patients were C/C homozygous for IVS3-48. The allelic frequency of IVS3-48C polymorphism in the healthy Japanese volunteers was 67.8% (103/152).     

Haplotype analysis in determination of the heredity of erythropoietic protoporphyria among Swiss families

Defects in the human ferrochelatase gene lead to the hereditary disorder of erythropoietic protoporphyria. The clinical expression of this autosomal dominant disorder requires an allelic combination of a disabled mutant allele and a low-expressed nonmutant allele. Unlike most other erythropoietic protoporphyria populations, mutations identified among Swiss erythropoietic protoporphyria families to date have been relatively homogeneous. In this study, genotype analysis was conducted in seven Swiss erythropoietic protoporphyria families, three carrying mutation Q59X, two carrying mutation insT213, and two carrying mutation delTACAG(580-584). Three different haplotypes of five known intragenic single nucleotide polymorphisms, namely -251 A/G, IVS1-23C/T, 798 G/C, 921 A/G, and 1520C/T, were identified. Each haplotype was shared by families carrying an identical mutation in the ferrochelatase gene indicating a single mutation event for each of the three mutations. These mutations have been present in the Swiss erythropoietic protoporphyria population for a relatively long time as no common haplotypes of microsatellite markers flanking the ferrochelatase gene were found, except of two conserved regions, telomeric of the insT213 allele and centromeric of the delTACAG(580-584)allele, each with a size > 3 cM. Among the nonmutant ferrochelatase alleles, patients from six erythropoietic protoporphyria families shared a common haplotype [-251G; IVS1-23T] of the first two single nucleotide polymorphisms. An exception was the haplotype [-251 A; IVS1-23C] identified in the index patient of one erythropoietic protoporphyria family. These results supported the recent findings that the low expressed allele is tightly linked to a haplotype [-251G; IVS1-23T] of two intragenic single nucleotide polymorphisms in the ferrochelatase gene.     

Genetic study in a Singaporean patient with erythropoietic protoporphyria

Erythropoietic protoporphyria (EPP) is a rare autosomal dominant disorder of haem biosynthesis resulting from a partial decrease in ferrochelatase (FECH) activity which leads to the excessive accumulation of protoporphyrin in blood, erythrocytes and tissues. Cutaneous manifestations of photosensitivity usually appear in early infancy upon the first sun exposures. This normally requires the co-inheritance of a common hypomorphic FECH allele and a deleterious FECH mutation. Here, we report the first Singaporean Chinese patient with EPP characterized at the molecular level.     

Mutations in the lipase‐H gene causing autosomal recessive hypotrichosis and woolly hair

Hypotrichosis is characterised by sparse scalp hair, sparse to absent eyebrows and eyelashes, or absence of hair from other parts of the body. In few cases, the condition is associated with tightly curled woolly scalp hair. The present study searched for disease‐causing sequence variants in the genes in four Pakistani lineal consanguineous families exhibiting features of hypotrichosis or woolly hair. A haplotype analysis established links in all four families to the LIPH gene located on chromosome 3q27.2. Subsequently, sequencing LIPH identified a novel non‐sense mutation (c.328C>T; p.Arg110*) in one and a previously reported 2‐bp deletion mutation (c.659_660delTA, p.Ile220ArgfsX29) in three other families.     

Concurrent Chondrodysplasia Punctata Type 2 (Conradi–Hunermann–Happle Syndrome) and Ichthyosis Vulgaris in Teenaged Twin Girls

We present concurrent X‐linked chondrodysplasia punctata and ichthyosis vulgaris in adolescent fraternal twin girls, notable for initial presentation with dry skin in adolescence, characterized by dark‐brown scale typical of ichthyosis vulgaris and blaschkolinear, atrophic, scaly plaques. This constellation of findings prompted further genetic investigation. Using a multigene approach to examine 39 genes associated with congenital ichthyosis, next‐generation sequencing revealed a novel heterozygous missense mutation at a mutational hotspot in the EBP gene c.439C>T (p.R147C) in conjunction with a single nonsense mutation in the FLG gene (p.R501X) in both sisters. These individuals highlight the clinical variability of Conradi–Hunermann–Happle syndrome, illustrate the possibility of co‐occurrence of rare and common forms of ichthyosis, and demonstrate the utility of multigene analysis.     

Ten novel mutations of the ADAR1 gene in Japanese patients with dyschromatosis symmetrica hereditaria

Dyschromatosis symmetrica hereditaria (DSH) is a pigmentary genodermatosis of autosomal-dominant inheritance. We have reported 20 different mutations of the adenosine deaminase acting on RNA 1 gene (ADAR1) in patients with DSH since we had clarified that the disease is caused by a mutation of the ADAR1 gene in 2003. In this study, we report 10 novel mutations responsible for DSH: p.Q102fsX123, p.T369fsX374, p.S664fsX677, p.R892L, p.I913R, p.R916Q, p.P990fsX1016, p.C1081S, p.C1169F, and p.K1187X.