Two Brothers with Mild Congenital Erythropoietic Porphyria Due to a Novel Genotype

OBSERVATION Two Brothers With Mild Congenital Erythropoietic Porphyria Due to a Novel Genotype

Ali A. Berry, MD; Robert J. Desnick, MD, PhD; Kenneth H. Astrin, PhD; Junard Shabbeer, PhD; Anne W. Lucky, MD; Henry W. Lim, MD

Background: Congenital erythropoietic porphyria (CEP) were carriers of the −76G→A mutation, whereas the is a rare autosomal recessive disease caused by the defi- mother and a 15-year-old brother were carriers of the cient activity of the heme biosynthetic enzyme, uropor- G225S mutation. Previous in vitro expression studies dem- phyrinogen III synthase (URO-synthase), and the accu- onstrated that the G225S mutation severely decreased mulation of the nonphysiologic and phototoxic porphyrin URO-synthase activity to 1.2% of normal, whereas the I isomers. Clinical manifestations range from severe mu- promoter mutation decreased the activity to approxi- tilation to mild erosions and blisters on sun-exposed areas. mately50% of wild type, accounting for the mild clini- Evaluation of the URO-synthase mutation and residual cal phenotype. enzyme activity has been correlated with the phenotypic expression of the disease. Conclusion: The mild disease phenotype in these patients is a further example of the clinical heterogeneity Observations: We describe 16- and 4-year-old broth- seen in CEP and is additional proof that in vitro enzyme ers with CEP with a mild phenotype due to a novel geno- expression studies provide dependable genotype- type, one allele having a promoter mutation (−76G→A) phenotype correlations. and the other having an exonic missense mutation (G225S). The father and a 4-year-old fraternal twin brother Arch Dermatol. 2005;141:1575-1579

ONGENITAL ERYTHROPOI- tions include transfusion-dependent etic porphyria (CEP, or chronic hemolysis and secondary hyper- Günther disease) is a rare splenism, erythrodontia, osteolytic skel- autosomal recessive dis- etal changes, and cutaneous photosen- ease caused by the defi- sitivity with blistering and extensive cientC activity of uroporphyrinogen III syn- scarring. Although most reported CEP cases thase (URO-synthase), the fourth enzyme have presented with early-onset severe dis- in the heme biosynthetic pathway.1 Nor- ease, there has been notable variation and mally, URO-synthase metabolizes hy- genetic heterogeneity in their phenotypic droxymethylbilane to uroporphyrinogen presentation.3 This ranges from moderate III, the tetrapyrrole precursor of heme.2,3 forms of the disease presenting in child- The deficiency of URO-synthase activity hood to milder adult-onset disease with leads to nonenzymatic conversion of hy- mild cutaneous manifestations.3-5 droxymethylbilane to uroporphyrinogen I, Recent advances in genetic analysis have a nonphysiologic substrate that is con- shed light on the genetic basis of the phe- Author Affiliations: verted to coproporphyrinogen I, and then notypic heterogeneity. Since the identifi- Department of Dermatology, these porphyrinogen I isomers are oxi- cation and localization of the URO- Henry Ford Hospital, Detroit, dized to uroporphyrin I (URO-I) and cop- synthase gene,6 more than 35 different Mich (Drs Berry and Lim); roporphyrin I (COPRO-I), which are pho- CEP-causing mutations have been iden- Department of Human totoxic compounds. Elevated levels of tified.7 These include missense and/or non- Genetics, Mount Sinai School URO-I and COPRO-I in erythrocytes re- sense mutations, splicing defects, small and of Medicine of New York sult in massive hemolysis, and the re- large deletions or insertions, and com- University, New York (Drs Desnick, Astrin, and leased porphyrins accumulate in periph- plex rearrangements. Except for 4 muta- Shabbeer); and Dermatology eral blood, skin, bone, and teeth. tions in the promoter region, all the mu- Associates of Cincinnati and The tissue deposition of URO-I and tations occurred in the coding region of Cincinnati Children’s Hospital, COPRO-I is responsible for the clinical the gene.3,7 Most mutations occurred in one Cincinnati, Ohio (Dr Lucky). manifestations of CEP. These manifesta- or a few unrelated families, with the ex-

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©2005 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 our patients further supports previous work indicating a strong genotype-phenotype correlation in this disease. Although the residual URO-synthase activity expressed by the severe G225S missense exonic mutation was only 1.2% of normal,9 the residual enzyme activity associated with −76G→A promoter substitu- tion was 54% of mean activity expressed by the normal allele in vitro, substantially higher than that seen in severe cases.3,10

REPORT OF CASES

CASE 1 A 16-year-old white adolescent boy was referred to the Department of Dermatology, Henry Ford Hospital, Detroit, Mich, for skin fragility, blister formation, and mild scarring of the face, arms, and legs since age 3 years. He reported having mild cutaneous photosensitivity and frequent reddish discoloration of the urine. Occasional splenomegaly had been detected, but no frank episodes of hemolysis had occurred. He had been evaluated for occasional abdominal pain, but no cause was found. No joint pains or neuropsychiatric symptoms were reported. His younger brother had a similar condition (see case 2), whereas his parents and other siblings had no symptoms of photosensitivity or any cutaneous findings. Figure 1. Periorbital hypertrichosis, as seen in case 1. Physical examination showed hypertrichosis over the periorbital area (Figure 1), several shallow scars on the forehead, and scattered crusted erosions with mild non- mutilating scarring over the dorsum of the hands (Figure 2) and extensor surfaces of the forearms. There was no hepatosplenomegaly. On the basis of these findings, cutaneous porphyria was suspected, and the patient’s plasma, erythrocyte, urine, and fecal porphyrin levels and erythrocyte uroporphyrinogen decarboxylase activity were evaluated. The results were consistent with the diagnosis of CEP because a marked increase in total urinary, plasma, erythrocyte, and fecal porphyrin levels was detected, with a predominance of URO-I and COPRO-I isomers in the urine and plasma and COPRO-I in the stool (Table). Uroporphy- rinogen decarboxylase activity was normal, thus exclud- ing the diagnosis of porphyria cutanea tarda or hepato- erythropoietic porphyria. Analysis of genomic DNA Figure 2. Erosions, some with overlying crusts, mild scarring, and revealed a novel combination of mutant URO-synthase postinflammatory hyperpigmentation on the dorsum of the hand, as seen in alleles, a paternally inherited promoter mutation case 1. (−76G→A) and maternally inherited missense exonic mutation (G225S).9-11 ception of the severe C73R missense mutation, which occurred in nearly 30% of the disease alleles.3,8 In vitro stud- CASE 2 ies3 that characterized the residual URO-synthase enzymatic activity expressed by different mutations re- The 4-year-old brother of the patient in case 1 was revealed that the level of residual activity directly corre- ferred to the Department of Dermatology, Henry Ford lated with the disease severity; namely, higher residual Hospital, complaining of pruritic and painful erosions and URO-synthase activity levels resulted in milder CEP phe- blistering with mild scarring on sun-exposed skin since notypes, and lower residual enzymatic activity levels re- 18 months of age. He was the product of a full-term preg- sulted in a more severe phenotype. nancy and uncomplicated delivery. He was born healthy In this article, we describe 2 brothers who have a and had not had any hemolysis or organomegaly. He had mild CEP phenotype and a previously unreported had no abdominal or joint pains. With a notable excep- genotype, −76G→A/G225S. The mild phenotype in tion of his 16-year-old brother (case 1), his parents and

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Porphyrin Precursors and Porphyrins Case 1 Case 2 Reference Range 5Ј-Aminolevulinic acid in urine, mg/24 h 2.5 0.9 0-7 Porphobilinogen in urine, mg/24 h 0.7 0.2 0-4 Total porphyrins in urine, nmol/24 h 8073 3590 0-300 Porphyrin pattern, nmol/L (I/III isomer ratio) Uroporphyrin 6135 (98) 22 764(98) 0-90 Coproporphyrin 1049 (98) 503 (98) 150-300 Heptacarboxyl porphyrin 242 72 0-15 Hexacarboxyl porphyrin 161 36 0-15 Pentacarboxyl porphyrin 484 215 0-15 Isocoproporphyrin 0 0 0 Total plasma porphyrin, µg/dL 12.3 8.6 0-0.9 Protoporphyrin in erythrocytes, µg/dL 342 376 0 Hematocrit in erythrocytes, % 43.6 36.9 36-47 Total porphyrin in feces, nmol/g dry weight 1398 836 0-200 Porphyrin pattern, nmol/g dry weight (I/III isomer ratio) Uroporphyrin 0 0 0-20 Coproporphyrin 1370 (98) 803 (98) 60-200 Pentacarboxyl porphyrin 28 25 0-20 Protoporphyrin 0 1 60-200

*Porphyrin analyses performed by Karl E. Anderson, MD, University of Texas Medical Branch, Galveston.

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Figure 4. Crusted erosions, milia, and postinflammatory hyperpigmentation on the dorsum of the hand in case 2. Note a small vesicle on the lateral aspect of the index finger. Figure 3. Erosions and crusting on the forehead in case 2. Note hypertrichosis on the temple.

2 other siblings, including a fraternal twin, did not have any similar complaints. G225S/WT –76G→A/WT Physical examination revealed crusted erosions on the nasal bridge and forehead (Figure 3). Erosions, super- ficial scars, hyperpigmentation, and milia were ob- served on the nape of the neck, dorsum of the hands –76G→A/G225S WT/G225S –76G→A/G225S –76G→A/WT (Figure 4), and extensor extremities. Hypertrichosis was noted on the face, forearms, and legs; no lesions were Case 1 Case 2 noted in the non–sun-exposed areas. There was no hepatosplenomegaly. Figure 5. Pedigree showing genotype and phenotype of patients and their Similar to case 1, cutaneous porphyria was suspected family. WT indicates wild type; black squares, affected individuals; half black squares and circle, carrier individuals; squares, male; and circle, female. and the patient’s plasma, erythrocyte, urine, and fecal porphyrin levels and erythrocyte uroporphyrinogen decarboxylase activity were evaluated. The results were con- ing further supported the diagnosis of CEP because the sistent with a diagnosis of CEP; a marked increase in total −76G→A/G225S genotype was identified. urinary, plasma, erythrocyte, and fecal porphyrin levels was detected with a predominance of URO-I and COPRO-I STUDIES OF FAMILY MEMBERS isomers in the urine and plasma and COPRO-I in the stool (Table). Erythrocyte uroporphyrinogen decarboxylase The results of mutation detection studies of all family activity was normal. As shown in Figure 5, genetic test- members are shown in Figure 5. One of the father’s URO-

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©2005 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 synthase alleles had the −76G→A promoter mutation, and expression of the −76G→A allele and the G225S allele one of the mother’s URO-synthase alleles contained the in Escherichia coli revealed residual URO-synthase ac- G225S missense mutation. A 15-year-old male sibling of tivities of 53.9% and 1.2%, respectively, of the mean in the affected brothers carried the G225S mutation, whereas vitro wild-type expressed activity.9,10 This suggests that the 4-year-old fraternal twin brother of the patient in case the mild phenotype seen in our patients is due to the 2 carried the promoter mutation. Except for the 2 af- compensatory residual activity of the URO-synthase ac- fected siblings, none of the family members had any signs tivity produced primarily by the −76G→A allele. The or symptoms of CEP. delay in onset of disease in our patients (3 years of age in case 1 and 18 months of age in case 2) may be due to TREATMENT the slow rate of accumulation of the porphyrin isomers in reaching a threshold pathogenic level. A similar phe- Both patients were advised to avoid sun exposure and to nomenon has been postulated for patients with myelo- use inorganic sunscreens that contained zinc oxide or ti- dysplastic disease who develop late-onset CEP, which is 5,13,14 tanium dioxide. They were also provided with inor- usually mild. ganic sunscreen formulated as a paste with coloring that The management of the cutaneous manifestations of could be matched to skin color.12 This was more accept- CEP consists of strict sun avoidance, use of inorganic sun- able and practical for use. The patients and their family screens, and prompt treatment of secondary bacterial skin members also received genetic counseling. They subse- infections. Inorganic sunscreens that contain zinc oxide quently reported better control of their cutaneous erup- or titanium dioxide are best if applied as a paste, but this tions. Except for limiting sun exposure, the affected broth- may be impractical and cosmetically unappealing for most ers have been able to lead normal lives. patients. Other treatment modalities with limited effi- cacy include oral beta carotene, intravenous hematin, plas- mapheresis, hydroxychloroquine, oral charcoal, and other COMMENT porphyrin binders.2,17 For severely affected patients who are transfusion dependent, erythrocyte transfusion,18 hy- Congenital erythropoietic porphyria is an inborn error droxyurea,19 and splenectomy20 appear effective, at least of heme biosynthesis that results from the markedly de- until adolescence; stem cell or bone marrow transplan- ficient, but not absent, activity of URO-synthase and the tation has been curative.20,21 In vitro gene transfer stud- resulting accumulation of uroporphyrinogen I and cop- ies have demonstrated restoration of URO-synthase ac- roporphyrinogen I, which are oxidized to the nonphysi- tivity, suggesting the future potential effectiveness of gene ologic porphyrin I isomers, URO-I and COPRO-I.1-3 therapy.22-24 Light in the Soret region band (400-410 nm) activates In summary, we describe 2 brothers with CEP who the phototoxic URO-I and COPRO-I isomers, resulting have an unusually mild phenotype due to a novel het- in skin fragility, blisters, milia, and mutilating scars. erozygous genotype that consists of a promoter muta- Other clinical manifestations include erythrodontia, tion (−76G→A) and an exonic missense mutation alopecia, and hypertrichosis. Patients with severe CEP (G225S). The mild disease in these patients is a further present with mutilating cutaneous involvement starting example of the clinical heterogeneity seen in CEP and is in the neonatal period, are transfusion dependent, additional proof that in vitro enzyme expression studies and have hypersplenism. Patients with late-onset dis- can correlate genotype to phenotype. ease usually present with only mild cutaneous lesions4,5 and may develop thrombocytopenia and myelodyspla- Accepted for Publication: August 21, 2005. sia5,13,14; they are not transfusion dependent. On the ba- Correspondence: Henry W. Lim, MD, Department of Der- sis of the in vitro expression of the individual mutant al- matology, Henry Ford Medical Center, New Center One, leles found in cases of CEP, it has been shown that the 3031 W Grand Blvd, Suite 800, Detroit, MI 48202 (hlim1 residual URO-synthase activity in milder cases is higher @hfhs.org). than the residual URO-synthase activity in severe Author Contributions: Study concept and design: Berry, cases.3,15,16 Desnick, and Lim. Acquisition of data: Berry, Desnick, As- To date, more than 35 URO-synthase mutations are trin, Shabbeer, Lucky, and Lim. Analysis and interpreta- listed in the Human Gene Mutation Database.3,7 Pa- tion of data: Desnick, Lucky, and Lim. Drafting of the manu- tients have been described who are homoallelic or het- script: Berry, Desnick, and Lim. Critical revision of the eroallelic for different mutations. For example, the manuscript for important intellectual content: Berry, most common mutation, C73R, has occurred in se- Desnick, Astrin, Shabbeer, Lucky, and Lim. Obtained fund- verely affected individuals when homoallelic (C73R/ ing: Desnick. Administrative, technical, and material sup- C73R) and in moderately affected patients when port: Berry, Desnick, Shabbeer, and Lim. Study supervi- heteroallelic (C73R/A104V).3 Thus, the genotype is di- sion: Desnick, Astrin, and Lim. rectly correlated with the amount of expressed URO- Financial Disclosure: None. synthase activity, as previously shown by in vitro stud- Funding/Support: This work was supported in part by ies, and is also directly correlated with disease grants from the National Institutes of Health, Bethesda, severity.3,9,10 In our patients, the novel genotype con- Md, including research grant RO1 DK26824 and grant sisted of 2 different mutations, a promoter region point 5 MO1 RR00071 to the Mount Sinai General Clinical mutation (−76G→A) in one allele and an exonic mis- Research Center from the National Center for Research sense mutation (G225S) in the other. Previous in vitro Resources.

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©2005 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 13. Murphy A, Gibson G, Elder GH, Otridge BA, Murphy GM. Adult-onset congenital REFERENCES erythropoietic porphyria (Günther’s disease) presenting with thrombocytopenia. J R Soc Med. 1995;88:357P-358P. 1. Romeo G, Levin EY. Uroporphyrinogen III cosynthetase in human congenital eryth- 14. Yamauchi K, Kushibiki Y. Pyridoxal 5-phosphate therapy in a patient with my- ropoietic porphyria. Proc Natl Acad Sci U S A. 1969;63:856-863. elodysplastic syndrome and adult onset congenital erythropoietic porphyria. Br 2. Anderson KE, Sassa S, Bishop DF, Desnick RJ. Disorders of heme biosynthesis: J Haematol. 1992;81:614-615. X-linked sideroblastic anemia and the porphyrias. In: Scriver CS, Beaudet AL, 15. Fontanellas A, Bensidhoum M, Enriquez de Salamanca R, Moruno Tirado A, de Sly WS, Valle D, eds. The Molecular and Metabolic Bases of Inherited Disease. Verneuil H, Ged C. A systematic analysis of the mutations of the uroporphyrino- New York, NY: McGraw-Hill; 2001:2961-3062. gen III synthase gene in congenital erythropoietic porphyria. Eur J Hum Genet. 3. Desnick RJ, Astrin KH. Congenital erythropoietic porphyria: advances in patho- 1996;4:274-282. genesis and treatment. Br J Haematol. 2002;117:779-795. 16. Desnick RJ, Glass IA, Xu W, Solis C, Astrin KH. Molecular genetics of congenital 4. Warner CA, Poh-Fitzpatrick MB, Zaider EF, Tsai SF, Desnick RJ. Congenital eryth- erythropoietic porphyria. Semin Liver Dis. 1998;18:77-84. ropoietic porphyria: a mild variant with low uroporphyrin I levels due to a mis- 17. Fritsch C, Bolsen K, Ruzicka T, Goerz G. Congenital erythropoietic porphyria. sense mutation (A66V) encoding residual uroporphyrinogen III synthase activity. J Am Acad Dermatol. 1997;36:594-610. Arch Dermatol. 1992;128:1243-1248. 18. Piomelli S, Poh-Fitzpatrick MB, Seaman C, Skolnick LM, Berdon WE. Complete 5. Kontos AP, Ozog D, Bichakjian C, Lim HW. Congenital erythropoietic porphyria suppression of the symptoms of congenital erythropoietic porphyria by long- associated with myelodysplasia presenting in a 72-year-old man: report of a case term treatment with high-level transfusions. N Engl J Med. 1986;314:1029- and review of the literature. Br J Dermatol. 2003;148:160-164. 1031. 6. Astrin KH, Warner CA, Yoo HW, Goodfellow PJ, Tsai SF, Desnick RJ. Regional 19. Guarini L, Piomelli S, Poh-Fitzpatrick MB. Hydroxyurea in congenital erythro- assignment of the human uroporphyrinogen III synthase (UROS) gene to chro- poietic porphyria. N Engl J Med. 1994;330:1091-1092. mosome 10q25.2- q26.3. Hum Genet. 1991;87:18-22. 20. Shaw PH, Mancini AJ, McConnell JP, Brown D, Kletzel M. Treatment of congen- 7. Stenson PD, Ball EV, Mort M, et al. Human Gene Mutation Database (HGMD): ital erythropoietic porphyria in children by allogeneic stem cell transplantation: 2003 update. Hum Mutat. 2003;21:577-581. a case report and review of the literature. Bone Marrow Transplant. 2001;27: 8. Strand LJ, Manning J, Marver HS. Acute intermittent porphyria: studies of the 101-105. enzymatic basis of disordered haem biosynthesis. S Afr Med J. September 25,1971: 21. Harada FA, Shwayder TA, Desnick RJ, Lim HW. Treatment of severe congenital 108-111. erythropoietic porphyria by bone marrow transplantation. J Am Acad Dermatol. 9. Xu W, Warner CA, Desnick RJ. Congenital erythropoietic porphyria: identifica- 2001;45:279-282. tion and expression of 10 mutations in the uroporphyrinogen III synthase gene. 22. Mazurier F, Moreau-Gaudry F, Salesse S, et al. Gene transfer of the uroporphy- J Clin Invest. 1995;95:905-912. rinogen III synthase cDNA into haematopoietic progenitor cells in view of a fu- 10. Solis C, Aizencang GI, Astrin KH, Bishop DF, Desnick RJ. Uroporphyrinogen III ture gene therapy in congenital erythropoietic porphyria. J Inherit Metab Dis. 1997; synthase erythroid promoter mutations in adjacent GATA1 and CP2 elements cause 20:247-257. congenital erythropoietic porphyria. J Clin Invest. 2001;107:753-762. 23. Kauppinen R, Glass IA, Aizencang G, Astrin KH, Atweh GF, Desnick RJ. Congen- 11. Shady AA, Colby BR, Cunha LF, Astrin KH, Bishop DF, Desnick RJ. Congenital ital erythropoietic porphyria: prolonged high level expression and correction of erythropoietic porphyria: identification and expression of eight novel mutations the heme biosynthetic defect by retroviral-mediated gene transfer into porphy- in the uroporphyrinogen III synthase gene. Br J Haematol. 2002;117:980-987. ric and erythroid cells. Mol Genet Metab. 1998;65:10-17. 12. Moseley H, Cameron H, MacLeod T, Clark C, Dawe R, Ferguson J. New sun- 24. Geronimi F, Richard E, Lamrissi-Garcia I, et al. Lentivirus-mediated gene trans- screens confer improved protection for photosensitive patients in the blue light fer of uroporphyrinogen III synthase fully corrects the porphyric phenotype in region. Br J Dermatol. 2001;145:789-794. human cells. J Mol Med. 2003;81:310-320.

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