Brain Advance Access originally published online on January 17, 2008
Brain 2008 131(3):760-761; doi:10.1093/brain/awm337
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Recessive hereditary methaemoglobinaemia, type II: delineation of the clinical spectrum
1Pôle des maladies du système nerveux, Fédération de neurologie, groupe hospitalier Pitié-Salpêtrière, AP-HP, 2Département Génétique et Développement, Institut Cochin, INSERM U567, Université Paris-V René-Descartes, 3Service de Neuropédiatrie, hôpital Trousseau, AP-HP, 4Service de Pédiatrie, Hôpital de Hautepierre, Strasbourg, France, 5Service de maladie Neurométaboliques, Hôpital Necker-enfants malades, AP-HP, and 6Unité de Neurobiologie des Processus Adaptatifs, CNRS UMR 7102, Université Paris VI Pierre et Marie Curie, Paris, France
Correspondence to: Dr E. Roze, MD, PhD, Pôle des Maladies du Système Nerveux, Fédération de Neurologie, Groupe hospitalier Pitié-Salpêtrière, 47-83 boulevard de l'Hôpital, 75651 Paris Cedex 13, France E-mail: emmanuel.roze{at}psl.aphp.fr
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Summary |
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Type II recessive hereditary methaemoglobinaemia (RHM) is a rare disease due to generalized NADH-cytochrome b5 reductase (cytb5r) deficiency. It results in mild cyanosis and severe neurological impairment. The clinical features and long-term outcome are poorly documented, and there are no systematic reviews. We examined six cases of type II RHM, four of which were new, together with 45 previously published cases, in order to establish the range of phenotypic expression. The clinical picture was very similar in most cases, with severe encephalopathy, microcephaly, generalized dystonia, movement disorders and mild cyanosis. The neurological prognosis was poor; in particular, none of the patients walked or spoke. In addition, the possibility of an atypical and milder phenotype was considered. We concluded that children with unexplained severe encephalopathy associated with generalized dystonia should be examined for cyanosis and have a methaemoglobinaemia assay performed. The diagnosis can be confirmed by very low cytb5r activity in both red and white blood cells. Here we report three novel mutations in the NADH-cytochrome b5 reductase gene. Prenatal diagnosis of this extremely severe disease should be proposed to affected families.
Key Words: cyanosis; dystonia; cerebral palsy; methaemoglobinaemia; cytochrome b5 reductase
Abbreviations: cytb5r, cytochrome b5 reductase; RHM, recessive hereditary methaemoglobinaemia.
Received October 11, 2007. Revised November 26, 2007. Accepted December 19, 2007.
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Introduction |
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Recessive hereditary methaemoglobinaemia (RHM) is an autosomal recessive metabolic disorder due to NADH-cytochrome b5 reductase (cytb5r, EC 1.6.2.2) deficiency. This enzyme exists in soluble and membrane-bound forms, both of which are ubiquitously expressed. The soluble erythrocytic cytb5r isoenzyme is involved in methaemoglobin reduction (Hultquist and Passon, 1971
), while the biological role of the soluble non-erythrocytic cytb5r isoenzyme is unclear (Leroux et al., 1977; Lostanlen et al., 1978). The membrane-bound microsomal enzyme participates in a fatty acid desaturation complex (Strittmatter et al., 1974) and in drug metabolism (Hildebrandt and Estabrook, 1971). All human cytb5r isoforms are produced from a single gene locus, DIA1 (Du et al., 1997; Leroux et al., 2001), now updated by HUGO to CYB5R3, located on chromosome 22 (Fisher et al., 1977; Junien et al., 1978). Two clinical types of RHM (type I and II) have been defined. Type I RHM is a benign hereditary form of methaemoglobinaemia in which cyanosis is the only clinical sign. In 1845, François reported the first patient with long-standing congenital cyanosis despite no obvious cardiac or pulmonary disorders (François, 1845).The disease was later found to be related to an erythrocytic enzyme deficiency. The enzyme, first called co-enzyme factor 1 (Gibson, 1948) or diaphorase (Scott and Griffith, 1959), was later identified as soluble cytb5 reductase (Hultquist and Passon, 1971). Type I RHM is characterized by cytb5r deficiency restricted to red blood cells. In type II RHM, permanent mild cyanosis is associated with severe neurological manifestations, including mental retardation, microcephaly, generalized dystonia and movement disorders. In 1948, Sacerdotti-Favini first described patients with congenital methaemoglobinaemia who, in addition to cyanosis, had progressive encephalopathy (Sacerdotti-Favini, 1948). A few isolated cases were reported between 1948 and 1972, followed by the largest series of eight new cases of type II RHM (Kaplan et al., 1974). Type II RHM was subsequently shown to be characterized by a profound enzyme deficiency in all tissues, involving both the soluble and membrane-bound cytb5r isoforms (Leroux et al., 1975). Several previous publications refer to this disease as ‘recessive congenital methaemoglobinaemia’. Considering that it is an autosomal recessive disorder, we thought ‘recessive hereditary methaemoglobinaemia’ was more appropriate and we propose that it should be the standard appellation. There is no treatment for type II RHM and its pathophysiology is unknown. Antenatal diagnosis is crucial in families with a history of type II RHM, as the risk of transmission is 25%.
To date, more than 40 mutations have been identified in the cytb5r gene of patients with type I and II RHM. The genotype–phenotype relationship is extensively discussed in recent papers (Percy et al., 2002; Grabowska et al., 2003; Percy et al., 2004; Leroux et al., 2005; Percy et al., 2005; Yilmaz et al., 2005; Nussenzveig et al., 2006). Mutations causing enzyme instability lead to type I RHM, whereas mutations that inactivate the enzyme are associated with type II RHM (Shirabe et al., 1992; Vieira et al., 1995; Dekker et al., 2001). Here we also report three novel mutations in the cytb5r gene.
In contrast with detailed biochemical or genetic studies, the clinical features and long-term outcome of patients with type II RHM are poorly documented. Here we report detailed clinical, biological, radiological and follow-up data on six cases of type II RHM, four of which are new, together with a systematic review of the literature. From this comprehensive clinical description of the disease, we aim to identify clinical clues for early recognition of the disease, and to propose tools for prognosis assessment and genetic counselling.
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Patients and Methods |
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The six patients with type II RHM were referred to a reference centre for rare neuropaediatric disorders in Paris (France), for clinical evaluation and management. A biochemical diagnosis was established in every case. Cytb5r activity in erythrocytes and leucocytes was measured according to Hegesh et al. (1968
). Genetic analysis was performed by PCR amplification and sequencing (Leroux et al., 2005).
All the patients were examined by at least one of the authors. The data were collected prospectively in cases 4–6 and from the medical records in cases 1–3. In addition to standard neurological examination, photographs and video recordings were made in four cases with the parents’ written informed consent. Cases 1 and 3 have been described elsewhere with respect to genetic findings, but with only a brief clinical description (Vieira et al., 1995; Leroux et al., 2005). Patients 4 and 5 had brain MRI including spectroscopy.
The NIH Pubmed database, and the SUDOC and PASCAL BIOMED (Paris V University) databases were scanned up to 2006 for reports including the key words: ‘methaemoglobinaemia’ and ‘cytochrome b5 reductase’. The reference lists of the reports thus retrieved were also scrutinized. Cases mentioned in more than one publication were considered only once. We enrolled all cases meeting one of the following criteria: (i) elevated methaemoglobinaemia (>1%); (ii) typical type II RHM clinical features, with or without erythrocyte and leucocyte cytb5r assay; ‘or’ (iii) low cytb5r activity in erythrocytes and leucocytes, regardless of clinical features; and (iv) no other obvious cause of encephalopathy. Only cases with a detailed clinical description, and methaemoglobinaemia and enzyme assays, were considered for statistical analysis. Cases purporting to be type II RHM, but with atypical clinical features and no leucocyte cyb5r assay were rejected. The authors of the published reports were contacted, and additional data were obtained for four patients. We analysed demographic, clinical, neuroradiological, biochemical and genetic findings.
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Results |
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Case reports
Case 1
This female patient was born to non-consanguineous parents of French and Spanish ancestry. There was no familial history. The pregnancy and delivery were normal. At birth, her weight and head circumference were –1.5 SD. At age 14 days, she had frequent vomiting and became cyanotic when crying. The head circumference was –2 SD. She had permanent mild cyanosis, which was more prominent on the labial mucosae and nails and worsened when she cried. There was also generalized dystonia with hyperextension of the four limbs and neck, and distal upper limb pronation. Spontaneous gesticulation was uncoordinated. Routine laboratory examinations and CSF analysis were normal. EEG showed diffuse slowing with no epileptic features. The visual evoked potential tests were normal. CT showed cortico-subcortical atrophy with prominent involvement of the frontal and temporal lobes. Methaemoglobinaemia was elevated at 15%. The diagnosis of type II RHM was confirmed by cytb5r assay in erythrocytes and leucocytes (estimated at 5% and 0%, respectively). At age 6 months, her condition worsened, with bouts of opisthotonos and permanent diffuse athetoid movements. She often cried and was agitated. She could smile and follow a point with her eyes, despite converging strabismus. At age 10 months, her psycho-motor performance was that of 3- to 4-month-old child. She could grasp objects but could not support her head. Her hypotrophy and microcephaly gradually worsened (weight –3SD, head circumference –4 SD). Vomiting was frequent and exacerbated her feeding difficulties. At age 20 months, she was still unable to support her head. She could produce simple sounds and recognize familiar faces. At age 3.5 years, her condition remained unchanged, with major hypotrophy, microcephaly, strabismus, permanent cyanosis and frequent vomiting. She died a few years later but the exact age of death was not mentioned in the medical records.
Subsequent genetic analysis of lymphoblastoid cell lines, previously published by Mota Vieira et al. (1995), showed that she was a compound heterozygote with the Cys204Arg mutation in one allele and an out-of-frame 3-bp deletion with loss of Met 273 (Met273Del) in the second allele.
Case 2
This female patient was born to French non-consanguineous parents with no familial history. Clinical examination at birth was normal except for cyanosis noted on the second day of life. Methaemoglobinaemia was elevated, at 32%. The diagnosis of type II RHM was confirmed by cytb5r assay in erythrocytes and leucocytes (both estimated at 0%).
In the first months of life, she often cried and was agitated. She first smiled at 4 months. She fed normally despite occasional vomiting. Cyanosis was discrete but worsened when she cried. At age 10 months, she had global hypertonia with flexion spasms prevailing on the upper limbs, and athetoid movements of the lower limbs, including toes reptation, and external rotation and dorsal flexion of the feet. Microcephaly was noted at age 11 months (–2 SD) but her growth was globally normal. In the following months, involuntary movements gradually decreased but her global hypertonia worsened. At age 2 years 8 months, she was still unable to sit, grab objects or support her head. She still had athetoid movements of the four limbs. EEG was normal.
She was lost to follow-up until age 23 years. She is now living in a specialized institute for severely disabled persons. She is hypotrophic (height –3.5 SD, weight –4 SD) and has severe microcephaly (head circumference –5 SD). Her communication skills are very poor: she does not answer to simple commands, is not interested by images, but recognizes familiar faces. She reacts to noise, expresses herself with simple sounds and follows with her eyes despite a diverging strabismus. She has severe generalized dystonia, which is prominent in the distal part of the limbs. She has choreo-athetoid movements of the four limbs and face, resulting in facial grimacing (Fig. 1a). These movements are mild at rest but increase on external stimulation or voluntary movement. She can achieve coarse dystonic prehension of objects. She has brisk tendon reflexes without the Babinski sign. Her skin is pale, with no obvious cyanosis except for the labial mucosae and nails (Fig. 1a and b). She has frequent spontaneously resolutive exacerbations of generalized cyanosis, with no clear triggering factor, for which she receives chronic riboflavin therapy. She has right hip subluxation and severe scoliosis requiring a corset. She cannot remain seated other than in her specially built seat, but she can support her head. She is totally dependent and unable to move around unaided. She eats mixed food despite moderate swallowing disorders.
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Genetic analysis showed compound heterozygosity for a novel mutation (Leu131Pro) in one allele and a previously published mutation, Gly76Ser (Percy et al., 2006b
), on the second allele. Both parents were heterozygotes, the father for Gly76Ser and the mother for Leu131Pro.
Case 3
This male patient was born to non-consanguineous Lebanese parents. A paternal cousin had unexplained psychomotor retardation and died in early childhood. There was a threat of premature delivery during the fifth month of pregnancy. At birth, measurements were normal except for microcephaly (head circumference –2 SD). In the first three months of life, he had cyanosis of the lips and nails, severe swallowing disturbances and converging strabismus. At 4 months, the microcephaly had worsened (head circumference –3 SD), and he had axial hypotonia with limb hypertonia. Brain CT showed cortical atrophy. EEG, visual and brainstem auditory evoked potentials were normal. Initial biological and metabolic analyses were inconclusive.
At age 36 months, he was hypotrophic (weight and height –3.5 SD) with severe microcephaly (head circumference –4 SD). Cyanosis was prominent on the labial mucosae. He had converging strabismus. Neurological examination showed axial hypotonia with dystonia of the four limbs and athetoid movements. Tendon reflexes were brisk with bilateral Babinski's sign. He could not sit, grasp or react to external stimulation, and only uttered sounds when crying. The methaemoglobin was 28.4% and cytb5r activity was 0% in erythrocytes and leucocytes. At age 4 years, he had severe hypotrophy with marked microcephaly (–6 SD), and a greyish-yellow skin colour. At age 10 years, he was bedridden and had major orthopaedic disorders such as lower limb retractions. His intellectual and neurological status were unchanged. Feeding was difficult because of major swallowing disturbances. He died at age 10,5 years of pneumonia.
Genetic analysis, which was previously published (Leroux et al., 2005), identified a homozygous out-of-frame 1-bp deletion with loss of Thr295 in exon 9 (Thr295Del). Both parents were heterozygous for the same mutation. This finding authorized prenatal diagnosis of a second pregnancy, at the parents’ request.
Case 4
This female patient was born to consanguineous Tunisian parents. Several family members had congenital malformations. The pregnancy and delivery were normal. Her birth weight and length were –1.5 SD and –2 SD, respectively, and she had microcephaly (–2 SD). In the first weeks of life, she was treated for a maternofoetal infection and had vomiting due to gastrooesophageal reflux. She often cried. She developed global hypotonia and profound cognitive impairment: she supported her head at age 11 months, smiled at 12 months, sat at 2 years and could move around on her rear at 3.5 years. She acquired dystonic gripping at age 4 years. At age 6 years, she underwent surgery for converging strabismus. At age 8 years, she had axial hypotonia, generalized dystonia with athetosic limb movements and occasional dystonic spasms. She made simple sounds. She was hypotrophic with microcephaly (weight and height –3.5 SD, head circumference –3.2 SD). Biological, genetic and radiological examinations failed to establish a diagnosis. EEG showed diffuse slowing with no epileptic features. She was admitted urgently for febrile pneumonia with a low SpO2 (85%) that was unresponsive to oxygen therapy. Marked cyanosis of the skin and mucosae was then noticed, suggesting a diagnosis of RHM. The methaemoglobin level was 17.5% and cytb5r activity was 0% and 3.2% of normal in erythrocytes and leucocytes, respectively. Brain MRI showed cerebral atrophy. Spectro-MRI was normal. At age 10 years, her height was –4 SD, her BMI was at the 75th percentile, her head circumference was –3 SD and her neurological status was relative stable. She still had brisk distal dystonic and choreo-athetosic movements. She was more rigid and had lost the ability to move around on her rear. She had become more reactive to environmental stimuli, reacting to her name, smiling and no longer crying. She communicated with sounds or shrieks. She had stopped vomiting.
Genetic analysis revealed a homozygous G-C transversion in intron 5 (IVS5, DS, G-C +8) resulting in out-of-frame exon 5 skipping. The same mutation had previously been published (Vieira et al., 1995) in an Algerian patient with type II RHM.
Case 5
This male patient was born to French parents with no family history. The pregnancy, delivery and birth measurements were normal. During the first months of life, he had generalized hypotonia with spontaneous dystonic leg extension spasms, failure to thrive, converging strabismus and frequent vomiting. He was hospitalized at age 9 months for episodes of discomfort and paleness, but no cause was found. The head circumference curve diverged from normal at age 5 months. At age 2 years, he had severe microcephaly (head circumference –3.5 SD) but normal weight and height. He had axial hypotonia with dystonia of the four limbs, and diffuse choreo-athetosic movements. He could grip objects with difficulty, in dystonic fashion. He made only simple sounds, had no eye tracking and could not sit. He developed West's syndrome with limb flexion spasms and partial epileptic seizures. EEG showed hypsarrhythmia. The epilepsy responded poorly to vigabatrin, hydrocortisone and topiramate. Lamotrigine attenuated the flexion spasms, which continued to occur daily. Visual evoked potentials were normal. Biological, genetic and radiological examinations failed to establish a diagnosis.
At age 2.5 years, he was admitted urgently for febrile pneumonia with a low O2 saturation of 90% that was unresponsive to oxygen therapy. Cyanosis of the lips and nails was noticed, suggesting a diagnosis of RHM. He had severe microcephaly (head circumference –3.5 SD), without hypotrophy (weight and height –1 SD). Neurological examination showed the same abnormalities as described above. His parents subsequently mentioned that they had previously noticed episodes of cyanosis when he had fever. The methaemoglobin level was 23.7% and cytb5r activity was 0% and 5.7% of normal in erythrocytes and leucocytes, respectively. MRI showed cranial dysmorphy, global cerebral atrophy and delayed myelination. Spectro-MRI was normal.
He was subsequently treated with vitamin C and riboflavin, which attenuated the cyanosis. At age 3.5 years, the microcephaly had worsened (head circumference –5 SD). The choreo-athetoid movements had diminished whereas dystonic postures of the extremities had increased. He reacted to familiar voices but could not track with the eyes because of severe converging strabismus and hypermetropia. He had to be fed carefully with mixed food because of a swallowing impairment.
A new homozygous mutation in exon 4 (Lys111Met) of the cytb5r gene was identified. Both parents were heterozygous for the same mutation. Genetic analysis and family inquiries confirmed the consanguinity of his maternal grandmother and paternal grandfather, who originated from nearby villages.
Case 6
This male patient was born to non-consanguineous French parents with no familial history. The pregnancy, delivery and birth measurements were normal. At birth, marked persistent generalized cyanosis was observed, with no cardiac or respiratory explanation. The methaemoglobin level was 23%, for which he was treated with methylene blue. The head circumference curve diverged from normal at age 5 months (–2 SD). However, his neurological status was considered normal until age 6 months, when he developed axial hypotonia, peripheral hypertonia with athetoid arm movements and converging strabismus. At age 11 months, he had a stable microcephaly (–2 SD) with normal weight and length. He could not sit, but made sounds, smiled and reacted to his name. His skin colour was ‘greyish white’, with very mild cyanosis of the lips and nails. The diagnosis of type II RHM was made at age 1 year, based on cytb5r activity at 0% and 16% of normal in erythrocytes and leucocytes, respectively. At age 18 months, the microcephaly had worsened (head circumference –6 SD). He smiled, his swallowing improved, he could grab objects, but he still could not sit. Cyanosis increased during crying episodes.
Genetic analysis revealed a novel homozygous G-A transition in intron 2, (IVS2, DS, G-A,+1) resulting in in-frame exon 2 skipping. Both parents were heterozygous for the same mutation.
Analysis of our cases and review of the literature
We collated 51 cases of type II RHM published from 1948 to 2006, including our six cases. The geographic origins of the patients summarized in Table 1 represent the cases published by the investigators, clinicians or biologists informed and interested in this pathology. At present, the RHM type II seems to be a worldwide extended genetic defect.
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Among these 51 cases, 23 patients, including our six cases, were selected for statistical analysis. The demographic, clinical and neuroradiological data of this group of patients are shown in Tables 2 and 3. Cyanosis and microcephaly were always present. The clinical spectrum was very homogeneous, except that patients 12 and 13 had an atypical phenotype (Takeshita et al., 1982
; Yawata et al., 1992). Apart from these two cases, all the patients had very severe psychomotor retardation, generalized dystonia with axial hypotonia and choreoathetoid movements, microcephaly and cyanosis. The main clinical features of this typical phenotype are presented in Table 4. Methaemoglobinaemia values ranged from 10 to 42% (mean ± SEM: 20.7% ± 3.2%). Cytb5r activity ranged from 0 to 16% of normal in erythrocytes (1.3% ± 0.8%) and 0 to 30% (4.3% ± 1.6%) of normal in leucocytes (Table 2).
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Another 28 RHM type II patients were identified in the literature, but were not selected for statistics (Table 5).
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Discussion |
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Clinical features
Neurological features: a highly homogeneous phenotype
During the first 2 months of life, 44% of patients had a ‘normal’ neurological examination. The full clinical phenotype manifested at about 6–9 months of life.
Fixed encephalopathy was always present and extremely severe. All the patients, including our 23-year-old patient, had psychomotor skills inferior to those of a 1-year-old child. At best, the patients could sit, grip objects in a dystonic palmar manner, react and smile at familiar faces and utter sounds. None of the patients walked or spoke. All the patients had generalized dystonia and axial hypotonia, sometimes associated with occasional dystonic storms and opisthotonos. Dystonia and movement disorders were mainly visible after age 6 months, when axial tone tended to improve. Spontaneous gesticulation was reported as permanent and uncoordinated, with proximo-distal and choreo-athetoid involuntary movements. These latter tend to decrease with aging, as usual in post-anoxic and metabolic movement disorders.
Microcephaly was always present and progressive. This feature is non-specific but reveals severe brain developmental deficiency. Growth retardation is frequent but inconsistent and is worsened by feeding difficulties associated with swallowing disturbances. However, none of the patients needed enteral nutrition, and vomiting gradually disappeared in our eldest patients (cases 2 and 4).
Strabismus was reported in 88% of cases. Three patients had refractory epilepsy (5, 8 and 11, cf. Table 3). Our case 5 is the first in which West's syndrome was a presenting feature of MHR type II. Behaviour abnormalities, such as frequent screaming and agitation, were reported in the first years of life. In our cases 2 and 3, they disappeared as the patients grew older. It is difficult to determine whether these are genuine behavioural disturbances or whether they result from movement disorders. Patients 2 and 3 had severe skeletal deformations such as scoliosis and musculo-tendinous retractions, likely due to chronic generalized dystonia.
Cyanosis, the key to diagnosis
In type II RHM, cyanosis is due the chocolate-brown colour of blood caused by the chronically high erythrocytic methaemoglobin level. Cyanosis may be very difficult to detect in this setting, however. The complexion is described as pale or ‘greyish-blue’. Cyanosis will be only seen on the labial mucosae and nails in dark-skinned patients, and this may explain the apparent lack of African patients. The cyanosis is enhanced during stressful events such as birth and infection, and may remain unnoticed for years. Yet, cyanosis is the most helpful diagnostic sign. In our series, all the patients were diagnosed after cyanosis was found. Failure to detect cyanosis may delay the diagnosis. For example, our Tunisian patient (case 4) was only diagnosed at age 8 years, when cyanosis became obvious because of a pulmonary infection.
Atypical cases
We collated seven atypical cases in five different families: four families are Japanese (cases 12 and 13, Tables 2 and 3 and cases 30–32, Table 5) and one is Cuban (cases 34 and 35, Table 5). None of the relevant publications provided detailed clinical data. The patients, aged from 13 to 43 years, were older than patients with the typical phenotype. Psychomotor retardation was milder (IQ 32 to 70) and, when mentioned, walking was described as ‘unsteady’ (Takeshita et al., 1982; Yawata et al., 1992) and speech as ‘altered’. One patient (Takeshita et al., 1982) had uncoordinated movements and neck dystonia. Leucocyte cytb5r activity was 5–30% of normal. We suggest that these patients may have a milder form of the disease, although this hypothesis needs to be confirmed considering the very small number of patients.
Prognosis
In the first reports published more than 30 years ago (Jaffe, 1966; Heusden et al., 1971; Kaplan et al., 1979), the life expectancy of patients with typical type II RHM did not appear to exceed 10 years of age. However, since 1979, only two deaths have been reported: one patient died at age 8 years (Aalfs et al., 2000) and our patient 3 died at age 10,5 years. Our four living patients, now aged from 2 to 23 years, appear to have no particular life-threatening disorders.
Survival appears to depend on two factors: (i) swallowing function, determining the risk of respiratory infections and failure to thrive; and (ii) medical, paramedical and familial management, especially prevention of decubitus complications. Our patient 2 is living in a specialized institute providing excellent medical care and, at age 23 years, holds the record for longevity with type II RHM.
Neuroradiological features
Detailed MRI data are available for two of our patients (cases 4 and 5) and for two published cases (Aalfs et al., 2000; Toelle et al., 2004). The most remarkable findings are cortical and subcortical atrophy, and delayed myelination reflecting global cerebral involvement. Although involvement of the basal ganglia would be expected, hypoplasia of the basal ganglia is mentioned in only one report (Aalfs et al., 2000). Spectro-MRI, performed in two of our cases, was normal in both. In most other publications, brain CT showed diffuse cortico-subcortical atrophy.
Biochemical findings
The methaemoglobin level ranges from 10 to 42%.
Cytb5r activity is routinely measured by the Hegesh method (Hegesh et al., 1968) using the methaemoglobin–ferrocyanure complex as substrate. In order to distinguish between type I and II RHM, cytb5r activity should be determined in both erythrocytes and leucocytes. In benign type I RHM, the activity of the soluble form of cytb5r is decreased only in erythrocytes, while enzyme activity in leucocytes is normal. In type II RHM, cytb5r activity is strongly diminished in both cell types, confirming the generalized cytb5r deficiency (Leroux et al., 1975). Kugler (Kugler et al., 2001) diagnosed type II RHM in a 49-year-old patient with cyanosis and ‘severe psychomotor retardation’ (no details provided). In this patient, elevated methaemoglobinaemia was associated with 20% of normal cytb5r activity in erythrocytes, while activity was not measured in leucocytes. The mutation identified in the cytb5r gene, Val253Met, has also been found in type I RHM in both the homozygous state (Dekker et al., 2001) and the heterozygous state (Grabowska et al., 2003; Percy et al., 2006b). Consequently, this patient probably had type I RHM associated with psychomotor retardation of another origin.
Correlation between the clinical phenotype and the genotype
During the last 15 years, molecular studies of the cytb5r gene have contributed to our understanding of the clinical heterogeneity of RHM types I and II. In addition, the development of a heterologous expression system has provided a powerful tool for studying the relationship between a given mutation and cytb5r function (Bewley et al., 2001, 2003; Davis and Barber, 2004; Percy et al., 2006a). More than 40 mutations have now been identified in the cytb5r gene, some of which are common to type I and type II RHM. Except for the first alternative exons 1M and 1S, belonging to different cytb5 transcripts (Leroux et al., 2001), the mutations are located in all the exons, starting with exon 2, and in the donor or acceptor splice sites of almost all the exons.
The genetic changes identified in type I RHM are missense mutations, except for two cases of heterozygous exon skipping (Dekker et al., 2001; Maran et al., 2005). They result in amino acid substitutions and decreased enzyme stability. In cells possessing the protein synthesizing machinery, the unstable enzyme is replaced in both soluble and membrane bound forms. By contrast, mature erythrocytes cannot synthetize proteins, and the lack of the soluble enzyme results in elevated methaemoglobin level, linked to continuous oxidative stress and nitrite exposure.
To date, 16 mutations have been identified in type II RHM. They lead to altered splicing, disruption of the active site of the enzyme or premature truncation of the protein. The enzyme is thus inactive in both its soluble and membrane-bound forms, in all cell types. (Shirabe et al., 1992; Vieira et al., 1995; Dekker et al., 2001). The three novel mutations described here are in keeping with this scheme.
In our patient 6, the novel homozygous G-A transition in the first nucleotide of intron 2 alters the consensus sequence of the donor splice site (IVS2, DS, G-A, +1) and causes in-frame exon 2 skipping. The lack of exon 2 suggests severe consequences at the protein level. The soluble form of the enzyme, translated from the ATG start site within exon 2, should be here completely absent, while the function of the membrane-bound form of the enzyme lacking the amino acids from Leu8 to Glu51 is probably seriously affected. Indeed, at birth this patient had marked generalized persistent cyanosis with a methaemoglobin level of 23%; no enzyme activity was detected in erythrocytes at age 1 year.
Patient 4 originated from Tunisia had a homozygous G-C transversion in intron 5 at the non-canonical position +8 of the donor splice site (IVS5, DS, G-C, +8) resulting in out-of-frame exon 5 skipping. The same mutation had previously been published (Vieira et al., 1995) in an Algerian patient with type II RHM.
The novel homozygous mutation identified in case 5 is the fourth homozygous missense mutation to be identified in type II RHM, the other three being Ser128Pro (Kobayashi et al., 1990), Arg241Gly (Toelle et al., 2004) and Cys204Arg (Maran et al., 2005). Analysis of the 3D rat cytb5r structure using Swiss-Pdb Wiever software (Guex and Peitsch, 1997) showed that the Leu131Pro mutation affects the enzyme's active site. The resulting general deficiency in cytb5 reductase activity is in keeping with the observed type II RHM phenotype.
Patient 2 had a novel heterozygous mutation (Leu131Pro) in exon 5 associated to a previously published mutation (Gly76Ser) located at the boundary of exon 3 with exon 4. The Gly76Ser mutation, found in the heterozygous state along with Val253Met in type I RHM, has been studied in a heterologous expression system. The Gly76Ser variant had only 11% of the catalytic efficiency of the wild-type enzyme (Percy et al., 2006b). In our case 2, the Gly76Ser mutation, in combination with Leu131Pro, led to a severe type II RHM phenotype. This provides an additional example of mutations in the cytb5 reductase gene that can be associated with both type I and type II RHM in compound heterozygotes. The resulting RHM phenotype is determined by the combination of the two allelic mutations and the final enzyme conformation. The Arg160Ter mutation has been found associated with Asp240Gly (Percy et al., 2005) in a patient with type I RHM, while its combination with Gln77Ter led to type II RHM (Aalfs et al., 2000). The nature of the mutations can also be important for the RHM phenotype. Mutation at codon 204, such as Cys204Tyr, was associated with type I RHM when present in the homozygous or heterozygous state (Wang et al., 2000; Wu et al., 2000), while Cys204Arg was associated with type II RHM (Vieira et al., 1995; Maran et al., 2005). Our patient 2 is the first compound heterozygote ever reported in type II RHM with two missense mutations (paternal Gly76Ser and maternal Leu131Pro).
Prenatal diagnosis
Type II RHM should be prevented through prenatal diagnosis in affected families, owing to its severity and 25% recurrence rate. Prenatal diagnosis can be based on cytb5r assay in foetal amniotic cells (Junien et al., 1981) or on the detection of previously identified mutations in trophoblast biopsies (Toelle et al., 2004; Leroux et al., 2005). Molecular prenatal diagnosis is reliable and can be done at an early stage of pregnancy, but only if the mutation in the cytb5r gene has been identified in each family at risk. Molecular prenatal diagnosis was successfully performed in the family of our case 3 (Leroux et al., 2005).
Pathophysiology and treatment
Although the genotype–phenotype correlation of RHM is becoming clearer, the pathophysiology of type II RHM is still completely unknown.
Type II RHM is characterized by a generalized cytb5 reductase deficiency (Leroux et al., 1975), including the membrane-bound microsomal isoform. This membrane-associated enzyme plays a fundamental role in the microsomal electron transport system and participates in fatty acid elongation and desaturation (Strittmatter et al., 1974), as well as fatty acid synthesis and drug metabolism (Hildebrandt and Estabrook, 1971). In contrast, its role in central nervous system development is unknown.
In 1966, a post-mortem study of a patient with type II RHM revealed a hypoplasic brain, moderately dilated lateral ventricles, a thin cerebral cortex with a below-normal number of neurons and retarded myelination. The basal ganglia were normal but marked degeneration of the cerebellar cortex, with loss of nearly all Purkinje cells, was observed (Jaffe, 1966).
The tissular fatty acid composition of tissues has been studied in only one autopsy case. In 1980, Hirono et al. analysed the lipid composition of myelin, white matter and grey matter in this patient. The cerebroside content of white matter was 48% of normal, with diminished unsaturation. The white-matter cholesterol and phospholipid content was 80% of normal (Hirono, 1980). In adipose tissue, the proportion of unsaturated fatty acids (linoleic, linolenic and arachidonic acid) was less than half the normal level, whereas there was an increase in saturated palmitic acid (Hirono, 1983). Low proportions of unsaturated fatty acids were observed in hepatic ethanolamine phosphoglycerides (Hirono, 1984).
These results suggest that reduced production of unsaturated fatty acids is involved in type II RHM, possibly leading to central dysmyelination and encephalopathy. However, these data were obtained by a single team in a single patient. Further studies are necessary to confirm the involvement of cytb5 reductase deficiency in the desaturation of fatty acids and their possible role in neurological development.
While cyanosis can be treated by methylene blue, riboflavin, or ascorbic acid, there is currently no way of preventing or treating the neurological deterioration associated with type II RHM.
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We are grateful to our patients and their families for their active cooperation. We thank Drs De Saint Martin, De Malefette, Saudubray, Devaux, Cuvelier, Tisseyre, Le Tual and Derosiere for helping to collect clinical data on our patients. We thank Dr Plessis for her help with the genetic analysis of patient 2. We are grateful to Drs Yilmaz, Toelle and Shotelersuk for providing additional clinical data on their patients and Constance Rouvière for helping in the manuscript preparation.
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  M J Percy and D Aslan NADH-cytochrome b5 reductase in a Turkish family with recessive congenital methaemoglobinaemia type I J. Clin. Pathol., October 1, 2008; 61(10): 1122 - 1123. [Abstract] [Full Text] [PDF]
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