Menkes disease is a rare X-linked recessive hereditary disorder first described by Menkes et al in 1962.1 The gene mutation results in clinical features including pili torti, unusual facies, mental/growth retardation and metabolic dysfunction. The pathogenic gene ATP7A was identified in 1993.2 It is located on chromosome X and encodes a transmembrane Cu2+ transporter. Here we reported the clinical manifestations and results of genetic study of a family with Menkes disease. In this family, a deletion mutation in ATP7A gene is responsible for the disease.
The proband (Figure 1) was the offspring of unconsanguineous parents. After a normal pregnancy, he was unremarkably delivered at 35 weeks gestational age. The infant appeared well until the age of 10 weeks, when the parents noticed his eyelid twitching 7 or 8 times per day; gaze directed upwards, transitory arm stiffness and body atony. Sometimes his right upper and lower limbs trembled for several seconds without signs of fever or changes of facial complexion. He was treated in the local hospital for two weeks and then admitted to our hospital. He had not laughed, pursued sounds or objects around him nor even raised his head since birth. On admission, he was conscious with a pale face and sagging cheeks. His pupils were equal in size and had brisk reaction to light, but movement of his eyeballs was absent. He had sparse, short, yellow and fragile pili torti. The muscle strength of his upper and lower extremities was graded V and muscle tone was normal. Tendon reflexes were present on all extremities and bilateral pathological reflexes were absent. Pain sensation was present symmetrically. No abnormal signs presented in his heart, lungs or abdomen. A physician recorded the infant's abnormal skin, sparse hair and suggested Menkes disease be considered. The ceruloplasmin level of 32.3 mg/L was one tenth the normal range (210.0-530.0 mg/L) and further historical data were gathered before the clinical diagnosis of Menkes disease was established.
The results of laboratory test (routine blood test, urine test) were unremarkable and X ray found periosteal reaction on left femur. Further tests (urine organic acid, blood lactate) were carried out to screen for possible metabolic disorders, but all results were within normal limits. The electroencephalogram (EEG) was abnormal with pseudoperiodic complexes of biphasic sharp waves and slow waves over the left hemisphere. Magnetic resonance imaging (MRI) showed cortical atrophy and an intensified signal in the basal ganglia, cerebellum and brainstem. Magnetic resonance angiography (MRA) showed contorted arteries, especially the middle cerebral arteries. After the in-hospital, treatment with Topamax was introduced. Serum ceruloplasmin of his father and mother was 247.0 mg/L and 377.0 mg/L, respectively and both of them had normal phenotype.
This study was approved by Ethics Committee of Beijing Children's Hospital and the parents of the proband gave their consent to it.
Genomic DNA isolated from peripheral blood of the 3 family members was subjected to polymerase chain reaction (PCR) amplification. ATP7A gene has 23 exons3 and 24 pairs of primers were designed to amplify all the encoding exons and their flanking intron sequences. Primers used to amplify segments spanning exon 9, 14 and 20 were as follows: exon 9 sense primer 5′-TGGATTAGCTAAAAGCCAAAGAA-3′, antisense primer 5′-TTGCCAAAATGAAAATACGTC-3′; exon 14 sense primer 5′-TGGAATCTCAGTATGTCCCAAT-3′, antisense primer 5′-CTCAGCTCATGGACCACAGA-3′; exon 20 sense primer 5′-GAACCCTGAGGAAAATTTTTGA-3′, antisense primer 5′-CCTCTCACCATACCAGTAGGC-3′.
PCR was performed in 50 μl mixture contained 1× PCR buffer, genomic DNA 50 ng, 10 pmol of each sense and antisense primer, 0.1 mmol/L dNTP, 20 mmol/L Mg2+ and 1.25 U Taq DNA polymerase. The thermal cycling condition was 35 cycles of 94°C for 40 seconds and 57°C for 45 seconds then completion at 72°C for 60 seconds. The PCR products were subjected to gel electrophoresis for purification and direct sequencing using ABI Prism 377 sequencer (Applied Biosystems, USA).
Direct sequencing of the PCR products found a deletion mutation of c.3045del T (numbering of nucleotide is from the A of the translation initiation codon ATG) in exon 14 of the proband (Figure 2 A and B). This nucleotide change results in frame shift and causes premature termination of the peptide chain only after 3 codons. The c.3045del T is a pathogenic mutation of ATP7A and is located in the cytoplasmic domain of ATP7A product. The deletion causes shortening of the peptide chain from 1500 amino acids to 1017. We have searched the Database (http://www.hgmd.cf.ac.uk/ac/gene.php?gene=ATP7A) to confirm that this deletion mutation is a novel pathogenic mutation of ATP7A. The proband's mother is heterozygous for this mutation (Figure 2 C and D). His father does not carry this mutation.
In addition, two single nucleotide polymorphisms (SNPs) were found. The c.2299 G>C SNP is located in exon 9 (GenBank SNP No. rs2227291), which resulted in the amino acid change of V767L (Figure 2E). His mother is heterozygous for this SNP (Figure 2F) and the proband inherited this SNP from his mother in the same allele with the deletion mutation of c.3045del T. The c.4048 G>A SNP is located in exon 20 (GenBank SNP number rs4826245), which resulted in the amino acid change of E1350K (Figure 2G). His mother is homozygous for this SNP (Figure 2H).
Hair detection under a light microscope in the proband
Hair of the proband was examined under a light microscope with a normal adult control (Figure 1 A and B). The proband's pale colored hair was thin, hollow and curled. This is the characteristic pili torti of Menkes disease.
ATP7A gene is located at Xq13.3 and has 23 exons. The normal gene product consists of 1500 amino acids and expresses widely in brain, kidney, lung and muscle but not liver tissues. ATP7A gene encodes a P-type ATPase transmembrane protein to transport Cu2+. Cu2+ is an essential microelement for body metabolism, serving as a cofactor to many enzymes and participating in numerous biochemical reactions.4 In Menkes disease, malfunction or absence of Cu2+ transportation ATPase caused by mutations in the gene results in insufficient absorption of Cu2+ from intestine to fulfill body's requirement. As a result, the biochemical changes in this disease is decreased Cu2+ concentration in plasma and brain.
The morbidity of Menkes disease is 1 case in about 50-250 thousands of live births. The development of the brain is very fast in the first year of life and needs more Cu2+ than available in Menkes disease. An unfortunate feature of Menkes disease is that the patient appears overall normal until 8-10 weeks postnatal when neurological symptoms appear in untreated infants and they will die in infant period or early childhood. Characteristic symptoms of this disease include:5-7 (1) hypopigmentation of pili torti; (2) unusual face appearances: buccal division downward sagging, big ears and higher palate; (3) mental and/or somatic retardation; (4) abnormal eyeball movement; (5) convulsion; (6) X ray: skeletal developmental disorder such as bony spur; (7) abnormal EEG, CT and/or MRI; (8) arteriography showing contorted arteries. The prominent manifestations in this proband were pili torti, unusual face appearance, epilepsy and mental/somatic retardation. In addition, abnormalities in MRI and MRA, low serum ceruloplasmin and abnormal EEG are all typical manifestations of Menkes disease.
As an X-linked recessive disease, these patients are always male, and female with normal phenotype are carriers. One third of cases are non-familial from new mutations. Much research about Menkes disease has been reported since the pathogenic gene was confirmed.8 About 70% of cases express little or no mRNA, indicating that most mutations are located in splice donor sites or promoter sites. Partial gene deletions are frequently mutations in ATP7A causing Menkes disease9 and exons 7-10, especially exon 8 is mutation hotspot.10 The c.3045del T in exon 14 in this study is pathogenic for the proband and his mother is a heterozygote carrying a mutant allele.
1. Menkes JH, Alter M, Steigleder GK, Weakley DR, Sung JH. A sex linked recessive disorder with retardation of growth, peculiar hair, and focal cerebral and cerebellar degeneration. Pediatrics 1962; 29: 764-779.
2. Vulpe C, Levinson B, Whitney S, Packman S, Gitschier J. Isolation of a candidate gene for Menkes disease
and evidence that it encodes a copper-transporting ATPase. Nat Genet 1993; 3: 7-13.
3. Tumer Z, Vural B, Tonnesen T, Chelly J, Monaco AP, Horn N. Characterization of the exon structure of the Menkes disease
gene using vectorette PCR. Genomics 1995; 26: 437-442.
4. Barnes N, Tsivkovskii R, Tsivkovskaia N, Lutsenko S. The copper-transporting ATPases, menkes and wilson disease proteins, have distinct roles in adult and developing cerebellum. J Biol Chem 2005; 280: 9640-9645.
5. Smith VV, Anderson G, Malone M, Sebire NJ. Light microscopic examination of scalp hair samples as an aid in the diagnosis of paediatric disorders: retrospective review of more than 300 cases from a single centre. J Clin Pathol 2005; 58: 1294-1298.
6. Kodama H, Murata Y, Kobayashi M. Clinical manifestations and treatment of Menkes disease
and its variants. Pediatr Int 1999; 41: 423-429.
7. Ozawa H, Nakamoto N, Kodama H. Clinical manifestations for early diagnosis of the patient with classical Menkes disease
. No To Hattatsu 2002; 34: 387-390.
8. Gu YH, Kodama H, Murata Y, Mochizuki D, Yanagawa Y, Ushijima H, et al. ATP7A
gene mutations in 16 patients with Menkes disease
and a patient with occipital horn syndrome. Am J Med Genet 2001; 99: 217-222.
9. Poulsen L, Horn N, Heilstrup H, Lund C, Tümer Z, Moller L B. X linked recessive Menkes's disease: identification of partial gene deletions in affected males. Clin Genet 2002; 62: 449-457.
10. Tumer Z, Lund C, Tolshave J, Vural B, Tonnesen T, Horn N. Identification of point mutations in 41 unrelated patients affected with Menkes's disease. Am J Hum Genet 1997; 60: 63-71.