Degenerative Disorders Primarily of Gray Matter

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Chapter 41 Degenerative Disorders Primarily of Gray Matter

This chapter groups together the seemingly disparate entities of Rett’s syndrome (RTT), Menkes’ disease, Alpers’ disease, and various forms of Batten’s disease. The first assumption is that they are neurodegenerative diseases that progress after a relatively normal period of early development. That is true for most, with the exception of Menkes’ disease, in which affected children are abnormal from birth. The second assumption is that cerebral and cerebellar cortex and deep gray structures are affected, with neuronal loss occurring in most and defective cellular function occurring in all. The third and most tenuous assumption is that the white matter or myelin is spared or only secondarily affected because of Wallerian degeneration. As more is learned about the cellular pathobiology of these disorders, it has become apparent that myelin and white matter are affected in a primary way and not as a result of neuronal loss or malfunction. Great strides have been made in defining the underlying genetics and molecular defects of these diseases. The next frontier is to understand the functions of the identified proteins and to devise intelligent, effective, and targeted therapies for these devastating disorders.

Rett’s Syndrome

Rett’s syndrome is an X-linked disease that primarily affects females. It is the second leading cause of mental retardation in females, with an incidence of 1 case per 10,000–22,000 females [Hagberg et al., 1985; Kozinetz et al., 1993]. All ethnicities are equally affected. The hallmarks of this syndrome are a period of normal development followed by regression of speech and development of stereotypical hand gestures. The genes that cause this syndrome are MECP2, which maps to the Xq28 locus, CDKL5 (cyclin-dependent kinase-like 5) gene (previously known as STK9) located in Xp22 [Scala et al., 2005; Kalscheue et al., 2003; Evans et al., 2005], Netrin G1 gene, located on chromosome 1 [Borg et al., 2005], and FOXG1 gene, located in 14q12 [Ariani et al., 2008]. The MECP2 protein is thought to be necessary for the maintenance of neurons during the later stages of development and after neuronal maturation is complete. The structure and function of the MECP2 protein continue to be the focus of intense scrutiny. Although this syndrome has many severe manifestations, approximately 50 percent of affected individuals live into the third decade of life. Despite the advances in knowledge about the cause and defects of Rett’s syndrome during the past decade, treatment remains primarily supportive.

History

Rett’s syndrome was first reported in 1966 by Dr. Andreas Rett [Rett, 1966]. This initial case report was followed in 1978 with a publication about Japanese female patients with a particular pattern of symptoms, including mental retardation and stereotypical hand-wringing [Ishikawa et al., 1978]. It was not until 1983, when Hagberg et al. [1983] published a case report of 35 female patients, that Rett’s syndrome gained international attention. Intense investigation of Rett’s syndrome over the past 20 years led to identification of the genetic defect in 1999 [Amir et al., 1999], 2005 [Borg et al., 2005; Scala et al., 2005; Kalscheue et al., 2003; Evans et al., 2005], and 2008 [Ariani et al., 2008].

Clinical Description

The diagnosis of Rett’s syndrome is based on a set of clinical observations accompanied by changes in various laboratory test results. The clinical criteria for classic Rett’s syndrome were established in the 1980s [Hagberg et al., 1985; Trevathan and Moser, 1988], and include loss of speech, seizures, mental retardation, and classic motor (specifically hand) movements. Criteria for atypical Rett’s syndrome were reported in 1993 [Hagberg and Gillberg, 1993]. More than 75 percent of patients have classic Rett’s syndrome, whereas 25 percent have atypical Rett’s syndrome variants [Hagberg, 2002].

In classic Rett’s syndrome (Table 41-1), the newborn initially appears developmentally normal. This period is followed by deceleration of head growth, loss of purposeful hand movements, development of stereotypic hand movements, and gait dyspraxia. These five criteria must be met for the diagnosis of classic Rett’s syndrome [Hagberg, 1995]. The chronology of these symptoms is critical for the diagnosis. Normal development is typical for the first 3–6 months of life. Deceleration in the rate of head growth occurs between 3 months and 4 years. Patients lose the ability to use their hands in a purposeful manner between 9 months and 2.5 years. Stereotypic hand movements appear between 1 and 3 years of age, and a dyspraxic gait manifests between 2 and 4 years of age if the patient is ambulatory.

Table 41-1 Obligatory Criteria for the Diagnosis of Rett’s Syndrome

Manifestation Age Comments
Period of normal neonatal development 0–6 mo Prenatal or perinatal period into the first 6 months of life, sometimes longer
Stagnation of rate of head circumference growth 3 mo–4 yr Normal at birth, then decelerates
Loss of purposeful hand skills 9 mo–2.5 yr Communicative dysfunction, social withdrawal, mental deficiency, loss of speech or babbling
Classic stereotypic hand movements 1–3 yr Hand-washing or hand-wringing and variants, including clapping and tapping, are common
Gait or posture dyspraxia
Absence of organomegaly, optic atrophy, retinal changes, or delayed intrauterine growth
2–4 yr Truncal “ataxia”

(Data from Hagberg B. Clinical manifestations and stages of Rett syndrome. Ment Retard Dev Disabil 2002;8:61–65, and from Percy AK. Clinical trials and treatment prospects. Ment Retard Dev Disabil 2002;8:106–111.)

Clinical manifestations of classic Rett’s syndrome are grouped into four stages: early onset (3–6 months of age), regression (1–4 years of age), stabilization, and late motor impairment (after the age of 3 years) [Jellinger, 2003]. The early-onset stage is characterized by developmental delay, deceleration of head growth [Neul and Zoghbi, 2004; Schultz et al., 1993], onset of autistic-like behavior, and classic hand-wringing [Jellinger, 2003]. Weight and height percentiles for age also decrease; the median values fall below the fifth percentile by age 7 years [Percy, 2002]. Although Rett’s syndrome can manifest earlier, clear signs of a central nervous system abnormality are usually not evident until 6 months of age [Akbarian, 2003]. The second stage of the syndrome is characterized by cognitive decline and regression [Hagberg, 2002]. Loss of speech and purposeful hand movements, emergence of stereotypic movements, seizures, breathing irregularities, other signs of autonomic instability, inattentive behavior, and hypotonia appear [Hagberg, 2002; Jellinger, 2003; Kerr et al., 2001]. The third stage consists of stabilization of symptoms; this stage differentiates Rett’s syndrome clinically from other pediatric neurodegenerative disorders. Sometimes there is a return of communication skills, with preservation of remaining ambulatory skills. This stage is also known as the pseudostationary stage because slow neuromotor regression continues [Hagberg, 1995]. In patients older than 3 years, bradykinesia and rigidity set in [Fitzgerald et al., 1990]. Stabilization can last years to decades. Late motor impairment begins when ambulation ceases; this signals the end of the stabilization or pseudostationary stage. This final stage of Rett’s syndrome is characterized by nonambulation and severe disability. The length of late motor impairment is variable and can last decades.

Common clinical manifestations of classic Rett’s syndrome include stereotypic hand movements, intense staring, breathing irregularities, bruxism, sleep disturbances and night laughter, scoliosis, lower limb spasticity and dystonia, seizures, swallowing dysfunction, constipation, gastroesophageal reflux, and small, bluish or red feet. The stereotypic hand movements occur while the individual is awake. These gestures are individualized, but they typically include continuous and repetitive twisting, wringing, knitting, and clapping motions. The intense eye communication may be compensatory for the loss of speech. This eye pointing has been observed in many individuals with Rett’s syndrome. The breathing irregularities are of two types: hyperventilation and breath-holding. Typically, they occur only while the individual is awake, but can also occur during sleep [d’Orsi et al., 2009a].

Periods of apnea can last 30–40 seconds, and they disrupt stretches of hyperventilation. Most individuals with Rett’s syndrome experience sleep disturbances. It has also been reported that up to 90 percent of young children interrupt sleep with night laughter.

Although it is not pathognomonic, many individuals with Rett’s syndrome have early growth retardation of their feet. The nails and skin demonstrate trophic changes, and the skin is cool to touch and discolored with a blue–red color. Autonomic dysregulation may produce some of these changes. The scoliosis in Rett’s syndrome is a double-curve deformation that develops during the first decade of life. Most commonly, the double curve has a longer upper curve and a shorter lower curve. The incidence of scoliosis increases with age, occurring in 8 percent of preschool patients and 80 percent of patients older than 16 years. Abnormalities of the lower extremities in Rett’s syndrome include asymmetric distal dystonia, mild spasticity, and feet that tend to orient in a flexed and supinated position.

Seizures are reported in 30–80 percent of individuals with Rett’s syndrome. The electroencephalogram (EEG) is always abnormal after the age of 2 years. Early-onset seizures were reported in patients with CDKL5 [Artuso et al., 2010] and Netrin G1 gene mutations [Borg et al., 2005]. Prevalence of drug-resistant epilepsy in RTT patients with MeCP2 mutations was 16 percent. No significant relationship was found between clinical severity of drug-resistant epilepsy and quantitative or qualitative EEG scores. In addition, no significant relationship was found between the drug-resistant epilepsy and the RTT genotype category, or a specific MECP2 genotype [Buoni et al., 2008]. Myoclonic status had been misdiagnosed as a movement disorder of gait impairment [Pelc and Dan, 2009; d’Orsi et al., 2009b]. Infrequent clinical manifestations of classic Rett’s syndrome include bloating, violent screaming, abnormal nociception, pain insensitivity [Devarakonda et al., 2009], hyperkalemic distal renal tubular acidosis [Assadi et al., 2006], and cardiac arrhythmias [Acampa and Guideri, 2006]. Bloating or air swallowing is generally mild, but 5–10 percent of individuals with Rett’s syndrome demonstrate severe bloating. Massive gastric dilatation, with total necrosis and perforation due to bloating, has been reported [Baldassarrea et al., 2006]. The gastrointestinal disturbances are attributed to changes within the autonomic nervous system. Screaming typically is encountered in teenage patients. The screaming may be associated with ill-defined pain, but no known pathology can be found. Occasionally, patients have abnormally prolonged responses or insensitivity to pain. Children and adults with Rett’s syndrome are at substantially increased risk of fracture. The lower limbs, especially the femur, are particularly susceptible and patients with the R270X and R168X mutations genotype are especially vulnerable. The presence of epilepsy also increased fracture risk [Downs et al., 2008]. Although a decreased life span is characteristic for this syndrome, many patients survive into adulthood [Sekul and Percy, 1992], with 50 percent remaining alive in their 30s [Akbarian, 2003].

The atypical Rett’s syndrome variants include a forme fruste variant, early seizure type variant, late childhood regression variant, preserved speech variant, and congenital Rett’s syndrome. Diagnosis of atypical Rett’s syndrome is complex. The criteria for variants of classic Rett’s syndrome, as outlined by Hagberg and Skjeldal [1994], are especially helpful (Box 41-1). Forme fruste is the most common atypical variant, accounting for about 80 percent of nonclassic Rett’s syndrome. There is a wide variability of function in forme fruste; it is a milder variant. It is seldom diagnosed before 8–10 years of age, and it is usually suspected in older individuals who are just beginning to develop symptoms of Rett’s syndrome. The early-onset seizure variant is linked to mutations in CDKL5 and Netrin G1 gene and manifests with early epilepsy onset between the first week and 5 months, hand stereotypies, severely impaired psychomotor development, and severe hypotonia [Artuso et al., 2010; Borg et al., 2005]. The late childhood regression form is characterized by a normal head circumference and by a more gradual and later onset (late childhood) of regression of language and motor skills. The preserved speech variant was first described in 1992 [Zappella, 1992]. It is characterized by the preservation of speech, but preserved head size and obesity are also common features [Zappella, 1992; Zappella et al., 2001]. There is some debate about whether the preserved speech variant is part of the autistic spectrum disorders, as well as the Rett’s syndrome spectrum [Percy et al., 1990]. Congenital Rett’s syndrome is rare. It differs from classic Rett’s syndrome because of the absence of the 3- to 6-month period of normal development [Hagberg and Skjeldal, 1994]. It is linked to mutations in FOXG1 gene [Ariani et al., 2008].

Box 41-1 Defining Variants of Rett’s Syndrome

Clinical Diagnostic Tests

Routine Laboratory Tests

Levels of lactate, pyruvate, and glutamate are increased in cerebrospinal fluid [Budden et al., 1990; Lappalainen et al., 1997a]. Cerebrospinal fluid testing yields decreased levels of β-phenylalanine, substance P, and gangliosides [Lekman et al., 1991; Matsuishi et al., 1997; Satoi et al., 2000]. There are increased levels of biogenic amines and creatine in the urine [Lekman et al., 1990]. Plasma levels of levels of β-endorphin and prolactin are decreased [Fanchetti et al., 1986]. The increased levels of lactate and pyruvate in the cerebrospinal fluid may result from hyperventilation [Budden et al., 1990], whereas the decreased levels of cerebrospinal fluid β-phenylalanine are caused by dysregulation of the dopaminergic pathways in patients with Rett’s syndrome [Satoi et al., 2000]. The levels of IgA and IgG antibodies to gluten and gliadin proteins found in grains and to casein found in milk are significantly increased in girls with Rett’s syndrome [Reichelt and Skjeldal, 2006].

Neurophysiologic Tests

The EEG is abnormal in Rett’s syndrome. Initial abnormalities are noticed in the rapid eye movement stage of sleep [Kudo et al., 2003]. During the stabilization stage of Rett’s syndrome, a slow spike-wave pattern resembling that in Lennox–Gastaut syndrome is observed [Glaze, 1987]. After 3 years of age, there is a decrease in alpha activity with a subsequent increase in theta activity [Bashina et al., 1994]. Evoked potential studies indicate intact visual and auditory peripheral pathways and dysfunction of central cortical pathways involved in processing and integration of sensory information in young girls with Rett’s syndrome. Somatosensory-evoked potentials can be characterized by “giant” responses, suggesting cortical hyperexcitability [Glaze, 2005]. There is a prolongation of somatosensory-evoked responses in older patients, suggesting involvement of the upper spinal cord and spinothalamic tracts [Bader et al., 1989]. Results of nerve conduction studies are consistent with an axonopathy and denervation indicative of lower motor dysfunction [Jellinger et al., 1990]. Impairment of the autonomic nervous system in Rett’s syndrome is suggested by an increased incidence of long QT intervals during electrocardiographic recordings and diminished heart rate variability [Glaze, 2005].

Neuroimaging Studies

Initial cranial computed tomographic (CT) scans and magnetic resonance imaging (MRI) are normal. As the patient ages and neurologic symptoms develop, generalized atrophy of the cerebral hemispheres and decreased volume of the caudate nucleus become apparent [Reiss et al., 1993]. Imaging of the basal ganglia reveals decreased volume of the caudate head [Dunn et al., 2002]. Commonly, there is a decrease in gray and white matter volumes, specifically within the frontal and temporal regions, and of the midbrain and cerebellum [Subramaniam et al., 1997]. Hypoperfusion of the prefrontal and temporoparietal regions is also reported [Lappalainen et al., 1997b]. Although imaging studies can help in making the diagnosis, there is no correlation between spectroscopic changes and clinical status [Gokcay et al., 2002]. One study reported an association between the level of hypoperfusion and early-onset Rett’s syndrome [Lappalainen et al., 1997b]. In more recent MR spectroscopy studies of RTT patients with MeCP2 mutations, NAA/Cr ratios decreased and myoinositol/Cr ratios increased with age. The mean glutamate and glutamine/Cr ratio was increased. The mean NAA/Cr ratio decreased in RTT patients with seizures and with increasing clinical severity score. Compared to patients with T158X, R255X, and R294X mutations, and C-terminal deletions, patients with the R168X mutation tended to have the greatest severity score and the lowest NAA/Cr ratio. Decreasing NAA/Cr and increasing myoinositol/Cr with age are suggestive of progressive axonal damage and astrocytosis in RTT, respectively, whereas increased glutamate and glutamine/Cr ratio may be secondary to increasing glutamate/glutamine cycling at the synaptic level [Horska et al., 2009].

Pathology

Brain

Gross findings include generalized atrophy of the frontal and temporal regions, the cerebellum, and especially the vermis. The corpus callosum decreases in size by as much as 30 percent [Oldfors et al., 1990; Reiss et al., 1993]. The brain is the only organ that is decreased in size compared with height [Armstrong et al., 1999]. Cerebellar volume is reported to remain relatively normal. The average weight of a brain from a patient with Rett’s syndrome is about 950 g, equivalent to the weight of a brain from a developmentally normal 1-year-old child [Armstrong, 2000]. More importantly, the brain weight does not continue to decrease with age, because Rett’s syndrome is not a progressive neurodevelopmental disorder in the classic sense.

There are many microscopic findings in brain tissue from Rett’s syndrome patients. Neuronal size is decreased, but cell density in the cerebral cortex, thalamus, basal ganglia, amygdala, and hippocampus is increased [Bauman et al., 1995]. The previous findings contrast with a report of an overall decrease in the number of neurons in the frontal cortex, the temporal cortex, and the cholinergic nucleus basalis of Meynert [Belichenko et al., 1994; Kitt and Wilcox, 1995]. Decreases in dendritic branching and dendritic number are found in the frontal, motor, and subicular areas [Armstrong, 1997; Armstrong et al., 1995; Cornford, 1994]. In addition to decreases in dendritic number and branches, shortening of the apical and basilar dendritic branches within these same regions of the brain has been reported [Armstrong et al., 1998]. Afferent neurons have decreased synaptic contacts. The striatum and internal pallidum exhibit hypochromia, whereas hypomyelination is observed in the substantia nigra pars compacta [Jellinger et al., 1988]. The neocortex has decreased expression of microtubule-associated protein 2, and disruption of the cytoskeleton within the neocortex is apparent [Kaufmann et al., 1995]. The caudate nucleus and putamen exhibit reduced levels of dopamine transporter protein [Wong et al., 1998]. Degenerative changes of the substantia nigra, caudate nucleus, and putamen have been demonstrated in neuropathological and neurochemical studies of RTT brains. Stereotypies and other movement disorders present in RTT could be interpreted as signs of dysfunction of the nigrostriatal-dopaminergic pathway [Kitt and Wilcox, 1995; Wenk, 1995].

There are conflicting results regarding the expression of nerve growth factor in Rett’s syndrome patients. One study documented no reduction in the cortical levels of nerve growth factor [Wenk and Hauss-Wgrzyniak, 1999], and others demonstrated large decreases in the expression of nerve growth factor and the neurotrophic tyrosine kinase type receptor, which binds to nerve growth factor with high affinity [Lipani et al., 2000]. Adults with Rett’s syndrome also have axonal degeneration, loss of motor neurons, loss of spinal ganglion cells, and decreased glutamate and gamma-aminobutyric acid type B (GABA B) receptor density [Oldfors et al., 1988; Blue et al., 1999]. Blue et al. [1999] reported age-specific alterations in amino acid neurotransmitter receptors within the basal ganglia of adults.

Electron microscopy of neurons depicts distinct abnormalities [Papadimitriou et al., 1988]. These changes include abnormal neurites that are filled with lysosomes and laminate bodies. Axonal swellings, large mitochondria, and membranous multilamellar bodies are seen. Although electron microscopy reveals intraneuronal inclusion bodies that contain lipofuscin-like material, there are no other characteristics of a lipid storage disorder.

Muscle

Type I and type II fiber atrophy is sometimes seen on muscle biopsy [Wakia et al., 1990]. Decreased cytochrome c oxidase and succinate cytochrome c reductase activities in muscle biopsies have also been reported [Coker and Melnyk, 1991]. The myocardium has no gross abnormalities. The atrioventricular node has an abnormal or immature rearrangement of muscle fibers within the conduction system [Armstrong, 1997]. Electron microscopy of muscle biopsy specimens reveals dumbbell-shaped mitochondria with foamy vacuoles [Ruch et al., 1989].

Genetics

Rett’s syndrome is an X-linked dominant disorder that has been mapped to the Xq28 locus [Ellison et al., 1992; Sirianni et al., 1998]. Although most cases of Rett’s syndrome are sporadic, genetic mapping was possible because familial inheritance does occur, and there is concordance in monozygotic twins [Jellinger, 2003]. Mutations within the methyl-CpG binding protein 2 gene (MECP2) cause 70–80 percent of reported cases of Rett’s syndrome in females [Auranen et al., 2001; Van den Veyver and Zoghbi, 2002]. This gene was identified in 1999 [Amir et al., 1999]. Most mutations in males lead to fetal demise. Although DNA mitochondrial mutations are found in some cases of Rett’s syndrome, there is no indication that mitochondrial DNA plays a part in the development of this syndrome [Nielson et al., 1993; Colantuoni et al., 2001]. Mutations within MECP2 have also been linked to childhood-onset schizophrenia, Angelman’s syndrome, and mild mental retardation [Watson et al., 2001].

The MECP2 protein has three known functional domains: an amino-terminal methyl-CpG binding domain [Lewis et al., 1992], a transcriptional repressor domain, and a carboxyl-terminal domain [Chandler et al., 1999]. The MECP2 protein binds to methylated CpG dinucleotides by the methyl-CpG binding domain [Nan et al., 1993]. The transcriptional repressor domain interacts with various co-repressor complexes and disrupts transcription [Nan et al., 1996, 1997]. The nuclear localization signal (NLS), consisting of amino acid residues 265–271, is contained within the transcriptional repressor domain. The biochemical function of the carboxyl-terminal region is unknown [Kriaucionis and Bird, 2003]. Seventy percent of the mutations within MECP2 are in eight hotspots affecting translation of the following amino acids: R106, R133, T158, R168, R255, R270, R294, and R306. Seven of these eight mutation hotspots affect arginine, which contains a CpG in its codon. These mutations may result from unrepaired deamination of 5-methylcytosine. This mechanism is thought to cause one-third of all point mutations that lead to human genetic disease [Cooper and Youssoufian, 1988]. Eighty percent of females with classic Rett’s syndrome have nonsense or frameshift mutations within the MECP2 gene [Van den Veyver and Zoghbi, 2002].

Genotype–Phenotype Correlation

Genotype–phenotype correlation has been attempted, but it is complicated by MECP2 gene X-chromosome inactivation. This inactivity allows a mother with a mutation of MECP2 to have a normal phenotype because of skewing of X-chromosome inactivation. If this mother has a daughter with the mutation of MECP2 but balanced X-chromosome inactivation, the daughter will have Rett’s syndrome [Amir et al., 2000]. Despite the problems with X-chromosome inactivation, many studies of genotype–phenotype correlations exist. It is reported that truncated mutations of MECP2 are more severe than missense mutations [Chae et al., 2002; Cheadle et al., 2000; Monros et al., 2001]. The location of the truncation generally does not affect the phenotype [Bienvenu et al., 2000; Giunti et al., 2001; Huppke et al., 2000; Satoi et al., 2000; Amir et al., 2000] reported that truncation mutations led to increased levels of homovanillic acid in cerebrospinal fluid and to increased respiratory problems. The same study reported an increased incidence of scoliosis in cases of missense mutations. Huppke et al. [2002] examined mutations from 123 patients with Rett’s syndrome. They determined that mutations affecting the NLS caused the most severe phenotype. They also reported that deletions within the carboxyl terminus caused the least severe clinical presentation. Truncations result in more severe disease than missense mutations, except when the truncation affects the carboxyl terminus. Single-amino acid mutations cause less severe phenotypes, presumably because they lead to mild impairment of protein function [Laccone et al., 2002].

Rett’s Syndrome Variants

Most of the mutations within MECP2 cause classic Rett’s syndrome. Twenty-nine cases of the preserved speech variant of Rett’s syndrome have mutations within the MECP2 gene [Conforti et al., 2003; Hoffbuhr et al., 2001; Huppke et al., 2000; Neul and Zoghbi, 2004; Nielsen et al., 2001; Obata et al., 2000; Weaving et al., 2003; Yamashita et al., 2001; Zappella et al., 2001]. These mutations were evenly distributed among the three known functional domains of the MECP2 gene: the methyl-CpG binding domain, the transcriptional repressor domain, and the carboxyl-terminal domain. Mutations resulting in less severe phenotypes (i.e., mutations within the carboxyl terminus or a truncation after the NLS motif) were common in patients with the preserved speech variant of Rett’s syndrome. Patients with the preserved speech variant who had mutations normally associated with severe disease had skewed X-chromosome inactivation (92:8 in one case), explaining the less severe phenotypes [Hoffbuhr et al., 2001; Zappella et al., 2001].

MECP2 Mutations in Males

Three outcomes occur in males: Rett’s syndrome, severe encephalopathy with neonatal fatality, and mild neuropsychiatric phenotypes [Geerdink et al., 2002; Villard et al., 2000; Wan et al., 1999; Zeev et al., 2002]. The classic form of Rett’s syndrome can occur in males [Jan et al., 1999]. Although similar mutations are seen in males and females with this disease, there is a report of a unique mutation within MECP2 that causes Rett’s syndrome in males [Ravn et al., 2003]. Male siblings of female Rett’s syndrome patients with identical MECP2 mutations develop a severe encephalopathy and die by 1–2 years of age. These mutations typically affect the methyl-CpG binding domain or the NLS portion of the MECP2 protein.

Rett’s syndrome is produced as a result of somatic mosaicism, meaning there is a mixed population of cells with the wild type of MECP2 and mutated MECP2 [Armstrong et al., 2001; Clayton-Smith et al., 2000; Topcu et al., 2002]. Males with Rett’s syndrome have a unique genetic composition. Klinefelter’s syndrome (46,XXY) allows phenotypic males to replicate the somatic mosaicism achieved by females and avoid neonatal fatality [Leonard et al., 2003; Schwartzman et al., 2001]. There are case reports of Rett’s syndrome occurring in a phenotypic male, in whom the SRY region of the Y chromosome that produces “maleness” is translocated on to an X chromosome, so that a phenotypic male is genotypically a female (46,XX) [Maiwald et al., 2002]. Mutations in the MECP2 gene also occur in males with mental retardation and no other symptoms of Rett’s syndrome. These mutations generally affect the carboxyl terminus of the methyl-CpG binding domain region of the MECP2 protein [Couvert et al., 2001; Kleefstra et al., 2002; Yntema et al., 2002a]. Whether these mutations contribute to the phenotypes observed, or are normal polymorphisms, is being explored [Laccone et al., 2002; Yntema et al., 2002b].

Cell Biology

Because Rett’s syndrome is caused primarily by mutations within the MECP2 gene, it is necessary to understand the function and interactions of the MECP2 protein. The MECP2 gene encodes a protein with three known functional domains: the methyl-CpG binding domain, the transcriptional repressor domain, and the carboxyl terminus. Human MECP2 has 48 amino acids (about 80 kDa) [Akbarian, 2003]. The methyl-CpG binding domain contains 85 amino acids [Nan et al., 1993] and binds to single- and double-methylated CpG dinucleotides [Bird and Wolfe, 1999; Lewis et al., 1992]. There is a correlation between the capability of the methyl-CpG binding domain to bind to pericentromeric heterochromatic regions of DNA and the ability of the protein to repress methylated promoters [Kudo et al., 2003]. Residues R111, R133C, and R134C within the methyl-CpG binding domain are thought to come into contact with methylated cystines. Mutations affecting R111 cause the MECP2 protein to lose its binding ability to heterochromatic DNA and its capability to repress transcription. Mutations resulting in R133C or R134C affect neither of the aforementioned protein properties. The MECP2 protein associates with chromatin remodeling complexes and aids in the regulation of the structure and function of chromatin.

The transcriptional repressor domain is 100 base pairs (bp) long, and it interacts with various co-repressor complexes [Nan et al., 1997]. One of these complexes is the Sin3A co-repressor complex. This complex contains histone deacetylases 1 and 2, which remove acetyl groups from histones and create a compressed form of chromatin that then inhibits or represses gene expression [Nan et al., 1998]. The action of the transcriptional repressor domain is partially reversed by trichostatin A, a histone deacetylase inhibitor. This finding suggests that repression by means of the transcriptional repressor domain is caused by histone deacetylation. MECP2 recruits these histone deacetylases and other chromatin remodeling complexes to methylated CpG dinucleotides. This leads to chromatin condensation that interferes with the binding of transcription complexes [Akbarian, 2003]. The transcriptional repressor domain has also been seen to bind to TFIIB (also designated GTF2B), SKI (a proto-oncogene), DNMT1 (which codes for a DNA methyltransferase), and SUV39H1 (which codes for a histone methyltransferase), although the importance of these interactions is unknown [Fuks et al., 2003; Kaludov and Wolffe, 2000; Kimura and Shiota, 2003]. Repression can be mediated in other ways, because the MECP2 protein binds to general transcription factors and interferes with the binding of transcription complexes. The transcriptional repressor domain is able to repress transcription when bound as far as 2000 bp from the transcription initiation site. The carboxyl terminus is thought to be involved in the binding of MECP2 to naked and nucleosomal DNA. Specifically, the carboxyl-terminal region of the MECP2 protein binds to DNA that is coiled around histone octamers [Chandler et al., 1999].

The MECP2 protein is mostly located within the nucleus of cells, and a small portion is seen within the perikarya [Kaufmann et al., 1995]. Although MECP2 binds throughout chromosomes, binding is most dense around pericentromeric heterochromatic regions of DNA. Forty percent of methyl-CpGs (i.e., binding sites for the methyl-CpG binding domain) are found within pericentromeric heterochromatic DNA. The immunoreactivity of MECP2 is increased around centromeric and perinucleolar heterochromatin. MECP2 does not associate with ribosomal DNA, despite its many methylations. MECP2 distribution is regulated by unknown factors and does not simply distribute to where methylated CpGs are found. The MECP2 protein is thought to play a role in the maintenance of neuronal nuclei in the later stages of development and within the mature brain [Akbarian, 2003]. It is hypothesized that MECP2 makes chromatin more stable and less accessible to transcription factors by anchoring chromatin fibers into the nuclear matrix.

There are reduced levels of dopamine, serotonin, and their metabolites, homovanillic acid and 5-hydroxy-indoleacetic acid, in Rett’s syndrome [Lekman et al., 1990]. Some researchers have noticed a decreased density of postsynaptic D2 receptors in older patients with Rett’s syndrome [Dunn, 2001], whereas others describe increased specific binding at D2 receptors. The latter finding implies that the decreased levels of dopamine are causing increased levels or density of postsynaptic receptors [Chiron et al., 1993; Dunn et al., 2002]. The D1 receptor density is unchanged [Wenk, 1995]. Jellinger et al. [1990] proposed that the different densities of postsynaptic receptors within the dopaminergic pathways might be age-specific. Increased choline concentrations and decreased choline acetyltransferase levels are thought to result from problems within the cholinergic system in the forebrain [Gokcay et al., 2002]. The frontal cortex and striatum have decreased levels of ferritin [Sofic et al., 1987]. Decreased levels of binding protein for the benzodiazepine receptor in the frontotemporal, parietal, and occipital regions of the brain also have been reported [Yamashita et al., 1998]. Significantly increased oxidative stress markers (intraerythrocyte non-protein-bound iron, plasma non-protein-bound iron, free F2-isoprostanes, esterified F2-isoprostanes, total F2-isoprostanes, and protein carbonyl concentrations) were evident in Rett’s syndrome subjects and associated with reduced arterial oxygen levels compared to controls. Biochemical evidence of oxidative stress was related to clinical phenotype severity and lower peripheral and arterial oxygen levels. Pulmonary image mismatch was found in the majority of the Rett’s syndrome population. These data identify hypoxia-induced oxidative stress as a key factor in the pathogenesis of classic Rett’s syndrome [De Felice et al., 2009].

Animal Models

In a model of MECP2-null mice, males and females were affected. Homozygous female mice and heterozygous male mice were developmentally normal for the first several weeks of life, but they died soon after neurologic symptoms appeared [Chen et al., 2001; Guy et al., 2001]. This model replicates the genetic component of Rett’s syndrome, but it was difficult to study because of the rapid deterioration and early death of the animals after symptoms appeared. Shahbazian et al. [2002] developed a mouse model that has a truncation mutation within the MECP2 gene with a less severe phenotype than the MECP2-null mice. The truncated protein mimics a commonly observed human mutation, and it is partially functional, containing the methyl-CpG binding domain and transcriptional repressor domain. This group observed that the mice appeared developmentally normal up to 6 weeks after birth. After this period of normal development, the mice developed neurologic symptoms, including tremors, motor impairment, hypoactivity, seizures, kyphosis, and the classic forearm movements associated with human Rett’s syndrome [Shahbazian et al., 2002b]. Random X-inactivation causes a variety of phenotypes due to a single genotype in females, which makes analysis of the mouse model difficult [Young and Zoghbi, 2004]. One solution is to use male mice, because they are not subject to random X-inactivation.

Luikenhuis et al. [2004] overexpressed MECP2 in postmitotic neurons of homozygous MECP2-null mice. These mice did not display any neurologic symptoms, and they were developmentally equivalent to the control population. In normal mice, MECP2-encoded RNA is not expressed until about 10 days after conception, and it reaches adult levels by 16 days after conception. It has been postulated that the defect in Rett’s syndrome involves neuronal maintenance and maturation, and therefore affects developmental stability. Ballas et al. found that the loss of MeCP2 occurs not only in neurons but also in glial cells of Rett brains, and that mutant astrocytes from a Rett mouse model, and their conditioned medium, failed to support normal dendritic morphology of either wild-type or mutant hippocampal neurons [Ballas et al., 2009]. This suggests that astrocytes in the Rett brain carrying MeCP2 mutations have a non-cell autonomous effect on neuronal properties, probably as a result of aberrant secretion of soluble factor(s).

Pathogenesis

The timeline of MECP2 expression suggests that it is needed in the later phase of cortical development in neonates and after maturation in adults. MECP2 is expressed in normal fetal brains until 20 weeks’ gestation, after which it disappears from the cerebellum. It disappears from the brainstem after the perinatal period. MECP2 does not reappear in the brain until after adolescence. Lack of MECP2 during any of these developmental periods may lead to synaptic and neuronal dysfunction of the catecholaminergic neurons in patients with Rett’s syndrome [Itoh and Takashima, 2002]. Mice with MECP2 overexpressed in postmitotic neurons are rescued from the Rett’s syndrome phenotype. A decrease in MECP2 in postmitotic neurons during the later stages of development is sufficient to cause Rett’s syndrome [Chen et al., 2001], but MECP2 deficiency in neuronal precursors is probably not a major contributor to the pathogenesis in Rett’s syndrome.

MECP2 causes transcriptional repression, and loss of function of this protein may cause an imbalance between transcription and gene silencing, leading to dysregulated gene expression and pathologic changes. Some groups have found no evidence for MECP2 transcriptional repression in neurons or glia. Gene expression studies found an increase in glial transcription, in contrast to the predicted decrease. Levels of presynaptic proteins, however, were decreased. MECP2 may be causing perturbations within the presynaptic signal transduction pathway [Colantuoni et al., 2001]. The critics of this theory point out that these studies were performed on postmortem brains, suggesting that relevant time points in gene expression may have been missed. Additional evidence argues against MECP2 deficiency unsilencing transcription and causing Rett’s syndrome. Affymetrix GeneChip analysis of MECP2-deficient human fibroblasts did not demonstrate any large-scale dysregulation of gene expression. There were only small differences in gene expression in presymptomatic, early symptomatic, and late symptomatic MECP2-deficient mouse brains [Tudor et al., 2002].

If MECP2 does not act as a transcriptional repressor, it may play a maintenance role during development. MECP2-encoded mRNA is undetectable in the mouse forebrain during mid-gestation [Coy et al., 1999]. Immunohistochemical studies in nonhuman primates and mice demonstrate MECP2 expression in neuronal nuclei correlates with neuronal maturity, and levels of expression are highest in the adult cerebral cortex [Akbarian et al., 2001]. In human cerebral cortex, MECP2 is seen only in Cajal–Retzius cells, the earliest maturing neurons [Marin-Padilla, 1998], at 14 weeks’ gestation. At 26 weeks’ gestation, MECP2-immunoreactive neurons are seen in the deeper, more differentiated cortical layers. MECP2 neuronal immunoreactivity increases with age. Only about 10 percent of cells are immunoreactive during the third trimester of pregnancy, but approximately 80 percent of neurons demonstrate immunoreactivity for MECP2 at 10 years of age [Shahbazian et al., 2002a]. These findings are also seen in rodents and nonhuman primates. The observation that MECP2 expression is decreased in immature neurons and elevated in mature neurons suggests a role for MECP2 in neuronal maintenance. MECP2 expression studies show high levels of the protein in mature neurons but not in glia or astrocytes.

MECP2 may also play a role in cell division. Removing pericentromeric heterochromatin or disrupting heterochromatin silencing by inhibition of histone deacetylases in Drosophila and yeast reduced chromosome transmission during cell division [Henikoff, 2001]. Because MECP2 associates with pericentromeric heterochromatin, it may influence cell division.

Treatment

Rett’s syndrome has no cure. The treatments available have been empirically derived and are designed to combat specific symptoms. Antiepileptic agents include carbamazepine, valproic acid, and lamotrigine. The use of l-DOPA and dopamine agonists to increase motor ability in Rett’s syndrome patients is controversial [Zappella, 1990]. A study of the treatment of Rett’s syndrome with folate and betaine did not find any objective evidence of improvement [Glaze et al., 2009]. Zinc sulfate, lithium, and antidepressants have been demonstrated to increase central brain-derived neurotropic factor (BDNF) levels or signaling in human as well as animal studies. Thus, it is proposed that these agents could have therapeutic potential for RTT subjects [Tsai, 2006]. The breathing irregularities associated with Rett’s syndrome can be treated with naltrexone, an opiate antagonist. Naltrexone (1–3 mg/kg/day) can reduce disorganized breathing and increases oxygen saturation levels [Percy, 2002]. Use of high-fat, high-calorie diets is recommended in the late stages of the disease, and it has been suggested that individuals with Rett’s syndrome require a higher protein intake [Motil et al., 1999]. Feeding by means of a gastrostomy tube is sometimes indicated [Jellinger, 2003]. Constipation in Rett’s syndrome has been managed with high-fiber foods, enemas, mineral oils, milk of magnesia, and polyethylene glycol or MiraLax, with varying degrees of success. Orthopedists and physical therapists routinely see individuals with Rett’s syndrome. The goals are to improve balance, enhance flexibility, and strengthen atrophying muscles. Bracing for scoliosis is necessary when a 25° curvature exists, and surgery is advised when the curvature exceeds 40°. Speech and occupational therapy are occasionally used to improve communication.

Small-scale clinical trials using l-carnitine and the ketogenic diet have been completed. l-Carnitine has been reported to improve the respiratory features of Rett’s syndrome [Ellaway et al., 2001], and the ketogenic diet may reduce seizure frequency during the first 3 months, but no long-term clinical trials have been reported. A comprehensive, life-span approach to the management of scoliosis in Rett’s syndrome is recommended that takes into account factors such as physical activity, posture, and nutritional and bone health needs. Surgery should be considered when the Cobb angle is approximately 40–50° and must be supported by specialist management of anesthesia, pain control, seizures, and early mobilization. Evidence- and consensus-based guidelines were successfully created and have the potential to improve care of a complex comorbidity in a rare condition and stimulate research to improve the current limited [Downs et al., 2009].

Menkes’ Disease

Menkes’ disease is an X-linked disorder caused by mutations in the ATP7A gene. The protein it encodes is necessary for absorption of copper from the intestinal epithelium and for transport of copper across the blood–brain barrier. The reported incidence is 1 case per 100,000 to 300,000 persons. Menkes’ disease has three variants: classic disease, mild disease, and occipital horn syndrome. In classic disease, a 2- to 3-month period of normal development is followed by severe neurologic regression characterized by seizures, hypotonia, visual impairment, and failure to thrive. Death ensues by 4 years of age. Kinky, coarse, and lightly pigmented hair is pathognomonic for this disease, although many other phenotypic features may be observed. Connective tissue disorders are common in all three variants because of dysfunction of the cupric enzyme lysyl oxidase. Two biochemical markers, decreased serum copper and ceruloplasmin, in conjunction with clinical manifestations, aid in the diagnosis. The only available treatment is replacement therapy using copper histidine. Early intervention is essential for the therapy to be neuroprotective.

History

The story of the discovery of Menkes’ disease begins in the 1930s, when veterinary physicians in Australia noticed the importance of copper for the normal development of sheep [Bennetts and Chapman, 1937]. They observed that mothers that grazed in copper-deficient pastures had offspring with cerebral demyelination, ataxia, and porencephaly. They concluded that copper deficiency in sheep was associated with ataxia and a demyelinating disease. The disease was described in 1962 by John Menkes [Menkes et al., 1962]. He described five males of English–Irish heritage who had “peculiar” hair, failure to thrive, and a neurodegenerative disorder. The syndrome was initially called Menkes’ kinky hair syndrome in reference to the unique appearance of the hair of these patients. The basis for Menkes’ disease was not known until the association was made with the illness in sheep [Danks et al., 1972, 1973]. The distinctive hair found in the copper-deficient sheep and in patients provided the necessary link. Serum testing revealed that patients who had this distinctive hair also had decreased serum copper and ceruloplasmin levels. Danks et al. [1972, 1973] then concluded that Menkes’ disease was a human example of a neurodevelopmental disorder caused by copper deficiency. In 1993, three groups used positional cloning to discover the ATP7A gene [Chelly et al., 1993; Mercer et al., 1993].

Clinical Description

Menkes’ disease typically occurs in males. Its occipital horn syndrome variant is also known as X-linked cutis laxa [Danks, 1995]. In classic Menkes’ disease, there is a period of normal development that typically lasts for 2–3 months [Kaler, 1994]. Developmental regression follows, with seizures, hypotonia, and failure to thrive [Kaler, 1998]. Patients exhibit severe mental retardation with symptoms of neurodegeneration [Mercer, 1998]. Most individuals with classic Menkes’ disease die between the ages of 7 months and 4 years [Bankier, 1995]. Occipital horn syndrome is characterized by a less severe genetic mutation that results in a connective tissue disorder.

The characteristic findings in classic Menkes’ disease are related to the patient’s hair. Commonly described as steel-woolish, the hair on the scalp and eyebrows is short, sparse, coarse, and twisted. The amount of hair is decreased, and it is generally shorter on the sides of the head. The color is often light, with white, silver, and gray being common. Light microscopy of hair reveals three characteristic findings [Kaler, 1998; Moore and Howell, 1985]:

Hypopigmentation is common but not the rule, and is caused by a deficiency of catechol oxidase. Patients typically have large jowls, sagging cheeks, large ears, and a high-arched palate. The skin appears loose at the nape of the neck, in the axillae, and on the trunk. Delayed tooth eruption and pectus excavatum are common, and the incidence of umbilical hernias is increased. Nephrocalcinosis and chronic renal failure have also been reported [Balestracci et al., 2009].

Classic Menkes’ disease has several distinctive neurologic findings. These patients exhibit truncal hypotonia with poor head control, hyperactive deep tendon reflexes, impaired visual fixation or tracking, and cortical adducted thumbs. There is increased appendicular tone, and asymmetric growth failure that appears shortly after neurodegeneration begins. EEGs are moderately or severely abnormal, and hypsarrhythmia occurs frequently [Venta-Sobero et al., 2004]. Ophthalmologic findings common in Menkes’ disease include myopia, strabismus, and problems with visual fixation and tracking.

Connective tissue disorders are common in the three variants. Pelvic ultrasound and cystograms reveal diverticula of the urinary bladder [Daly and Rabinovitch, 1981; Harke et al., 1977]. Associated vascular disorders include lumbar and iliac artery aneurysms [Adaletli et al., 2005]. Skull and skeletal radiographs are notable for Wormian bones in the skull, metaphyseal spurring of the long bones, and anterior flaring or multiple fractures of the ribs [Adams et al., 1974; Capesius et al., 1977; Koslowski and McCrossin, 1979; Stanley et al., 1976]. Congenital skull fracture, related to global osteopenia, subdural hematoma, intrauterine growth delay, and lethal outcome, is reported in neonatal Menkes’ disease [Veit-Sauca et al., 2009]. MRI demonstrates white matter changes and impaired myelination, cerebral blood vessel tortuosity, ventriculomegaly, and diffuse cerebral atrophy [Faerber et al., 1989; Johnsen et al., 1991]. White matter lesions localized in the deep periventricular white matter in the absence of diffuse cortical atrophy [Lee et al., 2007], and transient temporal lobe lesions related to vasogenic and cytotoxic edema [Ito et al., 2008] were reported.

Patients with occipital horn syndrome have hyperelastic skin and may develop other connective tissue disorders, including aortic aneurysms, hernias, bladder diverticula, and skeletal abnormalities, which most likely result from lysyl oxidase deficiency. The characteristic occipital horns are symmetric exostoses protruding from the occipital bone and pointing down. These may be present around 1–2 years of age, but are usually detected only around 5–10 years of age and continue to grow up to early adulthood [Tümer and Moller, 2009]. These individuals also have mild mental retardation and autonomic dysfunction that manifests as syncope, hypothermia, and diarrhea [Byers et al., 1980]. The severity of mild Menkes’ disease falls between that in the classic form and the much milder occipital horn syndrome [Procopis et al., 1981]. It is important for patients with milder variations of Menkes’ disease to have frequent vision examinations because ophthalmologic problems can greatly impair functioning of affected individuals.

Clinical and Biochemical Diagnoses

The clinical diagnosis of Menkes’ disease is supported by specific laboratory findings [Poulsen et al., 2002]. Early diagnosis of affected newborns is necessary for the institution of appropriate therapy and survival. A high index of suspicion based on clinical grounds is essential because supportive laboratory findings may be problematic in the first few months of life.

Initially, few or no neurologic manifestations occur [Gunn et al., 1984]. Kinky hair with light hair pigmentation is most suggestive of Menkes’ disease. The pili torti pathognomonic for Menkes’ disease are usually seen only in older patients. The diagnosis should be considered for a neonate born after premature labor and delivery with large cephalohematomas, unexplained hypothermia or hypoglycemia, jaundice requiring phototherapy, pectus excavatum, and inguinal or umbilical hernias [Kaler, 1998].

The classic biochemical markers of Menkes’ disease are low serum levels of copper and ceruloplasmin. Decreased intestinal copper absorption leads to low levels of copper in the plasma, liver, and brain. In contrast, copper stores are increased in the duodenum, kidney, spleen, pancreas, and skeletal muscle [Heydorn et al., 1975; Horn, 1984; Williams and Atkins, 1981].

Early laboratory diagnosis of neonates is complicated by the fact that copper and ceruloplasmin concentrations are normally low in healthy newborns, and these values can overlap with those typically found for older patients with Menkes’ disease [Lockitch et al., 1986, 1988]. Copper egress assay in cultured fibroblasts is the definitive diagnostic study at this age, but the test is lengthy and requires several weeks of cell culture. Rapid diagnosis of Menkes’ disease can be achieved by measurement of plasma catecholamines or polymerase chain reaction (PCR) detection of known deletions or point mutations in the ATP7A gene. The copper deficiency in Menkes’ disease affects the function of many enzymes requiring copper. Dopamine mono-oxygenase is one of the cuproenzymes that is dysfunctional in Menkes’ disease, resulting in abnormal plasma catechol concentrations in newborns and fetuses [Kaler et al., 1993a, 1993b, 1993c]. The plasma catecholamine profile is considered to be the most rapid and reliable way to diagnose Menkes’ disease during the neonatal period. Patients with Menkes’ disease have high plasma dopamine and low norepinephrine levels. Considered alone, neither dopamine nor norepinephrine levels have perfect sensitivity, whereas the ratio of dopamine to norepinephrine is high in all affected patients. Analogously, levels of the dopamine metabolite, dihydroxyphenylacetic acid, and the norepinephrine metabolite, dihydroxyphenylglycol, were imperfectly sensitive, whereas the dihydroxyphenylacetic acid to dihydroxyphenylglycol ratio is high in all patients. Plasma dihydroxyphenylalanine and the ratio of epinephrine to norepinephrine levels are high in affected neonates [Goldstein et al., 2009]. Increased urine ratios of homovanillic acid/vanillylmandelic acid (HVA/VMA) have been proposed as a screening tool for Menkes’ disease also [Matsuo et al., 2005]. PCR methods are helpful in the diagnosis of partial deletions and point mutations when they are already identified for a specific family. The DNA-based technologies used for screening ATP7A for point mutations are chemical cleavage mismatch detection [Das et al., 1994], single-strand conformational polymorphism analysis [Tumer et al., 1997], and dideoxy fingerprinting [Moller et al., 2000]. PCR-based methods for screening ATP7A for large partial deletions include multiplex PCR, genomic PCR, and reverse transcriptase PCR [Poulsen et al., 2002]. These PCR methods are useful in neonatal and prenatal diagnosis, and they are helpful for carrier screening.

Prenatal diagnosis and identification of carrier status are important for families that are at risk for Menkes’ disease. Assays looking for increased levels of copper in cultured fibroblasts also can be used for prenatal diagnosis and for testing of potential carriers [Goka et al., 1976; Poulsen et al., 2002; Tümer et al., 2003], but random X-inactivation renders carrier testing using this technique uninformative when negative. The only definitive tests that can exclude this disease are DNA-based assays.

An important component of all variants of Menkes’ disease is connective tissue involvement. Deoxypyridinoline is a cross-linking residue of type I collagen and is a good marker for lysyl oxidase activity, the cuproenzyme deficiency that is responsible for the connective tissue disorders observed in these patients. Deoxypyridinoline has been proposed as a marker for the presence of connective tissue disorders associated with Menkes’ disease [Kodama et al., 2003].

Genetics

Menkes’ disease is an X-linked disease, and one-third of cases are thought to represent new mutations [Haldane, 1935]. The gene responsible for Menkes’ disease, ATP7A, was discovered in 1993 [Chelly et al., 1993; Mercer et al., 1993; Vulpe et al., 1993]. The gene encodes the copper transporting ATPase known as ATP7A [Mercer, 1998]. ATP7A is part of a highly conserved family of cation-transporting ATPase proteins [Odermatt et al., 1993; Pederson and Carafoli, 1987]. This protein family includes the protein WD that is defective in Wilson’s disease. The ATP7A gene (formerly known as MNK) is located at Xq13.2–q13.3 [Tümer et al., 2003]. It has 23 exons and is about 140 kb long [Dierick et al., 1995]. ATP7A is 8.5 kDa. About 357 different mutations have been identified and are distributed as follows: nonsense mutations 18 percent, splice-site mutations 16 percent, missense mutations 17 percent, partial insertions 4 percent, minus mutations 5 percent, partial deletions 17 percent, chromosomal mutations 1 percent, and small indels 22 percent [Møller et al., 2009].

Occipital horn syndrome results from point mutations (75 percent), chromosomal rearrangements (about 1 percent), and large or partial deletions (about 15 percent) in the ATP7A gene [Liu et al., 1999; Tümer et al., 1999]. Severe classic Menkes’ disease RNA contains low levels of active ATP7A mRNA, whereas mild variants of Menkes’ disease have higher levels of active ATP7A mRNA. Occipital horn syndrome has 20–35 percent residual ATP7A mRNA in cultured cells, whereas classic Menkes’ disease has lower levels of mRNA in the cells [Kaler, 1994, 1998]. Decreased mRNA levels result from premature stop codons, missense mutations, frame shifts, and other deletions or mutations that affect RNA splicing [Das et al., 1995]. Patients with the identical deletion or mutation may have different clinical outcomes [Tümer et al., 2003]. This finding suggests that other modifying genes or proteins must play a role in the pathogenesis of this disease. Alternatively, other pathways for copper transport may exist at the cellular level.

Alternate splice products of ATP7A exist. One of the splice variants lacks exon 10, but the product is in-frame. This exon encodes transmembrane domains 3 and 4. This protein may act as a copper transporter, but it may not be able to function as a copper-transporting ATPase. It is also possible that alternate exons exist within intronic sequences.

Biochemistry

The genetic mutation of the ATP7A gene leads to problems with copper transport within the body. Normally, ATP7A allows efflux of copper from gut epithelium into the portal circulation, transport of copper across the blood–brain barrier, and transfer of copper that is reabsorbed by the kidney back into circulation. ATP7A protein deficiency results in an inability of the body to absorb copper from the gastrointestinal tract in amounts required to satisfy nutritional needs, as well as in impaired use and handling of the copper that is absorbed from the intestine. Copper is mobilized from the cytoplasm of cells so that it can be incorporated into secretory pathways [Tümer et al., 2003]. The mutation of a copper-transporting ATPase causes impaired cellular copper efflux, leading to increased intracellular copper concentrations. Patients with Menkes’ disease have high concentrations of copper in gut epithelial cells, and they absorb little copper from their diet. Copper accumulates in kidney tubules. At high levels, copper causes lipid peroxidation, protein cleavage, enzyme inhibition, and DNA damage. Normally, basal intracellular stores are maintained at low levels [Rae et al., 1999; Voskoboinik and Camakaris, 2002]. The disease is caused by a decreased amount of the ATP7A protein, or it can result from alterations to the protein that impair its ability to transport copper.

ATP7A has 6–8 transmembrane domains, and it transports Cu2+ ions using energy from adenosine triphosphate (ATP) hydrolysis [Vulpe et al., 1993]. The protein has several known motifs or domains within it, such as the ATP binding domain. The phosphorylation motif (DKTG) contains an aspartic acid that becomes phosphorylated during the protein’s cycle (present in all P-type ATPases). The cation transduction motif (CPC) features a conserved proline, which plays a role in the conformation changes that occur with cation transport [Silver et al., 1989]. There are six metal binding sites (MBSs) in the amino-terminal region of ATP7A. The consensus sequence of the MBS is GMxCxxC. Although human ATP7A has six different forms of MBS, microbial cells show that only one or two are necessary for a functional protein [Odermatt et al., 1993; Solioz et al., 1994]. Human ATP7A does not need an MBS, demonstrated by the fact that mutations in all six MBSs do not stop ATP7A from functioning [Forbes et al., 1999; Payne and Gitlin, 1998; Tsivkovskii et al., 2002]. The MBS may act as a sensor for intracellular copper levels, and it may play a regulatory role when concentrations are low [Goodyer et al., 1999; Strausak et al., 1999; Voskoboinik and Camakaris, 2002]. ATP7A also features a magnesium-binding motif (TGE), the “hinge” domain of the protein, and a phosphorylation motif (DKTG).

ATP7A-encoded mRNA is found is most cell types, but it is missing in liver. ATP7B, the protein mutated in Wilson’s disease (Table 41-2), transports copper in the liver [Vulpe et al., 1993; Paynter et al., 1994]. ATP7A and ATP7B are members of the P-type ATPases group IB family, as are bacterial heavy metal transporters [Tsivkovskii et al., 2002; Voskoboinik et al., 2001]. ATP7A has been localized to the trans-Golgi compartment under basal conditions [Petris et al., 1996]. This is consistent with the ability of ATP7A to supply cuproenzymes (i.e., lysyl oxidase) that are in secretory pathways. There is continuous recycling of ATP7A between the plasma membrane and the trans-Golgi compartment [Petris and Mercer, 1999]. ATP7A traffics to the plasma membrane with increased extracellular copper concentrations and is endocytosed back to the trans-Golgi compartment after extracellular levels decrease to normal. Copper-dependent vesicular trafficking moves ATP7A from the plasma membrane to the trans-Golgi network and back. Lower organisms have two copper ATPases, but this is unnecessary in humans because ATP7A traffics between the two areas where such ATPases are needed [Yuan et al., 1997]. Cu(I) may be the type of copper used by ATP7A as its substrate. It is unknown whether Cu(I) becomes Cu(II) before or after it is released in the Golgi lumen.

Table 41-2 Comparison of Menkes’ Disease and Wilson’s Disease

Characteristic Menkes’ Disease Wilson’s Disease
Inheritance pattern X-linked recessive Autosomal-recessive
Location Xq13.3 13q14.3
Incidence 1:300,000 1:100,000
Clinical manifestations Onset at birth
Cerebral degeneration
Global delay
Kinky hair
Pili torti
Abnormal facies
Hypopigmentation
Arterial rupture or thrombosis
Bone changes or cutis laxa
Dysarthria
Kayser–Fleischer rings
Laboratory test findings ↓Serum Cu
↓Serum ceruloplasmin
↑Intestinal or kidney Cu
↓ Liver Cu
↓ Serum Cu
↓ Serum ceruloplasmin
↑ Urinary Cu
↑ Liver Cu
Prognosis Lethal in classic cases
Death <3 yr
Can be treated effectively with chelating agents
Gene product 1500-amino acid copper-binding ATPase 1411-amino acid copper-binding ATPase
Location, expression All tissues except liver Liver, kidney, and placenta
Mutation Partial deletions in 15 percent; most others are point mutations Point mutations and small rearrangements

Menkes’ disease is caused by mutations that result in a substitution of highly conserved amino acids or those in highly conserved motifs. Any mutation that affects the structure and function of the ATP7A gene will lead to disease [Guy et al., 2001]. Mutations that induce Menkes’ disease include those that abolish the Mg2+ binding domain [Seidel et al., 2001] and those that change the cation transduction motif. Classic Menkes’ disease is usually caused by a premature truncation, typically occurring before the first transmembrane domain and resulting in loss of all catalytic activity. Occipital horn syndrome and mild Menkes’ disease usually result from missense or splice mutations. It is unknown how much catalytic activity is needed to result in a milder phenotype (i.e., ATP7A still able to absorb sufficient amounts of intestinal copper and enable its delivery to the requisite enzymes) [Tümer et al., 1997]. A case study of a patient with occipital horn syndrome showed that the splice mutation allowed 2–5 percent of ATP7A transcripts to be produced. This amount of protein was sufficient to allow partial absorption of copper from the gut epithelium and partial transport across the blood–brain barrier [Møller et al., 2000]. There was, however, too little protein for lysyl oxidase to function correctly. Between 2 and 5 percent of ATP7A activity is the proposed amount of protein necessary to decrease the severity of the Menkes’ disease phenotype.

Most of the clinical manifestations of Menkes’ disease can be explained by understanding which cuproenzymes are affected. Intracellular copper is necessary for oxidative reactions. Most clinical symptoms are caused by dysfunction of dopamine mono-oxygenase, peptidylglycine mono-oxygenase, cytochrome c oxidase, lysyl oxidase, and Cu/Zn superoxide dismutase (SOD), tyrosinase, and sulfhydryl oxidase.

Dopamine mono-oxygenase, also known as dopamine β-hydroxylase, is part of the catecholamine biosynthetic pathway. In Menkes’ disease, there is complete or partial deficiency of dopamine β-hydroxylase. This deficiency causes abnormal plasma and cerebrospinal fluid patterns. The degree of deficiency can be evaluated by looking at norepinephrine concentrations or the ratio of dihydroxyphenylalanine to dihydroxyphenylglycol. Cases of Menkes’ disease with deficient dopamine β-hydroxylase exist that have normal plasma and cerebrospinal fluid concentrations of norepinephrine. It is unknown why compensatory mechanisms are active in some patients but not in others. In the mouse model of Menkes’ disease, normal concentrations of norepinephrine are observed in certain areas of the brain [Prohaska and Bailey, 1994]. The dopamine β-hydroxylase deficiency causes temperature instability, hypoglycemia, eyelid ptosis, and loss of sympathetic adrenergic function [Biaggioni et al., 1990; Robertson et al., 1986].

Peptidylglycine mono-oxygenase is a cuproenzyme necessary for the removal of the carboxyl-terminal glycine residue from neuroendocrine precursors, including gastrin, cholecystokinin, vasoactive intestinal peptide, corticotropin-releasing factor, calcitonin, vasopressin, and thyrotropin-releasing hormone [Eipper et al., 1983, 1992]. When this enzyme is deficient, these neuroendocrine factors have 100- to 1000-fold decreased bioactivity. Affected animal models of Menkes’ disease have decreased peptidylglycine mono-oxygenase activity within their brains [Prohaska and Bailey, 1995]. The decreased activity of these neuroendocrine factors contributes to the phenotype of Menkes’ disease.

Cytochrome c oxidase is a copper-dependent enzyme that is deficient in Menkes’ disease. The decreased cytochrome c oxidase activity causes a subacute necrotizing encephalomyelitis without the severe lactic acidemia that is generally associated with complex IV defects [DiMauro et al., 1990; Robinson, 1989; Robinson et al., 1987]. Peripheral hypotonia and muscle weakness in patients with Menkes’ disease is partially caused by decreased activity of this enzyme.

The normal function of lysyl oxidase (protein lysine 6-oxidase) is to deaminate lysine and hydroxylysine during the first step of collagen cross-link formation [Siegel, 1979]. ATP7A may be needed to transport copper into the trans-Golgi for use by lysyl oxidase. Deficiency of lysyl oxidase decreases the strength of connective tissue in certain tissues or organs and leads to a host of connective tissue disorders that usually are associated with Menkes’ disease, including vascular tortuosity [Royce and Steinmann, 1990], bladder diverticula [Daly and Rabinovitch, 1981; Harke et al., 1977], and gastric polyps. ATP7A deficiency affects lysyl oxidase more profoundly than any of the other cuproenzymes [Gacheru et al., 1993]. It is possible that other cuproenzymes can acquire copper from cytoplasmic carriers without using ATP7A (an intermediate carrier), and this may explain why some mutations of ATP7A lead to the less severe occipital horn syndrome.

Cu/Zn SOD is also deficient in this disease [Rohmer et al., 1977]. Lowered levels of Cu/Zn SOD can increase susceptibility to damage by oxygen free radicals. It is not known whether decreased Cu/Zn SOD causes any developmental regression, because animal models without Cu/Zn SOD have normal development [Reaume et al., 1996] Postmortem studies of patients with Menkes’ disease have found increased levels of manganese SOD, which may be a compensatory change for the decreased levels of Cu/Zn SOD [Shibata et al., 1995]. It is unknown whether the decrease in this enzyme contributes to the phenotype observed in Menkes’ disease. Tyrosinase and sulfhydryl oxidase act on pigment formation and cross-linking of keratin, respectively; hence the manifestations of hypopigmentation, abnormal hair, and dry skin [Horn and Tümer 2002].