Occipital horn syndrome and a mild Menkes phenotype associated with splice site mutations at the MNK locus.

We have found mutations in the Menkes disease gene (MNK) which impair, but do not abolish, correct mRNA splicing in patients with less severe clinical phenotypes. In one family, four males aged 2-36 years with a distinctive Menkes variant have a mutation at the +3 position of a splice donor site near the 3' end of the Menkes coding sequence that is associated with exon skipping and a stable mutant transcript. In an unrelated 15-year-old male with typical occipital horn syndrome, a point mutation at the -2 exonic position of a splice donor site in the middle of the gene causes exon-skipping and activation of a cryptic splice acceptor site. In both mutations, maintenance of some normal splicing is demonstrable by RT-PCR, cDNA sequencing and ribonuclease protection.

Research challenges in central nervous system manifestations of inborn errors of metabolism.

The Research Challenges in CNS Manifestations of Inborn Errors of Metabolism workshop was designed to address challenges in translating potential therapies for these rare disorders, and to highlight novel therapeutic strategies and innovative approaches to CNS delivery, assessment of effects and directions for the future in the treatment of these diseases. Therapies for the brain in inborn errors represent some of the greatest challenges to translational research due to the special properties of the brain, and of inborn errors themselves. This review covers the proceedings of this workshop as submitted by participants. Scientific, ethical and regulatory issues are discussed, along with ways to measure outcomes and the conduct of clinical trials. Participants included regulatory and funding agencies, clinicians, scientists, industry and advocacy groups.

Favorably skewed X-inactivation accounts for neurological sparing in female carriers of Menkes disease.

Classical Menkes disease is an X-linked recessive neurodegenerative disorder caused by mutations in ATP7A, which is located at Xq13.1-q21. ATP7A encodes a copper-transporting P-type ATPase and plays a critical role in development of the central nervous system. With rare exceptions involving sex chromosome aneuploidy or X-autosome translocations, female carriers of ATP7A mutations are asymptomatic except for subtle hair and skin abnormalities, although the mechanism for this neurological sparing has not been reported. We studied a three-generation family in which a severe ATP7A mutation, a 5.5-kb genomic deletion spanning exons 13 and 14, segregated. The deletion junction fragment was amplified from the proband by long-range polymerase chain reaction and sequenced to characterize the breakpoints. We screened at-risk females in the family for this junction fragment and analyzed their X-inactivation patterns using the human androgen-receptor (HUMARA) gene methylation assay. We detected the junction fragment in the proband, two obligate heterozygotes, and four of six at-risk females. Skewed inactivation of the X chromosome harboring the deletion was noted in all female carriers of the deletion (n = 6), whereas random X-inactivation was observed in all non-carriers (n = 2). Our results formally document one mechanism for neurological sparing in female carriers of ATP7A mutations. Based on review of X-inactivation patterns in female carriers of other X-linked recessive diseases, our findings imply that substantial expression of a mutant ATP7A at the expense of the normal allele could be associated with neurologic symptoms in female carriers of Menkes disease and its allelic variants, occipital horn syndrome, and ATP7A-related distal motor neuropathy.

Diagnosis and therapy of Menkes syndrome, a genetic form of copper deficiency.

In the 25 y since copper deficiency was first delineated in persons with Menkes syndrome, advances in our understanding of the clinical, biochemical, and molecular aspects of this rare disorder have surpassed progress in the design of effective therapies. In contrast with purely nutritional copper deficiency, in which copper replacement can be curative, the nature of the basic defect in Menkes syndrome suggests that corrective efforts are likely to be more complicated, a point supported by the cumulative literature on this topic as well as by emerging molecular data. In this paper, certain clinical, biochemical, and molecular aspects of copper histidine treatment in 25 Menkes syndrome patients at the National Institutes of Health are reviewed. The delineation of a distinctive neurochemical pattern in plasma and cerebrospinal fluid, reflecting deficiency of the copper enzyme dopamine beta-monooxygenase, is arguably the most important finding in the study of Menkes syndrome. This abnormal pattern has proven extremely reliable as a rapid diagnostic test, enabling early identification of affected infants--a fundamental requirement for improving clinical outcomes. Of 11 patients identified by prenatal or prompt postnatal testing and treated within the first 10 d of age, one walked at 14 mo of age and has normal neurodevelopment at age 3 y and another infant's early progress appears promising. However, five patients died in infancy and neurodevelopmental outcome was suboptimal in four others. Consideration of additional therapeutic strategies seems necessary, therefore, for most patients and families facing this troublesome form of copper deficiency.

Brief clinical report: two children with de novo del(9p).

We describe two patients with characteristic manifestations of the del(9p) syndrome. Among the prominent manifestation are moderate mental retardation, trigonocephaly, flat nasal bridge, anteverted nostrils, long philtrum, square and hyperconvex nails, and apparently long digits. The metacarpophalangeal pattern profiles of the patients showed that the clinical impression of long fingers may be due to relative shortness of the metacarpals rather than to elongation of the phalanges.

Distinctive Menkes disease variant with occipital horns: delineation of natural history and clinical phenotype.

To delineate further the clinical spectrum of Menkes disease, an X-linked recessive disorder of copper transport, we studied 4 related males, ranging in age from 4-38 years, with a unique phenotype that combines manifestations of classical and mild Menkes disease and occipital horn syndrome (OHS). The propositus, and 18-year-old man, was evaluated following an intracerebral hemorrhage at age 15 years and was noted to have marked hypotonia, motor delay with mental retardation, bladder diverticula, failure to thrive, and diarrhea from infancy; seizures from age 3 years; and abnormal hair (pili torti) and face, cutis laxa, and multiple joint dislocations. Radiographic abnormalities included occipital exostoses, tortuous cerebral blood vessels with multiple branch occlusions, and hammer-shaped clavicles. Biochemical studies demonstrated reduced copper and ceruloplasmin levels in serum, and abnormal plasma catecholamine ratios. We reported previously the molecular defect in this family, a splice-site mutation that predicts formation of approximately 20% of the normal Menkes gene product [Kaler et al., 1994: Nat Genet 18:195-202]. Here, we detail the clinical course and physical features and radiographic findings in these 4 individuals, and compare their phenotype with classical and mild Menkes and OHS. Unusual Menkes disease variants such as this may escape recognition due to anomalies that appear inconsistent with the diagnosis, particularly prolonged survival and later onset of seizures. Males with mental retardation and connective tissue abnormalities should be evaluated for biochemical evidence of defective copper transport.

New autosomal recessive syndrome of sparse hair, osteopenia, and mental retardation in Mennonite sisters.

We report on 2 Mennonite sisters with a syndrome of sparse hair, osteopenia, mental retardation, minor facial abnormalities, joint laxity, and hypotonia. Their asymptomatic consanguineous parents (inbreeding coefficient F = 1/64) have 6 other offspring, 3 of whom died in infancy of type II osteogenesis imperfecta (OI), and 3 of whom are normal. We analyzed collagens synthesized by cultured fibroblasts from these 2 sisters and their parents and detected no major abnormalities. Results of chromosomal and metabolic evaluations including amino acid analysis of plasma, urine, and hair were unremarkable. A literature search and survey of a computerized syndrome identification database did not disclose an identical phenotype. The sisters bear superficial resemblance to several known syndromes which we excluded on clinical and/or biochemical grounds. We conclude that they represent a new autosomal recessive syndrome, distinct from type II OI and perhaps unique to the Mennonite population or to this particular family.

Novel method for molecular detection of the two common hereditary hemochromatosis mutations.

We describe a novel molecular screening technique for hereditary hemochromatosis through which HFE genotypes at codon positions 282 and 63 are simultaneously detected. The technique combines multiplex PCR and denaturing high-performance liquid chromatography (DHPLC) and allows automated high-throughput analysis. We used this method to genotype 43 previously characterized anonymous DNA specimens in blinded fashion and found multiplex PCR/DHPLC 100% accurate when compared with PCR/restriction enzyme digestion, yet far more efficient.

Application of a copper blotting method to the study of Menkes disease.

Menkes disease is an X-linked recessive disorder of copper metabolism. Deficient quantity or functional activity of a molecule involved in intracellular copper transport is believed to represent the basic defect. We applied an in vitro copper binding assay (copper blotting) to tissue proteins from Menkes patients and controls to evaluate differences in copper-binding. Proteins were separated by SDS-PAGE, electrotransferred to nitrocellulose, and probed with 67CuCl2. Copper-binding polypeptides were visualized by autoradiography. No major differences were observed between a Menkes patient and control subjects in copper blots of post-mortem liver, kidney, or brain--tissues affected clinically by the disturbance of copper metabolism in Menkes disease. We also applied the copper blotting technique to fibroblast proteins from an affected female in whom the gene responsible for Menkes disease is interrupted by a chromosomal translocation, and detected no differences in copper-binding proteins relative to normal controls. These experiments suggest that the gene product defective in Menkes disease is not detectable in copper blots, either because normal tissue levels are below the limits of detection of this method, or because the molecule involved does not bind copper under these conditions.

Non-random maternal X-chromosome inactivation associated with PHACES.

The acronym PHACES is used to describe the association of posterior fossa malformations, hemangiomas, arterial anomalies (cardiovascular or cerebrovascular), coarctation of the aorta and cardiac defects, eye abnormalities, and sternal or ventral defects. We report a female patient with an uncommon variant of this neurocutaneous disorder who manifested a sternal cleft; supraumbilical raphe; hemangiomas of the face, chest, and extremities; micrognathia and cerebrovascular anomalies. A literature review of PHACES patients with both sternal cleft and supraumbilical raphe showed a marked female predilection. Taken together with cases of sternal cleft, supraumbilical raphe and facial hemangiomas tabulated by Gorlin et al. (1994), 91% (40/44) of patients are female. One affected male died shortly after birth. We hypothesized that the gender bias in PHACES results from mutation in an X-linked dominant gene often lethal in males, and performed X-inactivation analysis of the polymorphic androgen receptor locus in this family. We documented consistently skewed X-inactivation (80%/20% in two independent analyses) in the unaffected mother and consistently random X-inactivation (47:53 and 61:39 in independent analyses) in the proband. These findings are consistent with favorably skewed X-inactivation producing a normal maternal phenotype, a phenomenon documented in X-linked dominant Rett syndrome.

Copper-replacement treatment for symptomatic Menkes disease: ethical considerations.

We describe a child with classical Menkes disease with a novel ATP7A mutation, intractable seizures, severe hypotonia and developmental delay, hypopigmentation of the skin and hair, and failure to thrive, who was treated with daily subcutaneous copper histidine injections for 2(1/2) years, beginning at 15 months of age. He became seizure-free and pigmentation of his skin and hair darkened, but he continued to have severe developmental delays. His condition remains stable 8 months after stopping treatment. We review the ethical aspects of offering copper treatment for Menkes disease infants diagnosed after neurological symptoms become manifest. These include (1) the prospect for any benefits, (2) the potential risks and discomforts, (3) the parents' wishes with respect to treatment, (4) the family's understanding of the treatment's potential futility, (5) the family's understanding of the investigational nature of this treatment, (6) the potential for treatment to have an adverse impact on unaffected family members, (7) whether the ultimate decision regarding treatment should rest with health care providers or with the patient's parents, and (8) the duration of treatment. The ethical issues encountered in providing possibly futile treatment in this difficult disorder seem relevant to other pediatric medical conditions as well.

Metabolic and molecular bases of Menkes disease and occipital horn syndrome.

Menkes disease and occipital horn syndrome (OHS) are related disorders of copper transport that involve abnormal neurodevelopment, connective tissue problems, and often premature death. Location of the gene responsible for these conditions on the X chromosome was indicated by pedigree analysis from the time of these syndromes' earliest descriptions. Characterization of an affected female with an X-autosomal translocation was used to identify the Menkes/OHS gene, which encodes a highly evolutionarily conserved, copper-transporting P-type ATPase. The gene normally is expressed in nearly all human tissues, and it localizes to the trans-Golgi network of cells. However, in over 70% of Menkes and OHS patients studied, expression of this gene has been demonstrated to be abnormal. Major gene deletions detectable by Southern blotting account for 15-20% of patients, and an interesting spectrum of other mutations is evident among 58 families whose precise molecular defects have been reported as of this writing. The center region of the gene seems particularly prone to mutation, and those that influence mRNA processing and splicing appear to be relatively common. Further advances in understanding the molecular and cell biological mechanisms involved in normal copper transport may ultimately yield new and better approaches to the management of these disorders.

Human manganese superoxide dismutase is readily detectable by a copper blotting technique.

Manganese-superoxide dismutase (MnSOD) in human fibroblast and liver lysates was found to bind copper avidly under the conditions of a copper blotting technique which also detects known copper-binding proteins. Competition studies suggest that the copper-binding site of the molecule under these conditions is distinct from its manganese-binding site. Copper blotting provides a sensitive way to detect MnSOD in human tissues, and may be generally applicable to studies of copper-binding by biological molecules.

Radiographic hand abnormalities in fifteen cases of Crouzon syndrome.

Fifteen patients with Crouzon syndrome were evaluated for abnormalities of hand bone length via metacarpophalangeal pattern profile analysis. Data from this group were compared to that from a normal control sample. A discriminant function, utilizing Z scores based on the lengths of three hand bones, was derived and distinguished between the two samples at a rate of 88.3%. The discriminant variables selected in the function represented hand bones that are prominently involved in ACS type V and ACPS type II, syndromes that feature craniofacial abnormalities not unlike those in Crouzon syndrome. The possibility of a common developmental mechanism affecting the hand skeleton in all three of these different conditions is raised.