M. E. Hodes

Genomic rearrangements resulting in PLP1 deletion occur by nonhomologous end joining and cause different dysmyelinating phenotypes in males and females.

In the majority of patients with Pelizaeus-Merzbacher disease, duplication of the proteolipid protein gene PLP1 is responsible, whereas deletion of PLP1 is infrequent. Genomic mechanisms for these submicroscopic chromosomal rearrangements remain unknown. We identified three families with PLP1 deletions (including one family described elsewhere) that arose by three distinct processes. In one family, PLP1 deletion resulted from a maternal balanced submicroscopic insertional translocation of the entire PLP1 gene to the telomere of chromosome 19. PLP1 on the 19qtel is probably inactive by virtue of a position effect, because a healthy male sibling carries the same der(19) chromosome along with a normal X chromosome. Genomic mapping of the deleted segments revealed that the deletions are smaller than most of the PLP1 duplications and involve only two other genes. We hypothesize that the deletion is infrequent, because only the smaller deletions can avoid causing either infertility or lethality. Analyses of the DNA sequence flanking the deletion breakpoints revealed Alu-Alu recombination in the family with translocation. In the other two families, no homologous sequence flanking the breakpoints was found, but the distal breakpoints were embedded in novel low-copy repeats, suggesting the potential involvement of genome architecture in stimulating these rearrangements. In one family, junction sequences revealed a complex recombination event. Our data suggest that PLP1 deletions are likely caused by nonhomologous end joining.

Genetics of Pelizaeus-Merzbacher Disease

Pelizaeus-Merzbacher disease (PMD) has been recognized as a clinical entity for more than a century. It has gradually become apparent that the disorder is a dysmyelination, in distinction to demyelinating conditions such as adrenoleukodystrophy. The failure to deposit myelin is due to decreased production of its chief protein, proteolipid protein (PLP). In about 30% of patients with the diagnosis of PMD there is a mutation in the coding portion of the proteolipid protein gene, PLP. This gene is located at Xq22 so the disease in these families shows an X-linked pattern of inheritance. The expression of the mutant gene is generally recessive, but some mutations are expressed frequently in females. At least some patients with PMD that do not show mutations in the coding region of PLP demonstrate linkage between the disease and PLP. As additional mutations in PLP are discovered, it is becoming apparent that the nosology of PLP-associated disease is changing. PMD now comprises a spectrum of disorders with similar but not necessarily identical clinical pictures. Some of these disorders may be certain forms of X-linked paraplegia, SPG2. Finally, some diseases that look like PMD may not be X-linked.

Heterogeneous Duplications in Patients with Pelizaeus-Merzbacher Disease Suggest a Mechanism of Coupled Homologous and Nonhomologous Recombination

We describe genomic structures of 59 X-chromosome segmental duplications that include the proteolipid protein 1 gene (PLP1) in patients with Pelizaeus-Merzbacher disease. We provide the first report of 13 junction sequences, which gives insight into underlying mechanisms. Although proximal breakpoints were highly variable, distal breakpoints tended to cluster around low-copy repeats (LCRs) (50% of distal breakpoints), and each duplication event appeared to be unique (100 kb to 4.6 Mb in size). Sequence analysis of the junctions revealed no large homologous regions between proximal and distal breakpoints. Most junctions had microhomology of 1-6 bases, and one had a 2-base insertion. Boundaries between single-copy and duplicated DNA were identical to the reference genomic sequence in all patients investigated. Taken together, these data suggest that the tandem duplications are formed by a coupled homologous and nonhomologous recombination mechanism. We suggest repair of a double-stranded break (DSB) by one-sided homologous strand invasion of a sister chromatid, followed by DNA synthesis and nonhomologous end joining with the other end of the break. This is in contrast to other genomic disorders that have recurrent rearrangements formed by nonallelic homologous recombination between LCRs. Interspersed repetitive elements (Alu elements, long interspersed nuclear elements, and long terminal repeats) were found at 18 of the 26 breakpoint sequences studied. No specific motif that may predispose to DSBs was revealed, but single or alternating tracts of purines and pyrimidines that may cause secondary structures were common. Analysis of the 2-Mb region susceptible to duplications identified proximal-specific repeats and distal LCRs in addition to the previously reported ones, suggesting that the unique genomic architecture may have a role in nonrecurrent rearrangements by promoting instability.

Branchio-oto-renal dysplasia and branchio-oto dysplasia: Two distinct autosomal dominant disorders

Three families are presented, one with branchio‐oto‐renal dysplasia (BOR) and two with branchio‐oto dysplasia (BO). The former syndrome is characterized by external ear malformations, cervical fistulae, mixed hearing loss and renal anomalies of varying severity. The latter syndrome differs in that there are no renal anomalies and that the sensorineural component of the hearing loss may be absent. The external ear malformations are quite variable in both syndromes. Evidence is presented which supports the idea that these two syndromes are not phenotypic variants of the same autosomal dominant mutation but distinct disease entities. The BOR syndrome appears to belong to a larger group of hereditary ear dysplasia‐renal adysplasia syndromes that must be carefully ruled out in all patients with familial branchial arch malformations as well as in the parents and siblings of infants with “Potter fades” in the presence of auricular malformation and renal adysplasia.

Transmission of a balanced homologous t(22q;22q) translocation from mother to normal daughter

The transmission of a t(22q;22q) translocation is reported. The mother had had multiple miscarriages and carried both t(22q;22q) and t(22p;22p) portions of the rearrangement in a portion of her cells. The phenotypically normal daughter, who was the proband and was referred because of multiple miscarriages, also carried the t(22q;22q) translocation.

The weaver mutation changes the ion selectivity of the affected inwardly rectifying potassium channel GIRK2

The weaver mutation in mice has recently been identified as a single base‐pair mutation in the Girk2 gene, which encodes a G‐protein‐activated inwardly rectifying potassium channel, GIRK2. The mutation results in a Gly to Ser substitution at residue 156, in the putative pore‐forming region of the potassium channel. In the present study, we used Xenopus oocytes to express mutant GIRK2, and to characterize the effects of the mutation on the channel. The mutation results in a loss of the normal high selectivity for K+ over Na+, with little effect on other channel properties such as activation by the mu opioid receptor. The resulting increase in basal Na+ permeability causes a marked depolarization of oocytes expressing the mutant GIRK2 protein. This result was observed even when the mutant GIRK2 was coexpressed with GIRK1, a situation more analogous to that seen in vivo. Thus, the increased Na+ permeability and resulting depolarization may contribute to the pathology of cerebellar granule cells and substantia nigra dopaminergic neurons observed in the weaver mice.

Possible localization of a major gene for cleft lip and palate to 4q

Two of the 13 PCR markers were most informative in tentatively identifying a major gene for cleft lip on the q arm of chromosome #4 with a maximum LOD score of 2.27 (Ø= 0)

Additional Copies of the Proteolipid Protein Gene Causing Pelizaeus-Merzbacher Disease Arise by Separate Integration into the X Chromosome

The proteolipid protein gene (PLP) is normally present at chromosome Xq22. Mutations and duplications of this gene are associated with Pelizaeus-Merzbacher disease (PMD). Here we describe two new families in which males affected with PMD were found to have a copy of PLP on the short arm of the X chromosome, in addition to a normal copy on Xq22. In the first family, the extra copy was first detected by the presence of heterozygosity of the AhaII dimorphism within the PLP gene. The results of FISH analysis showed an additional copy of PLP in Xp22.1, although no chromosomal rearrangements could be detected by standard karyotype analysis. Another three affected males from the family had similar findings. In a second unrelated family with signs of PMD, cytogenetic analysis showed a pericentric inversion of the X chromosome. In the inv(X) carried by several affected family members, FISH showed PLP signals at Xp11.4 and Xq22. A third family has previously been reported, in which affected members had an extra copy of the PLP gene detected at Xq26 in a chromosome with an otherwise normal banding pattern. The identification of three separate families in which PLP is duplicated at a noncontiguous site suggests that such duplications could be a relatively common but previously undetected cause of genetic disorders.

A new discontinuous buffer system for the electrophoresis of cationic proteins at near-neutral pH.

A simple, discontinuous buffer system for polyacrylamide gel electrophoresis near neutral pH is described. The buffer is MOPS (3-[N-morpholine]propanesulfonic acid), the leading ion K+ and the trailing ion histidine. The system offers improved resolution of cationic proteins.

Nonsense mutation in exon 3 of the proteolipid protein gene (PLP) in a family with an unusual form of Pelizaeus-Merzbacher disease

We report a G-->A transition at nucleotide 431 of the proteolipid protein gene (PLP) results in a nonsense codon in a family with an unusual form of Pelizaeus-Merzbacher disease (PMD). The mutation, which creates a second AluI restriction site, results in a nonsense mutation in PLP. The clinical picture resembles somewhat that of X-linked spastic paraplegia (SPG). It differs from this and both the classical and connatal forms of PMD in that it is relatively mild in form, onset is delayed beyond age 2 years, nystagmus is absent, tremors are prominent, mental retardation is not severe, some patients show dementia or personality disorders, the disease is progressive rather than static in some, and several females show signs of disease. The nonsense mutation, which is in exon 3B, should block the synthesis of normal PLP but spare DM20, the isoform whose persistence has been associated with mild forms of PLP-associated disease in both humans and mice.