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Many genetic neurological disorders exhibit variable expression within affected families, often exemplified by variations in disease age at onset. Epistatic effects (i.e. effects of modifier genes on the disease gene) may underlie this variation, but the mechanistic basis for such epistatic interactions is rarely understood. Here we report a novel epistatic interaction between SPAST and the contiguous gene DPY30, which modifies age at onset in hereditary spastic paraplegia, a genetic axonopathy. We found that patients with hereditary spastic paraplegia caused by genomic deletions of SPAST that extended into DPY30 had a significantly younger age at onset. We show that, like spastin, the protein encoded by SPAST, the DPY30 protein controls endosomal tubule fission, traffic of mannose 6-phosphate receptors from endosomes to the Golgi, and lysosomal ultrastructural morphology. We propose that additive effects on this pathway explain the reduced age at onset of hereditary spastic paraplegia in patients who are haploinsufficient for both genes.

Cri du chat syndrome (CdCS) and primary ciliary dyskinesia (PCD) are rare diseases that present with frequent respiratory symptoms. PCD can be caused by hemizygous DNAH5 mutation in combination with a 5p segmental deletion attributable to CdCS on the opposite chromosome. Chronic oto-sino-pulmonary symptoms or organ laterality defects in CdCS should prompt an evaluation for PCD., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Fragile X syndrome is the most common cause of inherited mental retardation and the second most common cause of mental impairment after trisomy 21. It occurs because of a failure to express the fragile X mental retardation protein. The most common molecular basis for the disease is the abnormal expansion of the number of CGG repeats in the fragile X mental retardation 1 gene (FMR1). Based on the number of repeats, it is possible to distinguish four types of alleles: normal (5 to 44 repeats), intermediate (45 to 54), premutation (55 to 200), and full mutation (>200). Today, the diagnosis of fragile X syndrome is performed through a combination of PCR to identify fewer than 100 repeats and of Southern blot analysis to identify longer alleles and the methylation status of the FMR1 promoter. We have developed a methylation-specific multiplex ligation-dependent probe amplification assay to analyze male fragile X syndrome cases with long repeat tracts that are not amplifiable by PCR. This inexpensive, rapid and robust technique provides not only a clear distinction between male pre- and full-mutation FMR1 alleles, but also permits the identification of genomic deletions, a less frequent cause of fragile X syndrome.

Mutations in the receptor expression enhancing protein 1 (REEP1) have recently been reported to cause autosomal dominant hereditary spastic paraplegia (HSP) type SPG31. In a large collaborative effort, we screened a sample of 535 unrelated HSP patients for REEP1 mutations and copy number variations. We identified 13 novel and 2 known REEP1 mutations in 16 familial and sporadic patients by direct sequencing analysis. Twelve out of 16 mutations were small insertions, deletions or splice site mutations. These changes would result in shifts of the open-reading-frame followed by premature termination of translation and haploinsufficiency. Interestingly, we identified two disease associated variations in the 3'-UTR of REEP1 that fell into highly conserved micro RNA binding sites. Copy number variation analysis in a subset of 133 HSP index patients revealed a large duplication of REEP1 that involved exons 2-7 in an Irish family. Clinically most SPG31 patients present with a pure spastic paraplegia; rare complicating features were restricted to symptoms or signs of peripheral nerve involvement. Interestingly, the distribution of age at onset suggested a bimodal pattern with the appearance of initial symptoms of disease either before the age of 20 years or after the age of 30 years. The overall mutation rate in our clinically heterogeneous sample was 3.0%; however, in the sub-sample of pure HSP REEP1 mutations accounted for 8.2% of all patients. These results firmly establish REEP1 as a relatively frequent autosomal dominant HSP gene for which genetic testing is warranted. We also establish haploinsufficiency as the main molecular genetic mechanism in SPG31, which should initiate and guide functional studies on REEP1 with a focus on loss-of-function mechanisms. Our results should be valid as a reference for mutation frequency, spectrum of REEP1 mutations, and clinical phenotypes associated with SPG31.

B cells use translesion DNA synthesis (TLS) to introduce somatic mutations around genetic lesions caused by activation-induced cytidine deaminase. Monoubiquitination at lysine(164) of proliferating cell nuclear antigen (PCNA(K164)) stimulates TLS. To determine the role of PCNA(K164) modifications in somatic hypermutation, PCNA(K164R) knock-in mice were generated. PCNA(K164R/K164R) mutants are born at a sub-Mendelian frequency. Although PCNA(K164R/K164R) B cells proliferate and class switch normally, the mutation spectrum of hypermutated immunoglobulin (Ig) genes alters dramatically. A strong reduction of mutations at template A/T is associated with a compensatory increase at G/C, which is a phenotype similar to polymerase eta (Poleta) and mismatch repair-deficient B cells. Mismatch recognition, monoubiquitinated PCNA, and Poleta likely cooperate in establishing mutations at template A/T during replication of Ig genes.

Neurofibromatosis 2 (NF2) is a genetic disorder caused by mutational inactivation of the NF2 gene and is characterized by bilateral vestibular schwannomas, spinal tumors, and other benign tumors of the nervous system. Previously, we found intragenic NF2 mutations in 99 of 188 unrelated NF2 patients by exon-scanning-based methods. Tumor analysis of 22 de novo NF2 patients led to the identification of 12 additional constitutive NF2 mutations. The remaining 77 patients were further examined for large alterations using the newly developed gene dosage assay multiplex ligation-dependent probe amplification (MLPA). One deletion of a single exon, seven deletions of multiple exons, seven deletions involving the 3' or 5' end of the NF2 gene, four deletions involving the whole NF2 gene, and one duplication of three exons were detected. For 47 of the 77 patients, mRNA of adequate quality could be obtained, enabling transcript analysis, which confirmed eight alterations detected by MLPA. In addition, in one family, the mRNA analysis detected an insertion of two exons of another gene. Thus, deletions, duplications, and insertions affecting the NF2 gene were found in 21 cases, which is 11% of the 188 unrelated NF2 patients studied, 16% of the 132 mutations identified, and 27% of the 77 cases in which no intragenic small mutations were detected by exon scanning. The combination of multiple screening techniques facilitated a mutation-detection rate of 100% for the 21 inherited cases in this study., ((c) 2005 Wiley-Liss, Inc.)