Lemuel Racacho

Functional alteration of PARL contributes to mitochondrial dysregulation in Parkinson's disease

Molecular genetics has linked mitochondrial dysfunction to the pathogenesis of Parkinsons disease by the discovery of rare, inherited mutations in gene products that associate with the mitochondria. Mutations in PTEN-induced kinase-1 (PINK1), which encodes a mitochondrial kinase, and PARKIN, encoding an E3 ubiquitin ligase, are the most frequent causes of recessive Parkinsons disease. Recent functional studies have revealed that PINK1 recruits PARKIN to mitochondria to initiate mitophagy, an important autophagic quality control mechanism that rids the cell of damaged mitochondria. PINK1 is post-translationally processed into a cleaved form whose levels are tightly regulated, although the significance of this processing is unknown. Here we demonstrate that the mitochondrial protease presenilin-associated rhomboid-like (PARL) can affect the proteolytic processing of PINK1 and that normal PINK1 localization and stability requires PARLs catalytic activity. PARL deficiency impairs PARKIN recruitment to mitochondria, suggesting PINK1s processing and localization are important in determining its interaction with PARKIN. We sequenced the PARL gene in Parkinsons disease patients and discovered a novel missense mutation in a functional domain of PARLs N-terminus. This PARL mutant is not sufficient to rescue PARKIN recruitment, suggesting that impaired mitophagy may be an underlying mechanism of disease pathogenesis in patients with PARL mutations.

A novel locus for inherited myoclonus-dystonia on 18p11

Objective Inherited myoclonus-dystonia (IMD) is a new term for an autosomal dominant disorder characterized by myoclonus and dystonia. Recently, IMD was linked to a region on chromosome 11q23 with two different mutations identified in the D2 dopamine receptor gene and linked to chromosome 7q with five different loss-of-function mutations identified in the &egr;-sarcoglycan gene. Methods These two regions and genes were excluded in a large Canadian family with IMD in whom 13 individuals are affected. A 25-cM genome scan of this large family with 32 individuals was performed. Results Two-point linkage analysis revealed a maximum lod score of 3.5 (recombination fraction 0.00; affected only) for the microsatellite marker GATA185C06-18 and a multipoint lod score of 3.9 across the 18p11 region. Haplotype analysis demonstrates that all the affected individuals shared a common haplotype between markers D18S1132 and D18S843 that defines the disease gene within a span of 16.9 cM. Conclusions These findings indicate that a novel IMD gene exists on chromosome 18p11.

Translated mutation in the Nurr1 gene as a cause for Parkinson's disease

Multiple genes have been now identified as causing Parkinsons disease (PD). In 2003, two mutations were identified in exon 1 of the Nurr1 gene in 10 of 107 individuals with familial PD. To date, investigators have only focused on screening for these known mutations of the Nurr1 gene. All individuals were recruited from two Parkinsons disease clinics in Canada. Following PCR amplification of each exon of the Nurr1 gene, samples underwent denaturing high‐performance liquid chromatography (DHPLC) analysis. Ten individuals also underwent direct sequencing as well as any samples where variants were identified. The Nurr1 gene was evaluated for 202 PD individuals, 37% of whom had at least one relative with PD and 100 control non‐PD individuals. Using DHPLC and direct sequencing, we did not detect any sequence variants in exon 1. Variants in amplicon 6 were seen and direct sequencing confirmed a known NI6P polymorphism in intron 6. Novel polymorphisms were also identified in exon 3 and intron 5. A novel mutation was identified in exon 3 in one nonfamilial PD individual. This heterozygous C‐to‐G transversion resulted in a serine‐to‐cysteine substitution and was not identified in any of the other 602 chromosomes screened. Mutations in the Nurr1 gene in our large cohort of familial and sporadic PD individuals are rare. The novel mutation in exon 3 is predicted to affect phosphorylation and functional studies to assess this are underway. This is the first coding mutation identified in the Nurr1 gene for Parkinsons disease.

Refinement of the DYT15 locus in myoclonus dystonia

Inherited myoclonus dystonia (MD) is an autosomal dominant disorder in which we previously mapped a novel locus to chromosome18p11 (OMIM number: 607488). Since no further informative STS markers were found within the flanking shared regions, we utilized single nucleotide polymorphisms (SNP) for fine‐mapping. All known or predicted genes within this region were directly sequenced. We identified three recombinant SNPs in the distal region but none from the proximal region. Our previous linked region has now been reduced to 3.18 Mb but direct sequencing of all seven known and four predicted genes with EST support did not identify any mutations.

Mutations in the ∈-sarcoglycan gene found to be uncommon in seven myoclonus-dystonia families

Myoclonus–dystonia syndrome (MDS) is a disorder for which the major cause appears to be mutations in the ε-sarcoglycan gene (SGCE). The authors have now performed mutation screening in 22 affected individuals from seven families with findings of typical MDS. A novel 5-bp deletion in exon 7 of the gene in one family and the previously reported R102X nonsense mutation in exon 3 in two other families were identified. Mutations in the SGCE gene were found in the minority of families screened in this series.

Brachydactyly A-1 mutations restricted to the central region of the N-terminal active fragment of Indian Hedgehog

Mutations in the gene Indian Hedgehog (IHH) that cause Brachydactyly A-1 (BDA1) have been restricted to a specific region of the N-terminal active fragment of Indian Hedgehog involving codons 95, 100, 131, and 154. We describe two novel mutations in codons 128 and 130, not previously implicated in BDA1. Furthermore, we identified an independent mutation at codon 131 and we also describe a New Zealand family, which carries the ‘Farabee’ founder mutation and haplotype. All of the BDA1 mutations occur in a restricted area of the N-terminal active fragment of the IHH and are in contrast to those mutations causing an autosomal recessive acrocapitofemoral dysplasia, whose mutations are located at the distal N- and C-terminal regions of IHH-N and are physically separated from the BDA1-causing mutations. The identification of multiple independent mutations in codons 95, 100, and now in 131, implicate a discrete function for this region of the protein. Finally, we present a clinical review of all reported and confirmed cases of BDA1, highlighting features of the disorder, which add to the spectrum of the IHH mutations.

Mutations in GDF5 presenting as semidominant brachydactyly A1

Brachydactyly A1 (BDA1) is an autosomal dominant disorder characterized by shortness of all middle phalanges of the hands and toes, shortness of the proximal phalanges of the first digit, and short stature. Missense mutations in the Indian Hedgehog gene (IHH) are known to cause BDA1, and a second locus has been mapped to chromosome 5p. In a consanguineous French Canadian kindred with BDA1, both IHH and the 5p locus were excluded. Microsatellites flanking GDF5 on chromosome 20q were found to cosegregate with the disease. Sequencing of the GDF5 coding region revealed that a mildly affected individual in the family was heterozygous, and that all of the severely affected individuals were homozygous for a novel missense c.1195C>T mutation that predicts a p.Arg399Cys substitution at a highly conserved amino acid. Functional analysis demonstrated that although the p.Arg399Cys mutant is able to stimulate chondrogenesis, it is much less effective than wild‐type GDF5. This data confirms genetic heterogeneity in BDA1, demonstrates that mutations upstream of IHH can result in BDA1, and shows that BDA1 can result from semidominant mutations in GDF5. Hum Mutat 31:1–8, 2010.

Large deletions account for an increasing number of mutations in SGCE

Myoclonus‐dystonia (M‐D) (MIM 159900) is a rare “dystonia plus” syndrome, characterized by rapid myoclonic jerks, predominantly in the neck and upper limbs, in combination with dystonia. Mutations in the gene ε‐sarcoglycan (SGCE) are known to be responsible for approximately one‐third of cases. We screened 21 probands diagnosed with M‐D for large deletions who were mutation negative as determined by PCR‐direct sequencing. Multiplex PCR and quantification of PCR products was performed using a modified application of denaturing high performance liquid chromatography (dHPLC). We have identified two novel large multiexonic deletions of SGCE, which were confirmed by amplifying and sequencing the deletion breakpoints. Five other families were found to harbor small mutations identified by direct sequencing. Analysis of the region surrounding the deletions demonstrates that both deletions are the result of nonhomologous recombination with homologous end joining. This is only the second report of intragenic deletions with SGCE and it highlights the need to include exonic copy number variation when performing mutational analysis of SGCE.

Two novel disease-causing variants in BMPR1B are associated with brachydactyly type A1

Brachydactyly type A1 is an autosomal dominant disorder primarily characterized by hypoplasia/aplasia of the middle phalanges of digits 2–5. Human and mouse genetic perturbations in the BMP-SMAD signaling pathway have been associated with many brachymesophalangies, including BDA1, as causative mutations in IHH and GDF5 have been previously identified. GDF5 interacts directly as the preferred ligand for the BMP type-1 receptor BMPR1B and is important for both chondrogenesis and digit formation. We report pathogenic variants in BMPR1B that are associated with complex BDA1. A c.975A>C (p.(Lys325Asn)) was identified in the first patient displaying absent middle phalanges and shortened distal phalanges of the toes in addition to the significant shortening of middle phalanges in digits 2, 3 and 5 of the hands. The second patient displayed a combination of brachydactyly and arachnodactyly. The sequencing of BMPR1B in this individual revealed a novel c.447-1G>A at a canonical acceptor splice site of exon 8, which is predicted to create a novel acceptor site, thus leading to a translational reading frameshift. Both mutations are most likely to act in a dominant-negative manner, similar to the effects observed in BMPR1B mutations that cause BDA2. These findings demonstrate that BMPR1B is another gene involved with the pathogenesis of BDA1 and illustrates the continuum of phenotypes between BDA1 and BDA2.

The ONDRISeq panel: custom-designed next-generation sequencing of genes related to neurodegeneration

The Ontario Neurodegenerative Disease Research Initiative (ONDRI) is a multimodal, multi-year, prospective observational cohort study to characterise five diseases: (1) Alzheimer’s disease (AD) or amnestic single or multidomain mild cognitive impairment (aMCI) (AD/MCI); (2) amyotrophic lateral sclerosis (ALS); (3) frontotemporal dementia (FTD); (4) Parkinson’s disease (PD); and (5) vascular cognitive impairment (VCI). The ONDRI Genomics subgroup is investigating the genetic basis of neurodegeneration. We have developed a custom next-generation-sequencing-based panel, ONDRISeq that targets 80 genes known to be associated with neurodegeneration. We processed DNA collected from 216 individuals diagnosed with one of the five diseases, on ONDRISeq. All runs were executed on a MiSeq instrument and subjected to rigorous quality control assessments. We also independently validated a subset of the variant calls using NeuroX (a genome-wide array for neurodegenerative disorders), TaqMan allelic discrimination assay, or Sanger sequencing. ONDRISeq consistently generated high-quality genotyping calls and on average, 92% of targeted bases are covered by at least 30 reads. We also observed 100% concordance for the variants identified via ONDRISeq and validated by other genomic technologies. We were successful in detecting known as well as novel rare variants in 72.2% of cases although not all variants are disease-causing. Using ONDRISeq, we also found that the APOE E4 allele had a frequency of 0.167 in these samples. Our optimised workflow highlights next-generation sequencing as a robust tool in elucidating the genetic basis of neurodegenerative diseases by screening multiple candidate genes simultaneously.