Heema Patel

Heterozygous missense mutations in BSCL2 are associated with distal hereditary motor neuropathy and Silver syndrome

Distal hereditary motor neuropathy (dHMN) or distal spinal muscular atrophy (OMIM #182960) is a heterogeneous group of disorders characterized by an almost exclusive degeneration of motor nerve fibers, predominantly in the distal part of the limbs. Silver syndrome (OMIM #270685) is a rare form of hereditary spastic paraparesis mapped to chromosome 11q12–q14 (SPG17) in which spasticity of the legs is accompanied by amyotrophy of the hands and occasionally also the lower limbs. Silver syndrome and most forms of dHMN are autosomal dominantly inherited with incomplete penetrance and a broad variability in clinical expression. A genome-wide scan in an Austrian family with dHMN-V (ref. 4) showed linkage to the locus SPG17, which was confirmed in 16 additional families with a phenotype characteristic of dHMN or Silver syndrome. After refining the critical region to 1 Mb, we sequenced the gene Berardinelli-Seip congenital lipodystrophy (BSCL2) and identified two heterozygous missense mutations resulting in the amino acid substitutions N88S and S90L. Null mutations in BSCL2, which encodes the protein seipin, were previously shown to be associated with autosomal recessive Berardinelli-Seip congenital lipodystrophy (OMIM #269700). We show that seipin is an integral membrane protein of the endoplasmic reticulum (ER). The amino acid substitutions N88S and S90L affect glycosylation of seipin and result in aggregate formation leading to neurodegeneration.

Sequence alterations within CYP7B1 implicate defective cholesterol homeostasis in motor-neuron degeneration

The hereditary spastic paraplegias (HSPs) are a genetically and clinically heterogeneous group of upper-motor-neuron degenerative diseases characterized by selective axonal loss in the corticospinal tracts and dorsal columns. Although numerous mechanisms involving defective subcellular transportation, mitochondrial malfunction, and increased oxidative stress have been proposed, the pathogenic basis underlying the neuronal loss is unknown. We have performed linkage analysis to refine the extent of the SPG5 disease locus and conducted sequence analysis of the genes located within this region. This identified sequence alterations in the cytochrome P450-7B1 (CYP7B1) associated with this pure form of HSP. In the liver, CYP7B1 offers an alternative pathway for cholesterol degradation and also provides the primary metabolic route for the modification of dehydroepiandrosterone neurosteroids in the brain. These findings provide the first direct evidence of a pivotal role of altered cholesterol metabolism in the pathogenesis of motor-neuron degenerative disease and identify a potential for therapeutic intervention in this form of HSP.

Derivation and feeder-free propagation of human embryonic stem cells under xeno-free conditions

BACKGROUND AIMS Human embryonic stem (hES) cells hold great potential for cell therapy and regenerative medicine because of their pluripotency and capacity for self-renewal. The conditions used to derive and culture hES cells vary between and within laboratories depending on the desired use of the cells. Until recently, stem cell culture has been carried out using feeder cells, and culture media, that contain animal products. Recent advances in technology have opened up the possibility of both xeno-free and feeder-free culture of stem cells, essential conditions for the use of stem cells for clinical purposes. To date, however, there has been limited success in achieving this aim. METHODS, RESULTS AND CONCLUSIONS Protocols were developed for the successful derivation of two normal and three specific mutation-carrying (SMC) (Huntingtons disease and myotonic dystrophy 1) genomically stable hES cell lines, and their adaptation to feeder-free culture, all under xeno-free conditions.

Defective mitochondrial mRNA maturation is associated with spastic ataxia.

In human mitochondria, polyadenylation of mRNA, undertaken by the nuclear-encoded mitochondrial poly(A) RNA polymerase, is essential for maintaining mitochondrial gene expression. Our molecular investigation of an autosomal-recessive spastic ataxia with optic atrophy, present among the Old Order Amish, identified a mutation of MTPAP associated with the disease phenotype. When subjected to poly(A) tail-length assays, mitochondrial mRNAs from affected individuals were shown to have severely truncated poly(A) tails. Although defective mitochondrial DNA maintenance underlies a well-described group of clinical disorders, our findings reveal a defect of mitochondrial mRNA maturation associated with human disease and imply that this disease mechanism should be considered in other complex neurodegenerative disorders.

Troyer syndrome revisited: A clinical and radiological study of a complicated hereditary spastic paraplegia

Abstract.Troyer syndrome, originally described in 1967 in an Old Order Amish population, is a complicated form of hereditary spastic paraplegia (HSP) inherited in an autosomal recessive fashion and slowly progressive. The cardinal features are spastic paraparesis, pseudobulbar palsy and distal amyotrophy, together with mild developmental delay and subtle skeletal abnormalities. We report a detailed evaluation of 21 cases of Troyer syndrome in the same Amish population, including three from the original study. Imaging of the brain revealed white matter abnormalities, particularly in the temporoparietal periventricular area. This study, coupled with the recent identification of the gene responsible (SPG20, encoding spartin), increases our understanding of this form of HSP.

A novel NIPA1 mutation associated with a pure form of autosomal dominant hereditary spastic paraplegia

The hereditary spastic paraplegias (HSPs) are a clinically and genetically heterogeneous group of neurodegenerative disorders characterised by lower limb spasticity and weakness. Mutations in NIPA1 (Nonimprinted in Prader-Willi/Angelman syndrome 1) have recently been identified as a cause of autosomal dominant pure HSP, with one mutation described in two unrelated families. NIPA1 has no known function but is predicted to possess nine transmembrane domains and may function as a receptor or transporter. Here we present a large British pedigree in which linkage analysis conclusively demonstrates linkage to the NIPA1 locus (maximum multipoint LOD score 4.6). Subsequent mutation analysis identified a novel missense substitution in a highly conserved NIPA1 residue (G106R) which further confirms a causative link between NIPA1 mutation and autosomal dominant hereditary spastic paraplegia.

Safety paradigm: genetic evaluation of therapeutic grade human embryonic stem cells

The use of stem cells for regenerative medicine has captured the imagination of the public, with media attention contributing to rising expectations of clinical benefits. Human embryonic stem cells (hESCs) are the best model for capital investment in stem cell therapy and there is a clear need for their robust genetic characterization before scaling-up cell expansion for that purpose. We have to be certain that the genome of the starting material is stable and normal, but the limited resolution of conventional karyotyping is unable to give us such assurance. Advanced molecular cytogenetic technologies such as array comparative genomic hybridization for identifying chromosomal imbalances, and single nucleotide polymorphism analysis for identifying ethnic background and loss of heterozygosity should be introduced as obligatory diagnostic tests for each newly derived hESC line before it is deposited in national stem cell banks. If this new quality standard becomes a requirement, as we are proposing here, it would facilitate and accelerate the banking process, since end-users would be able to select the most appropriate line for their particular application, thus improving efficiency and streamlining the route to manufacturing therapeutics. The pharmaceutical industry, which may use hESC-derived cells for drug screening, should not ignore their genomic profile as this may risk misinterpretation of results and significant waste of resources.

Strategy for the creation of clinical grade hESC line banks that HLA-match a target population.

Here, we describe a pre‐derivation embryo haplotyping strategy that we developed in order to maximize the efficiency and minimize the costs of establishing banks of clinical grade hESC lines in which human leukocyte antigen (HLA) haplotypes match a significant proportion of the population. Using whole genome amplification followed by medium resolution HLA typing using PCR amplification with sequence‐specific primers (PCR‐SSP), we have typed the parents, embryos and hESC lines from three families as well as our eight clinical grade hESC lines and shown that this technical approach is rapid, reliable and accurate. By employing this pre‐derivation strategy where, based on HLA match, embryos are selected for a GMP route on day 3–4 of development, we would have drastically reduced our cGMP laboratory running costs.

SPG3A mutation screening in English families with early onset autosomal dominant hereditary spastic paraplegia.

Mutations in the SPG3A gene encoding the novel GTPase atlastin have recently been implicated in causing autosomal dominant hereditary spastic paraplegia (ADHSP) in six unrelated families. The phenotype of affected individuals in all cases has been of an early onset uncomplicated form of the disease. One particular missense mutation, R239C, in exon 7 of SPG3A has been identified in three of these families. We performed mutation screening by direct sequencing of all 14 exons and flanking sequences of the SPG3A gene in affected individuals from 12 unrelated English families, all with an early onset uncomplicated ADHSP in whom spastin mutations had previously been excluded. The R239C mutation was found to co-segregate with the disease in one English ADHSP family confirming a widespread prevalence for this commonly occurring mutation. No additional SPG3A mutations were identified in the remaining 11 families suggesting that even within this specific sub-set of early onset uncomplicated ADHSP patients atlastin mutations are relatively rare.

Homozygosity at chromosome 8qter in individuals affected by mal de Meleda (Meleda disease) originating from the island of Meleda.

Background The inherited palmoplantar keratodermas (PPKs) are a clinically heterogeneous group of disorders characterized by thickening of the skin of the palms and the soles. These diseases also exhibit genetic heterogeneity and many autosomal dominant and recessive forms have been described. Mal de Meleda (Meleda disease, MD) is an autosomal recessive form of PPK first described on the Dalmatian island of Meleda. A gene for MD has recently been assigned to the most telomeric portion of chromosome 8q using two large Algerian families.