Identification of two novel loci for dominantly inherited familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, adult-onset motor neuron disease that arises as a dominantly inherited trait in approximately 10% of ALS cases. Mutations in one gene, cytosolic Cu/Zn superoxide dismutase (SOD1), account for approximately 25% of familial ALS (FALS) cases. We have performed a genetic linkage screen in 16 pedigrees with FALS with no evidence for mutations in the SOD1 gene and have identified novel ALS loci on chromosomes 16 and 20. The analysis of these genes will delineate pathways implicated as determinants of motor-neuron viability and provide insights into possible therapies for ALS.     

Recent advances in research on neuropathological aspects of familial amyotrophic lateral sclerosis with superoxide dismutase 1 gene mutations: neuronal Lewy body-like hyaline inclusions and astrocytic hyaline inclusions.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that primarily involves the motor neuron system. Of all patients with ALS, approximately 5%-10% of them are familial and most of the others are sporadic. Superoxide dismutase 1 (SOD1) gene mutations are shown to be associated with about 20% of familial ALS (FALS) patients. FALS is neuropathologically classified into two subtypes: classical FALS in which degeneration is restricted to only motor neurons and FALS which is characterized by the degeneration of the posterior column in addition to the lesion of the motor neuron system. The neuronal Lewy body-like hyaline inclusion (LBHI) is a characteristic neuropathological marker of mutant SOD1-linked FALS with posterior column involvement. Inclusions similar to the neuronal LBHIs have been discovered in astrocytes in certain patients with FALS exhibiting SOD1 gene mutations. The purpose of this review is to discuss the novel neuropathological significance of the astrocytic hyaline inclusions (Ast-HIs) and neuronal LBHIs in brain tissues from individuals with the posterior-column-involvement-type FALS with SOD1 gene mutations. In hematoxylin and eosin preparations, both Ast-HIs and neuronal LBHIs are eosinophilic inclusions and sometimes show eosinophilic cores with paler peripheral halos. Immunohistochemically, both inclusions are intensely positive for SOD1. At the ultrastructural level, both inclusions consist of approximately 15-25 nm-sized granule-coated fibrils and granular materials. Immunoelectron microscopically, these abnormal granule-coated fibrils and granular materials are positive for SOD1. Therefore, the FALS disease process originating from SOD1 gene mutations occurs in astrocytes as well as neurons and is involved in the formation of both inclusions.     

Transduction of familial amyotrophic lateral sclerosis-related mutant PEP-1-SOD proteins into neuronal cells

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by the selective death of motor neurons. Mutations in the SOD1 gene are responsible for a familial form of ALS (FALS). Although many studies suggest that mutant SOD1 proteins are cytotoxic, the mechanism is not fully understood. To investigate the role of mutant SOD1 in FALS, human SOD1 genes were fused with a PEP-1 peptide in a bacterial expression vector to produce in-frame PEP-1-SOD fusion proteins (wild type and mutants). The expressed and purified PEP-1-SOD fusion proteins were efficiently transduced into neuronal cells. Neurones harboring the A4V, G93A, G85R, and D90A mutants of PEP-1-SOD were more vulnerable to oxidative stress induced by paraquat than those harboring wild-type proteins. Moreover, neurones harboring the mutant SOD proteins had lower heat shock protein (Hsp) expression levels than those harboring wild-type SOD. The effects of the transduced SOD1 fusion proteins may provide an explanation for the association of SOD1 with FALS, and Hsps could be candidate agents for the treatment of ALS.     

Molecular Analyses of the Cu/Zn Superoxide Dismutase Gene in Patients with Familial Amyotrophic Lateral Sclerosis (ALS) in Japan

1. Amyotrophic lateral sclerosis (ALS) is a degenerative disorder characterized by selective damage to the neural system that mediates voluntary movement. Although the pathophysiologic process of ALS remains unknown, about 5 to 10% of cases are familial. According to genetic linkage studies, the familial ALS (FALS) gene has been mapped on chromosome 21 in some families and recent work identified some different missense mutations in the Cu/Zn superoxide dismutase gene in FALS families.2. We recently identified five mutations in six FALS families. The mutations identified in our FALS families are H46R, L84V, I104F, S134N, and V148I. The H46R mutation that locates in the active site of Cu/Zn SOD gene is associated with two Japanese families with very slow progression of ALS. On the other hand, the L84V mutation associated with a rapidly progressive loss of motor function with predominant lower motor neuron manifestations.3. In the family with the V148I, the phenotype of the patient varied very much among the affected members. One case had weakness of the lower extremities at first and died without bulbar paresis. The second case first noticed wasting of the upper limbs with bulbar symptoms, but the third had weakness of upper extremities without developing dysarthria nor dysphagia until death. These mutations account for 50% of all FALS families screened, although Cu/Zn SOD gene mutations are responsible for less than about 13–21% in the Western population.4. Our results indicate that the progression of disease with mutations of Cu/Zn SOD is well correlated with each mutation. The exact mechanism by which the abnormal Cu/Zn SOD molecules selectively affect the function of motor neurons is still unknown.     

Metabolic Dysfunction in Familial, but Not Sporadic, Amyotrophic Lateral Sclerosis

Abstract: Autosomal dominant familial amyotrophic lateral sclerosis (FALS) is associated with mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1). Previous studies have implicated the involvement of metabolic dysfunction in ALS pathogenesis. To further investigate the biochemical features of FALS and sporadic ALS (SALS), we examined SOD activity and mitochondrial oxidative phosphorylation enzyme activities in motor cortex (Brodmann area 4), parietal cortex (Brodmann area 40), and cerebellum from control subjects, FALS patients with and without known SOD mutations, SALS patients, and disease controls (Pick's disease, progressive supranuclear palsy, diffuse Lewy body disease). Cytosolic SOD activity, predominantly Cu/Zn SOD, was decreased ∼50% in all regions in FALS patients with SOD mutations but was not significantly altered in other patient groups. Marked increases in complex I and II–III activities were seen in FALS patients with SOD mutations but not in SALS patients. We also measured electron transport chain enzyme activities in a transgenic mouse model of FALS. Complex I activity was significantly increased in the forebrain of 60-day-old G93A transgenic mice overexpressing human mutant SOD1, relative to levels in transgenic wild-type animals, supporting the hypothesis that the motor neuron disorder associated with SOD1 mutations involves a defect in mitochondrial energy metabolism.     

Identification of new mutations in the Cu/Zn superoxide dismutase gene of patients with familial amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder affecting motor neurons. Although most cases of ALS are sporadic, approximately 10% are inherited as an autosomal dominant trait. Mutations in the Cu/Zn superoxide dismutase gene (SOD 1) are responsible for a fraction of familial ALS (FALS). Screening our FALS kindreds by SSCP, we have identified mutations in 15 families, of which 9 have not been previously reported. Two of the new mutations alter amino acids that have never been implicated in FALS. One of them affects a highly conserved amino acid involved in dimer contact, and the other one affects the active-site loop of the enzyme. These two mutations reduce significantly SOD 1 enzyme activity in lymphoblasts. Our results suggest that SOD 1 mutations are responsible for > or = 13% of FALS cases.     

Aberrant localization of FUS and TDP43 is associated with misfolding of SOD1 in amyotrophic lateral sclerosis

Background

Amyotrophic lateral sclerosis (ALS) is incurable and characterized by progressive paralysis of the muscles of the limbs, speech and swallowing, and respiration due to the progressive degeneration of voluntary motor neurons. Clinically indistinguishable ALS can be caused by genetic mutations of Cu/Zn superoxide dismutase (SOD1), TAR-DNA binding protein 43 (TDP43), or fused in sarcoma/translocated in liposarcoma (FUS/TLS), or can occur in the absence of known mutation as sporadic disease. In this study, we tested the hypothesis that FUS/TLS and TDP43 gain new pathogenic functions upon aberrant accumulation in the cytosol that directly or indirectly include misfolding of SOD1.

Methodology/Principal Findings

Patient spinal cord necropsy immunohistochemistry with SOD1 misfolding-specific antibodies revealed misfolded SOD1 in perikarya and motor axons of SOD1-familial ALS (SOD1-FALS), and in motor axons of R521C-FUS FALS and sporadic ALS (SALS) with cytoplasmic TDP43 inclusions. SOD1 misfolding and oxidation was also detected using immunocytochemistry and quantitative immunoprecipitation of human neuroblastoma SH-SY5Y cells as well as cultured murine spinal neural cells transgenic for human wtSOD1, which were transiently transfected with human cytosolic mutant FUS or TDP43, or wtTDP43.

Conclusion/Significance

We conclude that cytosolic mislocalization of FUS or TDP43 in vitro and ALS in vivo may kindle wtSOD1 misfolding in non-SOD1 FALS and SALS. The lack of immunohistochemical compartmental co-localization of misfolded SOD1 with cytosolic TDP43 or FUS suggests an indirect induction of SOD1 misfolding followed by propagation through template directed misfolding beyond its site of inception. The identification of a final common pathway in the molecular pathogenesis of ALS provides a treatment target for this devastating disease.     

Evidence of Increased Oxidative Damage in Both Sporadic and Familial Amyotrophic Lateral Sclerosis

Abstract: Some cases of autosomal dominant familial amyotrophic lateral sclerosis (FALS) are associated with mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1), suggesting that oxidative damage may play a role in ALS pathogenesis. To further investigate the biochemical features of FALS and sporadic ALS (SALS), we examined markers of oxidative damage to protein, lipids, and DNA in motor cortex (Brodmann area 4), parietal cortex (Brodmann area 40), and cerebellum from control subjects, FALS patients with and without known SOD mutations, SALS patients, and disease controls (Pick's disease, progressive supranuclear palsy, diffuse Lewy body disease). Protein carbonyl and nuclear DNA 8-hydroxy-2'-deoxyguanosine (OH⁸dG) levels were increased in SALS motor cortex but not in FALS patients. Malondialdehyde levels showed no significant changes. Immunohistochemical studies showed increased neuronal staining for hemeoxygenase-1, malondialdehyde-modified protein, and OH⁸dG in both SALS and FALS spinal cord. These studies therefore provide further evidence that oxidative damage may play a role in the pathogenesis of neuronal degeneration in both SALS and FALS.     

A Novel Locus for Familial Amyotrophic Lateral Sclerosis, on Chromosome 18q

Amyotrophic lateral sclerosis (ALS) is an adult-onset degenerative disorder characterized by the death of motor neurons in the cortex, brain stem, and spinal cord. Despite intensive research the basic pathophysiology of ALS remains unclear. Although most cases are sporadic, approximately 10% of ALS cases are familial (FALS). Mutations in the Cu/Zn superoxide dismutase (SOD1) gene cause approximately 20% of FALS. The gene(s) responsible for the remaining 80% of FALS remain to be found. Using a large European kindred without SOD1 mutation and with classic autosomal dominant adult-onset ALS, we have identified a novel locus by performing a genome scan and linkage analysis. The maximum LOD score is 4.5 at recombination fraction 0.0, for polymorphism D18S39. Haplotype analysis has identified a 7.5-cM, 8-Mb region of chromosome 18q21, flanked by markers D18S846 and D18S1109, as a novel FALS locus.     

Genetics of familial Amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a late onset, rapidly progressive and ultimately fatal neurodegenerative disease, caused by the loss of motor neurons in the brain and spinal cord. About 10% of all ALS cases are familial (FALS), and constitute a clinically and genetically heterogeneous entity. To date, FALS has been linked to mutations in 10 different genes and to four additional chromosomal loci. Research on FALS genetics, and in particular the discoveries of mutations in the SOD1, TARDBP, and FUS genes, has provided essential information toward the understanding of the pathogenesis of ALS in general. This review presents a tentative classification of all FALS-associated genes identified so far.     

Unique characteristics of the genetics epidemiology of amyotrophic lateral sclerosis in China

Continual discoveries of new genes and unraveling the genetic etiology in amyotrophic lateral sclerosis(ALS) have provided greater insight into the underlying pathogenesis in motor neuron degeneration, as well as facilitating the disease modeling and the testing of targeted therapeutics. While, the genetic etiology accounted for two-thirds of FALS and approximately 11% of SALS in Caucasians. However, the contributions of these causative genes to ALS vary among different populations. Furthermore, the prominent difference between Chinese population and other ethnics remains a source of ongoing debate. We systemically reviewed genetics literature of Chinese ALS populations and updated the mutation frequencies of the main ALS-implicated genes aiming to determine the genetic features of ALS in Chinese population. We also reviewed the associations between ALSimplicated single nucleotide polymorphisms(SNPs) and the risk of ALS in Chinese population. A total of 116 studies were included in this analysis(86 gene mutation study articles and 30 SNPs study articles). The results showed that the overall gene mutation rates of ALS-related causative genes were 55.0% in familial ALS(FALS) and 11.7% in sporadic ALS(SALS) in Chinese population. In Chinese FALS, the highest mutation frequency was found in SOD1 gene(25.6%), followed by FUS(5.8%), TARDBP(5.8%), DCTN1(3.6%) and C9 orf 72(3.5%). In Chinese SALS, the highest mutation frequency was also identified in SOD1 gene(1.6%), followed by ANXA11(1.4%), FUS(1.3%), SQSTM1(1.0%), OPTN(0.9%) and CCNF(0.8%).The associations between several SNPs and risk of ALS were also reported in Chinese population. The genetic features of ALS in Chinese population are significantly different from those in Caucasian population, indicating an association between genetic susceptibility and origin of population. Further explorations are required to understand the gene complexity of ALS, including the contribution of most minor genes and the molecular mechanisms in ALS pathologies.     

Astrocytes from familial and sporadic ALS patients are toxic to motor neurons

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease, with astrocytes implicated as contributing substantially to motor neuron death in familial (F)ALS. However, the proposed role of astrocytes in the pathology of ALS derives in part from rodent models of FALS based upon dominant mutations within the superoxide dismutase 1 (SOD1) gene, which account for <2% of all ALS cases. Their role in sporadic (S)ALS, which affects >90% of ALS patients, remains to be established. Using astrocytes generated from postmortem tissue from both FALS and SALS patients, we show that astrocytes derived from both patient groups are similarly toxic to motor neurons. We also demonstrate that SOD1 is a viable target for SALS, as its knockdown significantly attenuates astrocyte-mediated toxicity toward motor neurons. Our data highlight astrocytes as a non-cell autonomous component in SALS and provide an in vitro model system to investigate common disease mechanisms and evaluate potential therapies for SALS and FALS.     

Rapid Communication: Cu/Zn Superoxide Dismutase Activity in Familial and Sporadic Amyotrophic Lateral Sclerosis

Abstract: Amyotrophic lateral sclerosis (ALS) is a degenerative motor neuron disease that is inherited as an autosomal dominant trait in ~ 10% of cases. Recently we and others identified several single-base mutations in the Cu/Zn superoxide dismutase (SOD1) gene in patients with familial ALS (FALS). Using single-strand conformational polymorphism, we studied the C to G mutation in exon 2 of the SOD1 gene (resulting in a leucine to valine substitution in position 38) in affected and unaffected members of a large Belgian family with FALS. We measured the SOD1 activity in red blood cell lysates in 14 members of this family, including the only surviving clinically affected patient. SOD1 activity of the family members carrying the mutation was less than half that of members without the mutation. In addition, in 11 patients with sporadic ALS and 11 age- and sex-matched controls, red blood cell SOD1 activity was normal. These studies indicate that SOD1 activity is reduced in these FALS patients but not in sporadic ALS patients. Moreover, this SOD1 enzyme abnormality is detectable years before onset of clinical ALS in carriers of this FALS mutation.     

Superoxide Dismutase Activity, Oxidative Damage, and Mitochondrial Energy Metabolism in Familial and Sporadic Amyotrophic Lateral Sclerosis

The cause of neuronal death in amyotrophic lateral sclerosis (ALS) is unknown. Recently, it was found that some patients with autosomal-dominant familial ALS (FALS) have point mutations in the gene that encodes Cu/Zn superoxide dismutase (SOD1). In this study of postmortem brain tissue, we examined SOD activity and quantified protein carbonyl groups, a marker of oxidative damage, in samples of frontal cortex (Brodmann area 6) from 10 control patients, three FALS patients with known SOD1 mutations (FALS-1), one autosomal-dominant FALS patient with no identifiable SOD1 mutations (FALS-0), and 11 sporadic ALS (SALS) patients. Also, we determined the activities of components of the electron transport chain (complexes I, II-III, and IV) in these samples. The cytosolic SOD activity, which is primarily SOD1 activity, was reduced by 38.8% (p < 0.05) in the FALS-1 patients and not significantly altered in the SALS patients or the FALS-0 patient relative to the control patients. The mitochondrial SOD activity, which is primarily SOD2 activity, was not significantly altered in the FALS-1, FALS-0, or SALS patients. The protein carbonyl content was elevated by 84.8% (p < 0.01) in the SALS patients relative to the control patients. Finally, the complex I activity was increased by 55.3% (p < 0.001) in the FALS-1 patients relative to the control patients. These results from cortical tissue demonstrate that SOD1 activity is reduced and complex I activity is increased in FALS-1 patients and that oxidative damage to proteins is increased in SALS patients.     

Superoxide Dismutase Concentration and Activity in Familial Amyotrophic Lateral Sclerosis

Abstract: Some cases of autosomal-dominant familial amyotrophic lateral sclerosis (FALS) have been associated with mutations in SOD1 , the gene that encodes Cu/Zn superoxide dismutase (Cu/Zn SOD). We determined the concentrations (µg of Cu/Zn SOD/mg of total protein), specific activities (U/µg of total protein), and apparent turnover numbers (U/µmol of Cu/Zn SOD) of Cu/Zn SOD in erythrocyte lysates from patients with known SOD1 mutations. We also measured the concentrations and activities of Cu/Zn SOD in FALS patients with no identifiable SOD1 mutations, sporadic ALS (SALS) patients, and patients with other neurologic disorders. The concentration and specific activity of Cu/Zn SOD were decreased in all patients with SOD1 mutations, with mean reductions of 51 and 46%, respectively, relative to controls. In contrast, the apparent turnover number of the enzyme was not altered in these patients. For the six mutations studied, there was no correlation between enzyme concentration or specific activity and disease severity, expressed as either duration of disease or age of onset. No significant alterations in the concentration, specific activity, or apparent turnover number of Cu/Zn SOD were detected in the FALS patients with no identifiable SOD1 mutations, SALS patients, or patients with other neurologic disorders. That Cu/Zn SOD concentration and specific activity are equivalently reduced in erythrocytes from patients with SOD1 mutations suggests that mutant Cu/Zn SOD is unstable in these cells. That concentration and specific activity do not correlate with disease severity suggests that an altered, novel function of the enzyme, rather than reduction of its dismutase activity, may be responsible for the pathogenesis of FALS.     

Treatment with arimoclomol, a coinducer of heat shock proteins, delays disease progression in ALS mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative condition in which motoneurons of the spinal cord and motor cortex die, resulting in progressive paralysis. This condition has no cure and results in eventual death, usually within 1-5 years of diagnosis. Although the specific etiology of ALS is unknown, 20% of familial cases of the disease carry mutations in the gene encoding Cu/Zn superoxide dismutase-1 (SOD1). Transgenic mice overexpressing human mutant SOD1 have a phenotype and pathology that are very similar to that seen in human ALS patients. Here we show that treatment with arimoclomol, a coinducer of heat shock proteins (HSPs), significantly delays disease progression in mice expressing a SOD1 mutant in which glycine is substituted with alanine at position 93 (SOD1(G93A)). Arimoclomol-treated SOD1(G93A) mice show marked improvement in hind limb muscle function and motoneuron survival in the later stages of the disease, resulting in a 22% increase in lifespan. Pharmacological activation of the heat shock response may therefore be a successful therapeutic approach to treating ALS, and possibly other neurodegenerative diseases.     

Integrative role of cPLA with COX-2 and the effect of non-steriodal anti-inflammatory drugs in a transgenic mouse model of amyotrophic lateral sclerosis

Cyclooxygenase-2 (COX-2) is a key molecule in the inflammatory pathway in amyotrophic lateral sclerosis (ALS). Cytosolic phospholipase A (cPLA2) is an important enzyme providing substrate for cyclooxygenases. We therefore examined cPLA2 expression in human ALS and mutant Cu/Zn superoxide dismutase (SOD1) transgenic mice and its relation to COX-2. Immunohistochemistry and real-time RT-PCR revealed elevated cPLA2 protein and its mRNA levels in the lumbar spinal cord of mutant SOD1 mice. COX-2 immunoreactivity was increased in lumbar spinal cord sections from both familial ALS (FALS) and sporadic ALS (SALS) as compared to controls, and cPLA2 immunoreactivity was increased in a patient with FALS. Oral administration of the non-selective cyclooxygenase (COX) inhibitor, sulindac, extended the survival (by 10%) of G93A SOD1 mice as compared to littermate controls. Sulindac, as well as the selective COX-2 inhibitors, rofecoxib and celecoxib reduced cPLA2 immunoreactivity in the lumbar spinal cord of G93A transgenic mice. Sulindac treatment preserved motor neurons, and reduced microglial activation and astrocytosis, in the spinal cord of G93A SOD1 transgenic mice. These results suggest that cPLA2 plays an important role in supplying arachidonic acid to the COX-2 driven inflammatory pathway in ALS associated with SOD1 mutations.     

Elevated "Hydroxyl Radical" Generation In Vivo in an Animal Model of Amyotrophic Lateral Sclerosis

Abstract: Mutations in the enzyme copper/zinc superoxide dismutase-1 (SOD1) are associated with familial amyotrophic lateral sclerosis (FALS). The means by which the mutations cause FALS appears to be due to an adverse property of the mutant SOD1 protein that may involve increased generation of free radicals. We used in vivo microdialysis to measure the conversion of 4-hydroxybenzoic acid to 3,4-dihydroxybenzoic acid (3,4-DHBA) as a measure of "hydroxyl radical-like" production in transgenic amyotrophic lateral sclerosis (ALS) mice with the G93A mutation as well as littermate controls. The conversion of 4-hydroxybenzoic acid to 3,4-DHBA was significantly increased in the striatum of transgenic ALS mice at baseline but not in mice overexpressing wild-type human SOD1. Following administration of 3-nitropropionic acid 3,4-DHBA generation was significantly increased as compared with baseline, and the increase in the transgenic ALS mice was significantly greater than those in controls, whereas the increase in mice overexpressing wild-type human SOD1 was significantly attenuated. The present results provide in vivo evidence that expression of mutations in SOD1 can lead to increased generation of "hydroxyl radical-like" activity, which further implicates oxidative damage in the pathogenesis of ALS.     

Expression of Human A4V Mutant Cu,Zn Superoxide Dismutase in Schizosaccharomyces pombe: Investigations of its Toxic Properties

Cu,Zn superoxide dismutase (SOD1) is an antioxidant enzyme that catalyzes the removal of superoxide radicals generated in various biological oxidations. Amyotrophic lateral sclerosis (ALS) is one of the most common neurodegenerative disorders, occurring in families (FALS) and sporadically (SALS). FALS and SALS are distinguishable genetically but not clinically. More than 100 point mutations in the human SOD 1 gene have been identified that cause FALS. In order to determine the effects of mutant SOD protein, we first cloned wild-type and A4V mutant human SOD1 into Schizosaccharomyces pombe. This study shows viabilities and some antioxidant properties including SOD, catalase, proteasomal activity, and protein carbonyl levels of transformants in SOD1 deleted strain (MN415); and its parental strain (JY741) at different stress conditions. There was no more oxidative damage in the human mutant SOD carrying the transformant strain compared with other strains. These results may help to explain whether ALS progresses as a consequence of cellular oxidative damage.     

A new familial amyotrophic lateral sclerosis locus on chromosome 16q12.1-16q12.2

Familial amyotrophic lateral sclerosis (FALS) affects 5%-10% of cases of amyotrophic lateral sclerosis (ALS) and is inherited as an autosomal dominant condition with incomplete penetrance. One-fifth of these cases of FALS are associated with mutations in copper/zinc-dependent superoxide dismutase (SOD1), but the gene defect in the remaining 80% of familial cases is, as yet, unknown. We have carried out a preliminary genome screen, using a U.K. resource of families lacking SOD1 mutations, to identify other potential disease loci and have identified a putative locus on chromosome 16q12.1-q12.2. The region associated with disease was further refined in the major family that contributed to this result and was localized to D16S409-D16S3032, a 14.74-cM genetic interval that corresponds to a physical distance of 6.6 Mb, which coincides with a region independently identified by two further research groups in the United States and the United Kingdom.