A novel nuclear protein, 5qNCA (LOC51780) is a candidate for the myeloid leukemia tumor suppressor gene on chromosome 5 band q31.

Interstitial deletion or loss of chromosome 5, del(5q) or -5, is a frequent finding in myeloid leukemias and myelodysplasias, suggesting the presence of a tumor suppressor gene within the deleted region. In our search for this gene, we identified a candidate, 5qNCA (LOC51780), which lies within a consistently-deleted segment of 5q31. 5qNCA expresses a 7.2-kb transcript with a 5286-bp open reading frame which is present at high levels in heart, skeletal muscle, kidney, placenta, and liver as well as CD34+ cells and AML cell lines. 5qNCA encodes a 191-kD nuclear protein which contains a highly-conserved C-terminus containing a zinc finger with the unique spacing Cys-X2-Cys-X7-His-X2-Cys-X2-Cys-X4-Cys-X2-Cys and a jmjC domain, which is often found in proteins that regulate chromatin remodeling. Expression of 5qNCA in a del(5q) cell line results in suppression of clonogenic growth. Preliminary sequence results in AML and MDS samples and cell lines has revealed a possible mutation in the KG-1 cell line resulting in a THR to ALA substitution that has not been found in over 100 normal alleles to date. We propose 5qNCA is a good candidate for the del(5q) tumor suppressor gene based on its predicted function and growth suppressive activities, and suggest that further mutational and functional study of this interesting gene is warranted.

Highly abundant genes in the transcriptosome of human and baboon CD34 antigen-positive bone marrow cells.

Nonhuman primates are useful large animal model systems for the in vivo study of hematopoietic stem cell biology. To better understand the degree of similarity of the hematopoietic systems between humans and baboons, and to explore the relevance of such studies in nonhuman primates to humans, this study was designed to compare the global gene expression profile of bone marrow CD34(+) cells isolated from these 2 species. Human complementary DNA (cDNA) filter arrays containing 25 920 human cDNAs were surveyed for this purpose. The expression pattern and relative gene abundance of the 2 RNA sources were similar, with a correlation coefficient of 0.87. A total of 15 970 of these cDNAs were expressed in human CD34(+) cells, of which the majority (96%) varied less than 3-fold in their relative level of expression between human and baboon. Reverse transcriptase-polymerase chain reaction analysis of selected genes confirmed that expression was comparable between the 2 species. No species-restricted transcripts have been identified, further reinforcing the high degree of similarity between the 2 populations. A subset of 1554 cDNAs, which are expressed at levels 100-fold and greater than background, is described, which includes 959 expressed sequence tags and uncharacterized cDNAs, and 595 named genes, including many that are clearly involved in hematopoiesis. The cDNAs reported here represent a selection of some of the most highly abundant genes in hematopoietic cells and provide a starting point to develop a profile of the transcriptosome of CD34(+) cells.

Cytogenetic and molecular diagnosis of chromosome 5 deletions in myelodysplasia.

Deletions of chromosome 5, del(5q), are frequently observed in myelodysplasia (MDS). We evaluated molecular detection of loss of heterozygosity (LOH) as a diagnostic method to detect del(5q) in a series of 60 MDS cases at a single institution. LOH was compared to cytogenetics on the same clinical specimen, resolving ambiguous cases by fluorescent in situ hybridization (FISH) and additional LOH. There was poor concordance between molecular and cytogenetic results, but most discrepancies could be resolved by FISH and additional LOH. Molecular analysis was of low sensitivity because most cases contained a relatively high proportion of cells without del(5q), but it was accurate, while cytogenetics overestimated the proportion of cells with del(5q) and failed to detect some cases with complex rearrangements. Minor clones were detected both by FISH and LOH. Overall, we found an incidence of 23% (14 of 60 cases) for del(5q) in MDS. The results also suggest that there is a high degree of genetic heterogeneity in the cellular population of MDS. Although del(5q) is common in MDS, it may not be present in all cells, leading to diagnostic challenges.

Molecular genetics improves the management of hereditary non-polyposis colorectal cancer.

BACKGROUND: The syndrome of hereditary non-polyposis colorectal cancer (HNPCC) can be diagnosed fairly accurately using clinical criteria and a family history. Identifying HNPCC helps to prevent large-bowel cancer, or allows cancer to be treated at an early stage. Once the syndrome has been diagnosed a family member's risk can be judged approximately from a family tree, or it can now be predicted accurately if the causative mutation is known. OBJECTIVE: This study involved attempts to improve the management of a family with HNPCC over a period of 10 years. Clinical diagnostic criteria, colonoscopic surveillance, surgical treatment, genetic counselling, molecular genetic research, and finally predictive genetic testing were applied as they evolved during this time. SUBJECTS AND METHODS: A rural general practitioner first noted inherited large-bowel cancer in the family and began screening subjects as they presented, using rigid sigmoidoscopy at the local hospital. At the time that the disorder was recognised as being HNPCC (1987), screening by means of colonoscopy at our university hospital was aimed primarily at first-degree relatives of affected individuals. After realising how many were at risk, screening was brought closer to the family. A team of clinicians and researchers visited the local hospital to identify and counsel those at risk and to perform screening colonoscopy. Family members were recruited for research to find the gene and its mutation that causes the disease, to develop an accurate predictive test and to reduce the number of subjects undergoing surveillance colonoscopies. RESULTS: There are approximately 500 individuals in this family. In the 10 years of this study the number of subjects who have been counselled for increased genetic risk or who have requested colonoscopic surveillance for HNPCC in this kindred has increased from 20 to 140. After the causative mutation was found in the hMLH1 gene on chromosome 3, a test for it has reduced the number of subjects who need screening colonoscopy by over 70%. A protocol has been devised to inform family members, to acquire material for research in order to provide genetic counselling for (pre-test and post-test) risk, and to test for the mutation. Eventually, identifying those with the mutation should focus surveillance accurately. CONCLUSIONS: The benefits of restricting screening to subjects with the mutation that causes colorectal cancer and of performing operations to prevent cancer are hard to measure accurately. However, it is likely that at least half the family members will be able to avoid colonoscopic screening, some deaths from cancer should be prevented, and the cost of preventing and treating cancer in the family should fall substantially.

Myotilin is mutated in limb girdle muscular dystrophy 1A.

We have identified a mutation in the myotilin gene in a large North American family of German descent expressing an autosomal dominant form of limb girdle muscular dystrophy (LGMD1A). We have previously mapped this gene to 5q31. Symptoms of this adult onset disease are progressive weakness of the hip and shoulder girdles, as well as a distinctive dysarthric pattern of speech. Muscle of affected individuals shows degeneration of myofibers, variations in fiber size, fiber splitting, centrally located myonuclei and a large number of autophagic vesicles. Affected muscle also exhibits disorganization and streaming of the Z-line similar to that seen in nemaline myopathy. We have identified a C450T missense mutation in the myotilin gene that is predicted to result in the conversion of residue 57 from threonine to isoleucine. This mutation has not been found in 396 control chromosomes. The mutant allele is transcribed and normal levels of correctly localized myotilin protein are seen in LGMD1A muscle. Myotilin is a sarcomeric protein that binds to alpha-actinin and is localized in the Z-line. The observed missense mutation does not disrupt binding to alpha-actinin.

Delineation of a minimal interval and identification of 9 candidates for a tumor suppressor gene in malignant myeloid disorders on 5q31.

Interstitial deletion or loss of chromosome 5 is frequent in malignant myeloid disorders, including myelodysplasia (MDS) and acute myeloid leukemia (AML), suggesting the presence of a tumor suppressor gene. Loss of heterozygosity (LOH) analysis was used to define a minimal deletion interval for this gene. Polymorphic markers on 5q31 were identified using a high-resolution physical and radiation hybrid breakpoint map and applied to a patient with AML with a subcytogenetic deletion of 5q. By comparing the DNA from leukemic cells to buccal mucosa cells, LOH was detected with markers D5S476 and D5S1372 with retention of flanking markers D5S500 to D5S594. The D5S500-D5S594 interval, which covers approximately 700 kb, thus represents a minimal localization for the tumor suppressor gene. Further refinement of the physical map enabled the specification of 9 transcription units within the encompassing radiation hybrid bins and 7 in flanking bins. The 9 candidates include genes CDC25, HSPA9, EGR1, CTNNA1, and 5 unknown ESTs. Reverse-transcription polymerase chain reaction confirms that all of them are expressed in normal human bone marrow CD34(+) cells and in AML cell lines and thus represent likely candidates for the MDS-AML tumor suppressor gene at 5q31.

High-resolution physical map and transcript identification of a prostate cancer deletion interval on 8p22.

A genomic interval of approximately 1-1.5 Mb centered at the MSR marker on 8p22 has emerged as a possible site for a tumor suppressor gene, based on high rates of allele loss and the presence of a homozygous deletion found in metastatic prostate cancer. The objective of this study was to prepare a bacterial contig of this interval, integrate the contig with radiation hybrid (RH) databases, and use these resources to identify transcription units that might represent the candidate tumor suppressor genes. Here we present a complete bacterial contig across the interval, which was assembled using 22 published and 17 newly originated STSs. The physical map provides twofold or greater coverage over much of the interval, including 17 BACs, 15 P1s, 2 cosmids, and 1 PAC clone. The position of the selected markers across the interval in relation to the other markers on the larger chromosomal scale was confirmed by RH mapping using the Stanford G3 RH panel. Transcribed units within the deletion region were identified by exon amplification, searching of the Human Transcript Map, placement of unmapped expressed sequence tags (ESTs) from the Radiation Hybrid Database (RHdb), and from other published sources, resulting in the isolation of six unique expressed sequences. The transcript map of the deletion interval now includes two known genes (MSR and N33) and six novel ESTs.

Human mortalin (HSPA9): a candidate for the myeloid leukemia tumor suppressor gene on 5q31.

Human mortalin (HSPA9) was originally identified by its close homology to murine mortalins, which play important roles in cellular senescence. The two murine genes, mot-1 and mot-2, differ in only two amino acid residues, but have opposite functions in cellular immortalization. HSPA9 was recently localized to chromosome 5, band q31, a region that is frequently deleted in myeloid leukemias and myelodysplasia (MDS), making it a candidate tumor suppressor gene, which is consistent with the biological function of its murine homologue. To evaluate mortalin in this capacity, its expression in normal and leukemic cell lines was investigated, and its genomic structure was determined in order to facilitate mutation detection. RT-PCR and Northern blot analysis revealed a broad distribution in normal tissues and in leukemia cell lines, producing a single 2.8 kb transcript. Genomic characterization showed that the gene spans 18 kb, and consisted of 17 exons with boundaries that were almost identical to its murine counterpart. Using intron-based primers to flank each exon, sequence of the complete protein-coding regions was obtained for three AML cell lines, including two lines with chromosome 5 loss (KG-1 and HL-60) and one without (AML-193) compared to normal DNA. No mutations were identified although one conservative nucleotide sequence variant was observed in exon 16. We have shown that mortalin is highly conserved in genomic structure as well as sequence, and the designed primers will be suitable for future studies to detect mutations in clinical samples.

A radiation hybrid breakpoint map of the acute myeloid leukemia (AML) and limb-girdle muscular dystrophy 1A (LGMD1A) regions of chromosome 5q31 localizing 122 expressed sequences.

We have constructed a high-resolution map of a 6-Mb interval of human chromosome 5, band q31, incorporating 175 sequence tagged sites, of which 33 are genetic polymorphisms and 122 are nonredundant expressed sequences. The map was assembled initially as a YAC contig, incorporating data from radiation hybrid maps. To improve resolution and to identify errors in the databases, a radiation hybrid breakpoint map was developed for the interval, which included hybrids from both Stanford G3 and GeneBridge 4 panels. This novel approach facilitated the integration of one RH panel with another and enabled the identification and localization of new, previously unmapped ESTs from the radiation hybrid databases. ESTs were assembled into overlapping transcription units and ordered with respect to polymorphic markers in the region, resulting in a comprehensive map that incorporates markers from multiple different types of maps. This map of 5q31 will facilitate gene discovery efforts for several disorders, including limb-girdle muscular dystrophy type 1A and the genes deleted in acute myeloid leukemias and myelodysplasia. The study demonstrates the utility of a radiation hybrid breakpoint panel for correction of map errors and for the efficient identification of new transcript units in a large genomic interval.

Use of a CEPH meiotic breakpoint panel to refine the locus of limb-girdle muscular dystrophy type 1A (LGMD1A) to a 2-Mb interval on 5q31.

Limb-girdle muscular dystrophy type 1A (LGMD1A) is an autosomal dominant disease characterized by progressive weakness of the hip and shoulder girdle. The gene for LGMD1A had been localized to a 7-cM interval at 5q31 in a single large family (Family 39). To refine the localization of LGMD1A further and to aid in its identification, a high-resolution physical map of the locus was used to identify and provisionally localize 25 polymorphic markers. A subset of these markers was then ordered genetically, using a CEPH meiotic breakpoint panel, resulting in an integrated physical-genetic map of the locus. Relevant markers were genotyped on the members of Family 39 who contained informative recombination events, resulting in a further narrowing of LGMD1A to an interval bounded by D5S479 and D5S594, estimated to be 2 Mb in size. Integration of the genetic and physical map permits the identification of several transcription units from within the narrowed LGMD1A interval, including one that is muscle specific, representing candidate genes for this familial dystrophy.

Exclusion of identified LGMD1 loci from four dominant limb-girdle muscular dystrophy families.

The limb-girdle muscular dystrophies are a clinically and genetically heterogeneous group of disorders. Recent linkage analyses and positional cloning studies have identified numerous loci responsible for the recessive and dominant forms, underscoring the inherent heterogeneity. In this report, we investigate four large autosomal dominant limb-girdle pedigrees and exclude these pedigrees from linkage to these loci. In addition, there is no evidence for linkage to any of the seven recessive LGMD loci.

Modification of bacterial artificial chromosome clones using Cre recombinase: introduction of selectable markers for expression in eukaryotic cells.

Bacterial artificial chromosome clones (BACs) are widely used at present in human genome physical mapping projects. To extend the utility of these clones for functional genomic studies, we have devised a method to modify BACs using Cre recombinase to introduce a gene cassette into the loxP sequence, which is present in the vector portion of the BAC clone. Cre-mediated integration is site specific and thus maintains the integrity of the genomic insert sequences, while eliminating the steps that are involved in restriction digest-based DNA cloning strategies. The success of this method depends on the use of a DNA construct, RETRObac, which contains the reporter marker green fluorescent protein (GFP) and the selectable marker neomycin phosphotransferase (neo), but does not contain a bacterial origin of replication. BAC clones have been modified successfully using this method and the genomic insert shows no signs of deletions or rearrangements. Transfection efficiencies of the modified BACs into human or murine cell lines ranged from 1% to 6%. After culture in media containing G418 for 3 weeks, approximately 0. 1% of cells previously sorted for GFP expression acquired stable antibiotic resistance. Introduction of a human BAC clone that contains genomic p53 sequences into murine NIH3T3 cells led to expression of human p53 mRNA as determined by RT-PCR, demonstrating that sequences contained on the BAC are expressed. We believe that GFP-neo modified BAC clones will be a valuable resource in efforts to study biological effects of known genes as well as in efforts to clone and analyze new genes and regulatory regions.

Evidence for anticipation in autosomal dominant limb-girdle muscular dystrophy.

Anticipation, an increase in severity or decrease in age of onset (AO) inherent in the transmission of the disease gene from affected parent to affected child, has been increasingly described in human disease. To assess anticipation in a large kindred in which autosomal dominant limb-girdle muscular dystrophy (LGMD1A) is segregating, age of disease onset was collected from patient interviews of affected family members. A total of 25 parent-offspring pairs, in which the parents are three (3R), four (4R), or five (5R) generations removed from a common founding ancestor, were available for analysis. Life table analyses showed significant decreases in age at first reported symptoms in the offspring of the 3R (chi2=5.55, p=0.02) and 4R (chi2=7.81, p=0.005) parents. Pairwise analyses confirmed this decrease with a median decrease of 13 years in transmission to offspring from 3R parents and 18 years in transmission to offspring from 4R parents. The finding of anticipation in this pedigree suggests that the mutation in LGMD1A may be the result of the expansion of an unstable trinucleotide repeat.

Localization of the candidate tumor suppressor gene ING1 to human chromosome 13q34.

A novel gene ING1 was recently cloned and defined as a candidate tumor suppressor gene. Reduced expression and rearrangements of ING1 are found in several tumor cell lines, ING1 overexpression is associated with cell growth arrest and ING1 suppression promotes neoplastic transformation (1). Using radiation hybrid mapping technique ING1 was assigned to subtelomeric region of the long arm of human chromosome 13 (13q34) which is known to be frequently rearranged in squamous carcinomas of head and neck.

Molecular subsets and prognostic factors in acute lymphoblastic leukemia.

Leukemia is a heterogeneous collection of diseases with different molecular origins. The molecular features may provide physicians with markers that can be used to monitor disease. In acute lymphoblastic leukemia (ALL), molecular monitoring methods can be very useful in developing and improving treatment. Some argue that one of the main problems in adult ALL is that it is homogeneous but, in fact, it is a very diverse condition. However, it can't be seen in the surface markers or in the nucleus of these diseases, so there is a need to develop ways to identify these cases. Good and potentially curable subsets of ALL patients are beginning to be identified by combining traditional phenotypic and immunologic markers with molecular and genetic approaches. As treatments improve, definition of therapeutic and prognostic subsets and their significance to clinical outcome is imperative. While progress has been made in the use of molecular techniques to detect residual disease, we must still acquire experience in using these markers to monitor treatment efficacy, with the eventual aim of improving outcome.

Value of molecular monitoring during the treatment of chronic myeloid leukemia: a Cancer and Leukemia Group B study.

PURPOSE: Disappearance of the Philadelphia chromosome during treatment for chronic myeloid leukemia (CML) has become an important therapeutic end point. To determine the additional value of molecular monitoring during treatment for CML, we performed a prospective, sequential analysis using quantitative Southern blot monitoring of BCR gene rearrangements of blood and marrow samples from Cancer and Leukemia Group B (CALGB) study 8761. PATIENTS AND METHODS: Sixty-four previously untreated adults with chronic-phase CML who were enrolled onto CALGB 8761, a molecular-monitoring companion study to a treatment study for adults with chronic-phase CML (CALGB 9013). Treatment consisted of repetitive cycles of interferon alfa and low-dose subcutaneous cytarabine. Blood and marrow Southern blot quantitation of BCR gene rearrangements was compared with marrow cytogenetic analysis before the initiation of treatment and of specified points during therapy. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis was performed to detect residual disease in patients who achieved a complete response by Southern blot or cytogenetic analysis. RESULTS: Quantitative molecular monitoring by Southern blot analysis of blood samples was found to be equivalent to marrow monitoring at all time points. Twelve of 62 (19%) follow-up samples studied by Southern blot analysis had a complete loss of BCR gene rearrangement in matched marrow and blood specimens. Southern blot monitoring of blood samples was also found to be highly correlated to marrow cytogenetic evaluation at all points, although there were four discordant cases in which Southern blot analysis of blood showed no BCR gene rearrangement, yet demonstrated from 12% to 20% Philadelphia chromosome-positive metaphase cells in the marrow. RT-PCR analysis detected residual disease in five of six patients in whom no malignant cells were detected using Southern blot or cytogenetic analyses. CONCLUSION: Quantitative Southern blot analysis of blood samples may be substituted for bone marrow to monitor the response to therapy in CML and results in the need for fewer bone marrow examinations. To avoid overestimating the degree of response, marrow cytogenetic analysis should be performed when patients achieve a complete response by Southern blot monitoring. This approach provides a rational, cost-effective strategy to monitor the effect of treatment of individual patients, as well as to analyze large clinical trials in CML.

Polymerase chain reaction-based diagnosis of del (5q) in acute myeloid leukemia and myelodysplastic syndrome identifies a minimal deletion interval.

Loss of all or part of the long arm of human chromosome 5 is a recurrent abnormality in patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), especially after chemotherapy for a prior malignancy. It is one of the worst prognostic indicators in AML, associated with chemotherapy resistance and short survival. These deletions center at band 5q31, which has thus been proposed as the location of a tumor suppressor gene; this site is to be distinguished from that observed in 5q- syndrome, centering at 5q32. To define the molecular extent and the clinical prevalence of 5q31 deletions, we collected a series of AML and MDS cases of mixed karyotype, taking care to exclude MDS cases with 5q- syndrome. The samples were analyzed for loss of heterozygosity (LOH) using a panel polymerase chain reaction (PCR)-based microsatellite markers from 5q, comparing malignant cells with normal tissue derived from lymphoblastoid cell lines or buccal mucosa scrapings. Losses were detected in seven of 29 matched samples, including four of 17 with MDS, and three of 12 with AML; six of these seven also had a cytogenetically-visible del(5q) or -5. The one case without a cytogenetic deletion showed molecular loss of three contiguous markers, with retention of flanking markers interleukin-9 (IL-9) and D5S414, and thus contained a small deletion that is below cytogenetic resolution. PCR failed to detect 5q loss in two cases with large cytogenetic deletions, but both had been treated and contained low percentages of malignant cells in the samples. This study thus led to the identification of a case with a minimal deletion for the 5q31 tumor suppressor gene, specified by IL-9-D5S414, that is approximately 1 Mb (2 cM) in extent. Additionally, we demonstrate that PCR-based allelotyping is a reliable method for the detection of chromosomal deletion in myeloid malignancy, providing the specimens contain a high proportion of malignant cells. These studies will help to identify the tumor-suppressor gene at 5q31, and will help to develop molecular methods for diagnosis and monitoring of these disorders.

Loss of heterozygosity from the short arm of chromosome 8 is associated with invasive behavior in breast cancer.

Loss of heterozygosity (LOH) from the short arm of chromosome 8 (8p) is frequent in many human cancers, including breast, colon, prostate, and bladder cancers. LOH occurs in two regions of 8p, 8p21 and 8p22, and suggests the presence of two separate tumor suppressor genes. In breast cancers, 8p LOH occurs in both early and late clinical stage tumors, while in colon, prostate, and bladder cancers, there is an association between 8p LOH and advanced clinical stage. We investigated this discrepancy by comparing 8p LOH in infiltrating ductal carcinomas (IDC) to breast cancers of earlier clinical stage, i.e., tumors with no invasion [ductal carcinoma in situ (DCIS)-only tumors]. We used three markers which sample several reported loci of 8p LOH. We microdissected tumor from paraffin blocks of 39 IDC and 23 DCIS-only breast cancers and amplified tumor/normal DNA pairs for the microsatellite markers D8S254 (8p22), D8S133 (8p21.3), and NEFL (8p21). All cases of IDC were informative with at least one marker, with a combined rate of LOH of 46%. The results for each marker were [no. LOH/no. informative (%)]: D8S254, 8/26 (31%); D8S133 12/31 (39%), and NEFL, 9/25 (36%). In the DCIS-only group, all 23 were informative for at least one marker, but 8p LOH was absent. We conclude that 8p LOH from 8p21-22 is frequent in IDC of the breast, but absent in DCIS-only cases, and may play a role in breast cancer progression by conferring invasive ability.

Immunomagnetic separation can enrich fixed solid tumors for epithelial cells.

Immunomagnetic separation is a highly specific technique for the enrichment or isolation of cells from a variety of fresh tissues and microorganisms or molecules from suspensions. Because new techniques for molecular analysis of solid tumors are now applicable to fixed tissue but sometimes require or benefit from enrichment for tumor cells, we tested the efficacy of immunomagnetic separation for enriching fixed solid tumors for malignant epithelial cells. We applied it to two different tumors and fixation methods to separate neoplastic from non-neoplastic cells in primary colorectal cancers and metastatic breast cancers, and were able to enrich to a high degree of purity. Immunomagnetic separation was effective in unembedded fixed tissue as well as fixed paraffin-embedded tissue. The magnetically separated cells were amenable to fluorescence in situ hybridization and polymerase chain reaction amplification of their DNA with minimal additional manipulation. The high degree of enrichment achieved before amplification contributed to interpretation of loss of heterozygosity in metastatic breast cancers, and simplified fluorescence in situ hybridization analysis because only neoplastic cells were hybridized and counted. Immunomagnetic separation is effective for the enrichment of fixed solid tumors, can be performed with widely available commercial antibodies, and requires little specialized instrumentation. It can contribute to interpretation of results in situations where enrichment by other methods is difficult or not possible.

Evidence for locus heterogeneity in autosomal dominant limb-girdle muscular dystrophy.

Limb-girdle muscular dystrophy (LGMD) is a diagnostic classification encompassing a broad group of proximal myopathies. A gene for the dominant form of LGMD (LGMD1A) has recently been localized to a 7-cM region of chromosome 5q between D5S178 and IL9. We studied three additional dominant LGMD families for linkage to these two markers and excluded all from localization to this region, providing evidence for locus heterogeneity within the dominant form of LGMD. Although patterns of muscle weakness were similar in all families studied, the majority of affected family members in the chromosome 5-linked pedigree have a dysarthric speech pattern, which is not present in any of the five unlinked families. The demonstration of heterogeneity within autosomal dominant LGMD is the first step in attempting to subclassify these families with similar clinical phenotypes on a molecular level.