Evidence For Locus Heterogeneity Research Articles

Clinical Application of Massively Parallel Sequencing in the Diagnosis of Hereditary Hemolytic and Dyserythropoietic Anemias

The Hereditary Hemolytic Anemias (HHAs) are a genetically heterogeneous group of anemias characterized by decreased red blood cell (RBC) survival because of defects in hemoglobin, RBC membrane proteins or enzymes. The diagnosis of this group of disorders is complex and challenging requiring analysis of the morphology of RBCs, hemoglobin electrophoresis, and a battery of phenotypic assays. The phenotypic analysis is often problematic in transfusion dependent patients or at times of presentation with a hemolytic crisis as transfused blood or reticulocytosis confounds diagnostic testing. Molecular genetic testing has grown in popularity in the diagnosis of hereditary hemolytic anemias as it is not affected by transfusions or other clinical variables and provides additional insight into the mechanism of the disease. We have developed a Next Generation Sequencing (NGS) panel for HHA due to RBC membrane disorders and enzymopathies and congenital dyserythropoietic anemias (CDA). CDAs, although collectively rare, are included in the panel as they are occasionally misdiagnosed as hereditary spherocytosis (HS) due to their clinical characteristics of hemolysis, increased osmotic fragility, and splenomegaly albeit with inadequate reticulocytosisWe reviewed the results of 282 sequential HHA/CDA panels testing for patients with suspected HHA or CDA diagnosis, performed and interpreted at Cincinnati Children's Hospital Medical Center between 1/2013-5/2016. Forty-three samples were omitted from the final analysis due to diagnosis of other disorders, indicating that negative results were true-negatives. For the analysis of the remaining 239 panels, all results were reviewed and categorized based on the type of testing ordered: comprehensive HHA/CDA (32 genes), RBC membrane disorders (13 genes), RBC enzyme disorders (14 genes), or CDA (6 genes). The protein-coding exons plus 25 bases of exon-intron junction as well as promoter sequences were included in the design. Genomic DNA was isolated from blood and target regions were enriched using the Haloplex technology. Enriched samples were then sequenced on an Illumina MiSeq benchtop sequencer with 150 base pair, paired-end reads. Sequencing reads were aligned to the human genome reference sequence and analysis of coverage and variants was completed using NextGENe software. All positive findings were confirmed by Sanger sequencing.These 239 panels included 159 (66.5%) comprehensive HHA/CDA panels, 41 (17.2%) RBC membrane disorder panels, 10 (4.2%) RBC enzyme disorder panels, and 29 (12.1%) CDA panels. Overall, a diagnosis was confirmed or identified in 135 (56.5%) patients with specific genotype of hereditary spherocytosis in 52 patients; hereditary elliptocytosis in 15 patients; hereditary pyropoikilocytosis in 7 patients; hereditary stomatocytosis/xerocytosis in 12 patients; South East Asian Ovalocytosis in 1 patient; G6PD deficiency in 15 patients; pyruvate kinase deficiency in 17 patients; other rare RBC enzymopathies in 6 patients; and CDA in 10 patients. The clinical performance of RBC membrane disorder and RBC enzyme disorder panels were comparable between 68-70% in reaching a final diagnosis, while CDA panel confirmed final diagnosis in only 20% of suspected cases. The overall low prevalence, complexity of diagnosis with findings of dyserythropoiesis in bone marrow studies in patients with severe HHA, and evidence of locus heterogeneity in CDA might explain this result. Among patients with suspected RBC membrane disorders, approximately 14% were eventually diagnosed with hereditary xerocytosis (HX). HX diagnosis is critical to make in such patients since splenectomy is contraindicated due to the high risk of life-threatening thrombophilia complications.In more than half (56.5%) of all cases with suspected hereditary hemolytic anemia, genetic testing provided or confirmed the diagnosis and optimized patients' clinical management. Further genetic counseling and testing for other at-risk family members was made possible by achieving molecular diagnosis. Genetic testing substantially altered management in approximately 14% of cases with suspected RBC membrane disorders due to the diagnosis of HX. In conclusion, genetic testing has a significant clinical utility and may facilitate and improve diagnosis, prognosis and management considerations in patients with hereditary hemolytic or dyserythropoietic anemia. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

No evidence of locus heterogeneity in familial microcephaly with or without chorioretinopathy, lymphedema, or mental retardation syndrome.

BackgroundMicrocephaly with or without chorioretinopathy, lymphedema, or mental retardation syndrome (MCLMR) is a rare autosomal dominant disorder with variable expressivity. It is characterized by mild-to-severe microcephaly, often associated with intellectual disability, ocular defects and lymphedema. It can be sporadic or inherited. Eighty-seven patients have been described to carry a mutation in KIF11, which encodes a homotetrameric motor kinesin, EG5.MethodsWe tested 23 unreported MCLMR index patients for KIF11. We also reviewed the clinical phenotypes of all our patients as well as of those described in previously published studies.ResultsWe identified 14 mutations, 12 of which are novel. We detected mutations in 12 affected individuals, from 6 out of 6 familial cases, and in 8 out of 17 sporadic patients. Phenotypic evaluation of patients (our 26 + 61 earlier published = 87) revealed microcephaly in 91%, eye anomalies in 72%, intellectual disability in 67% and lymphedema in 47% of the patients. Unaffected carriers were rare (4 out of 87: 5%). Family history is not a requisite for diagnosis; 31% (16 out of 52) were de novo cases.ConclusionsAll inherited cases, and 50% of sporadic cases of MCLMR are due to germline KIF11 mutations. It is possible that mosaic KIF11 mutations cause the remainder of sporadic cases, which the methods employed here were not designed to detect. On the other hand, some of them might have another mimicking disorder and genetic defect, as microcephaly is highly heterogeneous. In aggregate, KIF11 mutations likely cause the majority, if not all, of MCLMR.

Genetic Predisposition for Type 1 Diabetes Mellitus - The Role of Endoplasmic Reticulum Stress in Human Disease Etiopathogenesis

Genetic Predisposition for Type 1 Diabetes Mellitus - The Role of Endoplasmic Reticulum Stress in Human Disease EtiopathogenesisThe increasing incidence of diabetes mellitus worldwide has prompted a rapid growth in the pace of scientific discovery of the mechanisms involved in the etiopathogenesis of this multifactorial disease. Accumulating evidence suggests that endoplasmic reticulum stress plays a role in the pathogenesis of diabetes, contributing to pancreatic beta cell loss and insulin resistance. Wolfram syndrome is an autosomal recessive neurodegenerative disorder accompanied by insulin-dependent diabetes mellitus and progressive optic atrophy. The pathogenesis of this rare neurodegenerative genetic disease is unknown. A Wolfram gene (WFS1 locus) has recently been mapped to chromosome 4p16.1, but there is evidence for locus heterogeneity, including the mitochondrial genome deletion. Recent positional cloning led to identification of the second WFS locus, a mutation in the CISD2 gene, which encodes an endoplasmic reticulum intermembrane small protein. Our results were obtained by the analysis of a families belonging to specific population, affected by Wolfram syndrome. We have identified the newly diagnosed genetic alteration of WFS1 locus, a double non-synonymous and frameshift mutation, providing further evidence for the genetic heterogeneity of this syndrome. Newly identified mutations may contribute to the further elucidation of the pathogenesis of Wolfram syndrome, as well as of the complex mechanisms involved in diabetes mellitus development.

Identification and characterization of a novel P2Y12 variant in a patient diagnosed with type 1 von Willebrand disease in the European MCMDM-1VWD study

AbstractWe investigated whether defects in the P2Y12 ADP receptor gene (P2RY12) contribute to the bleeding tendency in 92 index cases enrolled in the European MCMDM-1VWD study. A heterozygous mutation, predicting a lysine to glutamate (K174E) substitution in P2Y12, was identified in one case with mild type 1 von Willebrand disease (VWD) and a VWF defect. Platelets from the index case and relatives carrying the K174E defect changed shape in response to ADP, but showed reduced and reversible aggregation in response to 10 μM ADP, unlike the maximal, sustained aggregation observed in controls. The reduced response was associated with an approximate 50% reduction in binding of [3H]2MeS-ADP to P2Y12, whereas binding to the P2Y1 receptor was normal. A hemagglutinin-tagged K174E P2Y12 variant showed surface expression in CHO cells, markedly reduced binding to [3H]2MeS-ADP, and minimal ADP-mediated inhibition of forskolin-induced adenylyl cyclase activity. Our results provide further evidence for locus heterogeneity in type 1 VWD.

A reanalysis of 409 European‐Ancestry and African American schizophrenia pedigrees reveals significant linkage to 8p23.3 with evidence of locus heterogeneity

The detection and replication of schizophrenia risk loci can require substantial sample sizes, which has prompted various collaborative efforts for combining multiple samples. However, pooled samples may comprise sub-samples with substantial population genetic differences, including allele frequency differences. We investigated the impact of population differences via linkage reanalysis of Molecular Genetics of Schizophrenia 1 (MGS1) affected sibling-pair data, comprising two samples of distinct ancestral origin: European (EA: 263 pedigrees) and African-American (AA: 146 pedigrees). To exploit the linkage information contained within these distinct continental samples, we performed separate analyses of the individual samples, allowing for within-sample locus heterogeneity, and the pooled sample, allowing for both within-sample and between-sample heterogeneity. Significance levels, corrected for the multiple tests, were determined empirically. For all suggestive peaks, stronger linkage evidence was obtained in either the EA or AA sample than the combined sample, regardless of how heterogeneity was modeled for the latter. Notably, we report genomewide significant linkage of schizophrenia to 8p23.3 and evidence for a second, independent susceptibility locus, reaching suggestive linkage, 29 cM away on 8p21.3. We also detected suggestive linkage on chromosomes 5p13.3 and 7q36.2. Many regions showed pronounced differences in the extent of linkage between the EA and AA samples. This reanalysis highlights the potential impact of population differences upon linkage evidence in pooled data and demonstrates a useful approach for the analysis of samples drawn from distinct continental groups.

Narrowing the DYT6 dystonia region and evidence for locus heterogeneity in the Amish–Mennonites

The DYT6 gene for primary torsion dystonia (PTD) was mapped to chromosome 8p21-q22 in two Amish-Mennonite families who shared a haplotype of marker alleles across a 40 cM linked region. The objective of this study was to narrow the DYT6 region, clinically characterize DYT6 dystonia in a larger cohort, and to determine whether DYT6 is associated with dystonia in newly ascertained multiplex families. We systematically examined familial Amish-Mennonite dystonia cases, identifying five additional members from the original families, as well as three other multiplex Amish-Mennonite families, and evaluated the known DYT6 haplotype and recombination events. One of the three new families carried the shared haplotype, whereas the region was excluded in the two other families, suggesting genetic heterogeneity for PTD in the Amish-Mennonites. Clinical features in the five newly identified DYT6 carriers were similar to those initially described. In contrast, affected individuals from the excluded families had a later age of onset (46.9 years vs. 16.1 years in the DYT6), and the dystonia was both more likely to be of focal distribution and begin in the cervical muscles. Typing of additional markers in the DYT6-linked families revealed recombinations that now place the gene in a 23 cM region surrounding the centromere. In summary, the DYT6 gene is in a 23 cM region on chromosome 8q21-22 and does not account for all familial PTD in Amish-Mennonites.

Genetic Heterogeneity in Autosomal Dominant Pseudohypoaldosteronism Type I: Exclusion of Claudin-8 as a Candidate Gene

Background/Aims: Pseudohypoaldosteronism type I (PHAI) is an inherited disorder characterized by renal salt wasting, hyperkalemic metabolic acidosis, and hyperaldosteronism. Its known causes are mutations in the mineralocorticoid receptor and the epithelial sodium channel (ENaC), but there are reports of genetic heterogeneity. Claudin-8 is a tight junction protein that acts as a paracellular cation barrier in the distal nephron. The aim of this study was to test the hypothesis that mutations in claudin-8, which would be expected to induce a distal tubule cation leak, can be a cause of PHAI. Methods: We identified 10 patients with autosomal dominant PHAI in whom mutations in the mineralocorticoid receptor and ENaC had been excluded. The claudin-8 gene and upstream region was sequenced in all patients. Results: No disease-associated claudin-8 mutations were identified. A novel polymorphic allele in the 3′-untranslated region was identified in 2 patients, but was also found in 15% of individuals in a panel of normal controls. Conclusion: We present further evidence for locus heterogeneity in PHAI. Mutations in claudin-8 are unlikely to be a cause of PHAI. Further studies of other claudins in this disease are warranted.

Linkage of autosomal dominant common variable immunodeficiency to chromosome 5p and evidence for locus heterogeneity.

Common variable immunodeficiency (CVID, OMIM 240500) and selective immunoglobulin A deficiency (IgAD) are the most frequent primary immunodeficiencies in humans. Of the cases with CVID/IgAD, 20%-25% are familial, but the only previous claims of linkage or association are to the HLA region on chromosome 6p. We report the results of a genome-wide scan in three multiplex families with CVID, IgAD, and dysgammaglobulinemia, where affection is inherited in an autosomal dominant pattern. Two of the families are consistent with linkage to the telomeric region of chromosome 5p, whereas the third is consistent with linkage to the HLA region. Using a locus heterogeneity model and a conservative penetrance model, we obtained a LOD score of 3.35 for the 5p region. We sequenced the exons of one promising candidate gene within this region (PDCD6, also known as ALG-2) but found no causative mutation.

Family MRX9 revisited: further evidence for locus heterogeneity in MRX.

Nonspecific X-linked mental retardation (MRX) patients are characterized by mental retardation, without additional distinguishing features. Consequently, MRX families can only be distinguished by mapping studies; yet, due to imprecise mapping studies performed in the past, the number of genes causing MRX is debatable, and a more precise localization for families is necessary to estimate this number. MRX 9 has been mapped to the pericentromeric region Xp21-q13. We refined the mapping of the MRX9 family to Xp11.22-Xp11.4. A sequencing analysis of three likely candidate genes in Xp11, SREB3, synapsin I, and TM4SF2, revealed no mutations.

A Mutation in COL9A1 Causes Multiple Epiphyseal Dysplasia: Further Evidence for Locus Heterogeneity

Multiple epiphyseal dysplasia (MED) is an autosomal dominantly inherited chondrodysplasia. It is clinically highly heterogeneous, partially because of its complex genetic background. Mutations in four genes, COL9A2, COL9A3, COMP, and MATR3, all coding for cartilage extracellular matrix components (i.e., the alpha2 and alpha 3 chains of collagen IX, cartilage oligomeric matrix protein, and matrilin-3), have been identified in this disease so far, but no mutations have yet been reported in the third collagen IX gene, COL9A1, which codes for the alpha1(IX) chain. MED with apparently recessive inheritance has been reported in some families. A homozygous R279W mutation was recently found in the diastrophic dysplasia sulfate transporter gene, DTDST, in a patient with MED who had a club foot and double-layered patella. The series consisted of 41 probands with MED, 16 of whom were familial and on 4 of whom linkage analyses were performed. Recombination was observed between COL9A1, COL9A2, COL9A3, and COMP and the MED phenotype in two of the families, and between COL9A2, COL9A3, and COMP and the phenotype in the other two families. Screening of COL9A1 for mutations in the two probands from the families in which this gene was not involved in the recombinations failed to identify any disease-causing mutations. The remaining 37 probands were screened for mutations in all three collagen IX genes and in the COMP gene. The probands with talipes deformities or multipartite patella were also screened for the R279W mutation in DTDST. The analysis resulted in identification of three mutations in COMP and one in COL9A1, but none in the other two collagen IX genes. Two of the probands with a multipartite patella had the homozygous DTDST mutation. The results show that mutations in COL9A1 can cause MED, but they also suggest that mutations in COL9A1, COL9A2, COL9A3, COMP, and DTDST are not the major causes of MED and that there exists at least one additional locus.

Multilocus linkage analysis of the German asthma data.

We analyzed data from the German Asthma Genetics Group with three methods that utilize pedigree-specific nonparametric linkage scores to facilitate the search for multiple independent and interacting susceptibility loci. The three methods included a conditional analysis, logistic regression, and neural networks. Although there were differences, the three methods identified many of the same susceptibility loci. The most consistent evidence was provided for loci on chromosomes 1, 2, 6, 9, and 15. Both the conditional and the logistic regression analyses suggested an epistatic relationship between loci on chromosomes 2 and 9. The logistic regression analysis further revealed evidence for locus heterogeneity between loci on chromosomes 6 and 15. Finally, the neural network analysis identified a potential locus on chromosome 17 that was not identified in the other analyses.

Exhaustive mutation analysis of the NF1 gene allows identification of 95% of mutations and reveals a high frequency of unusual splicing defects

Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant disorders and is caused by mutations in the NF1 gene. Mutation detection is complex due to the large size of the NF1 gene, the presence of pseudogenes and the great variety of possible lesions. Although there is no evidence for locus heterogeneity in NF1, mutation detection rates rarely exceed 50%. We studied 67 unrelated NF1 patients fulfilling the NIH diagnostic criteria, 29 familial and 38 sporadic cases, using a cascade of complementary techniques. We performed a protein truncation test starting from puromycin-treated EBV cell lines and, if no mutation was found, continued with heteroduplex, FISH, Southern blot and cytogenetic analysis. We identified the germline mutation in 64 of 67 patients and 32 of the mutations are novel. This is the highest mutation detection rate reported in a study of typical NF1 patients. All mutations were studied at the genomic and RNA level. The mutational spectrum consisted of 25 nonsense, 12 frameshift, 19 splice mutations, six missense and/or small in-frame deletions, one deletion of the entire NF1 gene, and a translocation t(14;17)(q32;q11.2). Our data suggest that exons 10a-10c and 37 are mutation-rich regions and that together with some recurrent mutations they may account for almost 30% of the mutations in classical NF1 patients. We found a high frequency of unusual splice mutations outside of the AG/GT 5¢ and 3¢ splice sites. As some of these mutations form stable transcripts, it remains possible that a truncated neurofibromin is formed. Hum Mutat 15:541–555, 2000. © 2000 Wiley-Liss, Inc.

Pseudoxanthoma elasticum: Mutations in the MRP6 gene encoding a transmembrane ATP-binding cassette (ABC) transporter

Pseudoxanthoma elasticum (PXE), the prototypic heritable connective tissue disorder affecting the elastic structures in the body, manifests with cutaneous, ophthalmologic, and cardiovascular findings, with considerable morbidity and mortality. The molecular basis of PXE has remained unknown, but the disease locus has recently been mapped to an approximately 500-kb interval on chromosome 16p13.1, without evidence for locus heterogeneity. In this study, we report pathogenetic mutations in MRP6, a member of the ABC transporter gene family, in eight kindreds with PXE. The mutation detection strategy consisted of heteroduplex scanning of coding sequences in the MRP6 gene, which were amplified by PCR by using genomic DNA as template, followed by direct nucleotide sequencing. A total of 13 mutant MRP6 alleles were disclosed in the eight probands with PXE. These genetic lesions consisted of either single base pair substitutions resulting in missense, nonsense, or splice site mutations, or large deletions resulting in allelic loss of the MRP6 locus. Examination of clinically unaffected family members in four multiplex families identified heterozygous carriers, consistent with an autosomal recessive inheritance pattern. Collectively, identification of mutations in the MRP6 gene provides the basis to examine the pathomechanisms of PXE and allows development of DNA-based carrier detection, prenatal testing, and preimplantation genetic diagnosis in families with a history of this disease.

Combined Analysis of Hereditary Prostate Cancer Linkage to 1q24-25: Results from 772 Hereditary Prostate Cancer Families from the International Consortium for Prostate Cancer Genetics

A previous linkage study provided evidence for a prostate cancer-susceptibility locus at 1q24-25. Subsequent reports in additional collections of families have yielded conflicting results. In addition, evidence for locus heterogeneity has been provided by the identification of other putative hereditary prostate cancer loci on Xq27-28, 1q42-43, and 1p36. The present study describes a combined analysis for six markers in the 1q24-25 region in 772 families affected by hereditary prostate cancer and ascertained by the members of the International Consortium for Prostate Cancer Genetics (ICPCG) from North America, Australia, Finland, Norway, Sweden, and the United Kingdom. Overall, there was some evidence for linkage, with a peak parametric multipoint LOD score assuming heterogeneity (HLOD) of 1.40 (P=.01) at D1S212. The estimated proportion of families (alpha) linked to the locus was.06 (1-LOD support interval.01-.12). This evidence was not observed by a nonparametric approach, presumably because of the extensive heterogeneity. Further parametric analysis revealed a significant effect of the presence of male-to-male disease transmission within the families. In the subset of 491 such families, the peak HLOD was 2.56 (P=.0006) and alpha =.11 (1-LOD support interval.04-.19), compared with HLODs of 0 in the remaining 281 families. Within the families with male-to-male disease transmission, alpha increased with the early mean age at diagnosis (<65 years, alpha =.19, with 1-LOD support interval.06-.34) and the number of affected family members (five or more family members, alpha =.15, with 1-LOD support interval.04-.28). The highest value of alpha was observed for the 48 families that met all three criteria (peak HLOD = 2.25, P=.001, alpha=.29, with 1-LOD support interval.08-.53). These results support the finding of a prostate cancer-susceptibility gene linked to 1q24-25, albeit in a defined subset of prostate cancer families. Although HPC1 accounts for only a small proportion of all families affected by hereditary prostate cancer, it appears to play a more prominent role in the subset of families with several members affected at an early age and with male-to-male disease transmission.

Localization of the Netherton Syndrome Gene to Chromosome 5q32, by Linkage Analysis and Homozygosity Mapping

Netherton syndrome (NS [MIM 256500]) is a rare and severe autosomal recessive disorder characterized by congenital ichthyosis, a specific hair-shaft defect (trichorrhexis invaginata), and atopic manifestations. Infants with this syndrome often fail to thrive; life-threatening complications result in high postnatal mortality. We report the assignment of the NS gene to chromosome 5q32, by linkage analysis and homozygosity mapping in 20 families affected with NS. Significant evidence for linkage (maximum multipoint LOD score 10.11) between markers D5S2017 and D5S413 was obtained, with no evidence for locus heterogeneity. Analysis of critical recombinants mapped the NS locus between markers D5S463 and D5S2013, within an <3.5-cM genetic interval. The NS locus is telomeric to the cytokine gene cluster in 5q31. The five known genes encoding casein kinase Ialpha, the alpha subunit of retinal rod cGMP phosphodiesterase, the regulator of mitotic-spindle assembly, adrenergic receptor beta2, and the diastrophic dysplasia sulfate-transporter gene, as well as the 38 expressed-sequence tags mapped within the critical region, are not obvious candidates. Our study is the first step toward the positional cloning of the NS gene. This finding promises a better understanding of the molecular mechanisms that control epidermal differentiation and immunity.

Exhaustive mutation analysis of the NF1 gene allows identification of 95% of mutations and reveals a high frequency of unusual splicing defects.

Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant disorders and is caused by mutations in the NF1 gene. Mutation detection is complex due to the large size of the NF1 gene, the presence of pseudogenes and the great variety of possible lesions. Although there is no evidence for locus heterogeneity in NF1, mutation detection rates rarely exceed 50%. We studied 67 unrelated NF1 patients fulfilling the NIH diagnostic criteria, 29 familial and 38 sporadic cases, using a cascade of complementary techniques. We performed a protein truncation test starting from puromycin-treated EBV cell lines and, if no mutation was found, continued with heteroduplex, FISH, Southern blot and cytogenetic analysis. We identified the germline mutation in 64 of 67 patients and 32 of the mutations are novel. This is the highest mutation detection rate reported in a study of typical NF1 patients. All mutations were studied at the genomic and RNA level. The mutational spectrum consisted of 25 nonsense, 12 frameshift, 19 splice mutations, six missense and/or small in-frame deletions, one deletion of the entire NF1 gene, and a translocation t(14;17)(q32;q11.2). Our data suggest that exons 10a-10c and 37 are mutation-rich regions and that together with some recurrent mutations they may account for almost 30% of the mutations in classical NF1 patients. We found a high frequency of unusual splice mutations outside of the AG/GT 5 cent and 3 cent splice sites. As some of these mutations form stable transcripts, it remains possible that a truncated neurofibromin is formed.

In this Issue

Sentinel lymph node biopsy has been recently advocated as a way to determine whether lymph node dissection would be necessary as part of the surgical treatment of melanoma. This concept is based on the notion that the “sentinel lymph node”, the first lymph node of the draining site of the primary tumor, is never bypassed in malignant melanoma. Thus, lack of metastases in the sentinel lymph node has been considered a good prognostic finding. In this issue, Lukowsky et al. (p. 554) have evaluated this concept by examining the regional lymph nodes, sentinel and nonsentinel, in 24 patients with malignant melanoma by histopathology and immunohistochemistry, and compared these results for the presence of tyrosinase mRNA as determined by RT-PCR. Because the tyrosinase gene is expressed exclusively in melanocytic cells, detection of the corresponding transcripts would signify the presence of metastases in the tissue. As expected, 10 of 20 patients with histologic evidence of metastasis in the sentinel lymph node were also positive for tyrosinase mRNA, as were three additional patients without morphologic evidence, attesting to the sensitivity of the RT-PCR approach. Surprisingly, however, in seven patients RT-PCR was clearly positive for tyrosinase mRNA in nonsentinel lymph nodes, whereas the sentinel lymph node was negative. Assuming the specificity of the PCR for tyrosinase, it appears that the sentinel lymph node biopsy may miss micrometastases that have spread beyond the primary tumor and somehow bypassed the sentinel lymph node. These results clearly warrant further studies to validate the concept of sentinel lymph node biopsy. They also suggest that incorporation of RT-PCR into the armamentoire to examine regional lymph nodes in patients with malignant melanoma increases the sensitivity for detection of metastases Collagens comprise a superfamily of proteins, and as many as 20 different mammalian collagens have been studied thus far to the extent that they have been designated by a Roman numeral. Among these 20 collagens, at least a dozen have been demonstrated in the skin. In this issue, Peltonen et al. (p. 635) have examined the localization of another collagen, type XIII, which has recently been shown to be a transmembrane protein. Using a variety of immunologic and molecular biologic techniques, the authors demonstrate that type XIII collagen is expressed in the epidermis in various contact sites. Specifically, colocalization studies suggested that type XIII collagen is not a member of the desmosome, but is likely to be associated with adherens type junctions. Previously, another collagen, type XVII collagen, has been shown to be a keratinocyte gene product, in type 2 transmembrane orientation and a component of hemidesmosomes. Type XVII collagen clearly confers stability to the dermal–epidermal basement membrane zone, as mutations in the corresponding gene (COL17A1) underlie a non-Herlitz type of junctional epidermolysis bullosa, known as generalized atrophic benign EB (GABEB). The functional role of type XIII collagen within the epidermis is currently unknown, but the authors speculate that, in analogy with type XVII collagen, aberrations in this collagen may be associated with heritable and/or acquired blistering skin diseases. The spectacular progress recently made in understanding the molecular basis of various heritable skin diseases is exemplified by dystrophic epidermolysis bullosa (DEB). At the beginning of this decade nothing was known of the specific mutations resulting in various forms of DEB, and the prevailing notion suggested that somehow the expression of proteolytic enzymes, primarily collagenase, was altered, resulting in dissolution of anchoring fibrils and other collagenous structures. Following the cloning of type VII collagen as the candidate gene for DEB, a number of reports have detailed specific mutations in COL7A1 in different variants of DEB. In this issue, Whittock et al. (p. 673) compare the efficiency of three different mutation detection strategies, i.e., protein truncation test, fluorescence chemical cleavage of mismatch, and conformation sensitive gel electrophoresis, for their sensitivity to detect mutations in COL7A1 in patients with DEB. The results indicate that none of the these methods alone is able to detect all putative mutations, but combined use of these techniques yields an overall sensitivity of 87%. In fact, the authors were able to detect 147 of 169 mutations in a total of 93 patients with DEB, 57 of the mutations being previously unreported. These novel mutations, together with previously published ones, brings the total number of COL7A1 mutations in DEB to over 200. Examination of the mutation database reveals that the majority of the genetic lesions are family specific with a few recurrent mutations being detected. There is thus far no evidence for locus heterogeneity, and the gene for collagenase has been excluded from DEB locus by genetic linkage analyses. These observations have diagnostic importance with prognostic implications, and they form the basis for DNA-based prenatal testing and preimplantation genetic diagnosis, as well as for gene therapy in the future.

Genetic mapping of a novel familial form of infantile hemangioma.

Infantile hemangiomas are the most common tumor of infancy, occurring with an incidence of up to 10% of all births. They are benign but highly proliferative lesions involving aberrant localized growth of capillary endothelium. Although most hemangiomas occur sporadically and as single lesions, or in conjunction with pleiotropic genetic syndromes, we have previously identified six kindreds where hemangiomas appear to segregate as an autosomal dominant trait with high penetrance. Four such families contain affected individuals in three or more generations. In the current study, blood samples from five of these families were collected and used in a whole genome linkage search at 10-cM resolution. We established evidence for linkage to 5q in three families, and evidence for locus heterogeneity. The three 5q-linked families were further genotyped to generate haplotype information and narrow the candidate interval. Based on recombination breakpoint analysis, the interval exists between markers D5S2490 and D5S408, spanning 55 cM, and corresponding to 5q31-33. Using information from affected and unaffected individuals, the interval spans 38 cM between markers D5S1469 and D5S211. Three candidate genes involved with blood vessel growth map to this region: fibroblast growth factor receptor-4 (FGFR4), platelet-derived growth factor receptor-beta (PDG-FRB), and fms-related tyrosine kinase-4 (FLT4). The genes and gene products associated with familial hemangiomas may be involved somatically in the more common sporadic cases.

Locus heterogeneity in autosomal dominant congenital external ophthalmoplegia (CFEOM).

Congenital external ophthalmoplegia (CFEOM) is an uncommon autosomal dominant condition that has previously been mapped to the pericentromeric region of chromosome 12 in seven families with no evidence of locus...

3rd workshop of the European CMT consortium: 54th ENMC International Workshop on genotype/phenotype correlations in Charcot-Marie-Tooth type 1 and hereditary neuropathy with liability to pressure palsies 28-30 November 1997, Naarden, The Netherlands.

3rd workshop of the European CMT consortium: 54th ENMC International Workshop on genotype/phenotype correlations in Charcot-Marie-Tooth type 1 and hereditary neuropathy with liability to pressure palsies 28-30 November 1997, Naarden, The Netherlands.