The epitheliogenesis imperfecta locus maps to equine chromosome 8 in American Saddlebred horses

Epitheliogenesis imperfecta (EI) is a hereditary junctional mechanobullous disease that occurs in newborn American Saddlebred foals. The pathological signs of epitheliogenesis imperfecta closely match a similar disease in humans known as Herlitz junctional epidermolysis bullosa, which is caused by a mutation in one of the genes (LAMA3, LAMB3 and LAMC2) coding for the subunits of the laminin 5 protein (laminin alpha3, laminin beta3 and laminin gamma2). The LAMA3 gene has been assigned to equine chromosome 8 and LAMB3 and LAMC2 have been mapped to equine chromosome 5. Linkage disequilibrium between microsatellite markers that mapped to equine chromosome 5 and equine chromosome 8 and the EI disease locus was tested in American Saddlebred horses. The allele frequencies of microsatellite alleles at 11 loci were determined for both epitheliogenesis imperfecta affected and unaffected populations of American Saddlebred horses by genotyping and direct counting of alleles. These were used to determine fit to Hardy-Weinberg equilibrium for control and EI populations using Chi square analysis. Two microsatellite loci located on equine chromosome 8q, ASB14 and AHT3, were not in Hardy-Weinberg equilibrium in affected American Saddlebred horses. In comparison, all of the microsatellite markers located on equine chromosome 5 were in Hardy-Weinberg equilibrium in affected American Saddlebred horses. This suggested that the EI disease locus was located on equine chromosome 8q, where LAMA3 is also located.     

Digenic junctional epidermolysis bullosa: mutations in COL17A1 and LAMB3 genes

Junctional epidermolysis bullosa (JEB), a genetically heterogeneous group of blistering skin diseases, can be caused by mutations in the genes encoding laminin 5 or collagen XVII, which are components of the hemidesmosome-anchoring filament complex in the skin. Here, a family with severe nonlethal JEB and with mutations in genes for both proteins was identified. The index patient was compound heterozygous for the COL17A1 mutations L855X and R1226X and was heterozygous for the LAMB3 mutation R635X. As a consequence, two functionally related proteins were affected. Absence of collagen XVII and attenuated laminin 5 expression resulted in rudimentary hemidesmosome structure and separation of the epidermis from the basement membrane, with severe skin blistering as the clinical manifestation. In contrast, single heterozygotes carrying either (1) one or the other of the COL17A1 null alleles or (2) a double heterozygote for a COL17A1 and a LAMB3 null allele did not have a pathological skin phenotype. These observations indicate that the known allelic heterogeneity in JEB is further complicated by interactions between unlinked mutations. They also demonstrate that identification of one mutation in one gene is not sufficient for determination of the genetic basis of JEB in a given family.     

Laminin 5 mutations in junctional epidermolysis bullosa: molecular basis of Herlitz vs non-Herlitz phenotypes

Junctional epidermolysis bullosa (JEB) is a group of heritable blistering diseases in which tissue separation occurs within the lamina lucida of the cutaneous basement membrane zone. Clinically, two broad subcategories have been recognized: The Herlitz variant (H-JEB; OMIM 226700) is characterized by early demise of the affected individuals, usually within the first year of life, while non-Herlitz (nH-JEB; OMIM 226650) patients show a milder phenotype with life-long blistering, yet with normal lifespan. In this study, we have examined a cohort of 27 families, 15 with Herlitz and 12 with non-Herlitz JEB, for mutations in the candidate genes, LAMA3, LAMB3, and LAMC2, encoding the subunit polypeptides of laminin 5. The mutation detection strategy consisted of PCR amplification of all exons in these genes, followed by heteroduplex scanning and nucleotide sequencing. We were able to identify pathogenic mutations in both alleles of each proband, the majority of the mutations being in the LAMB3 gene. Examination of the mutation database revealed that most cases with Herlitz JEB harbored premature termination codon (PTC) mutations in both alleles. In non-Herlitz cases, the PTC mutation was frequently associated with a missense mutation or a putative splicing mutation in trans. In three cases with putative splicing mutations, RT-PCR analysis revealed a repertoire of splice variants in-frame, predicting the synthesis of either shortened or lengthened, yet partly functional, polypeptides. These observations would explain the relatively mild phenotype in cases with splicing mutations. Collectively, these findings, together with the global laminin 5 mutation database, contribute to our understanding of the genotype/phenotype correlations explaining the Herlitz vs non-Herlitz phenotypes.     

Paternal uniparental disomy for chromosome 1 revealed by molecular analysis of a patient with pycnodysostosis.

Molecular analysis of a patient affected by the autosomal recessive skeletal dysplasia, pycnodysostosis (cathepsin K deficiency; MIM 265800), revealed homozygosity for a novel missense mutation (A277V). Since the A277V mutation was carried by the patient's father but not by his mother, who had two normal cathepsin K alleles, paternal uniparental disomy was suspected. Karyotyping of the patient and of both parents was normal, and high-resolution cytogenetic analyses of chromosome 1, to which cathepsin K is mapped, revealed no abnormalities. Evaluation of polymorphic DNA markers spanning chromosome 1 demonstrated that the patient had inherited two paternal chromosome 1 homologues, whereas alleles for markers from other chromosomes were inherited in a Mendelian fashion. The patient was homoallelic for informative markers mapping near the chromosome 1 centromere, but he was heteroallelic for markers near both telomeres, establishing that the paternal uniparental disomy with partial isodisomy was caused by a meiosis II nondisjunction event. Phenotypically, the patient had normal birth height and weight, had normal psychomotor development at age 7 years, and had only the usual features of pycnodysostosis. This patient represents the first case of paternal uniparental disomy of chromosome 1 and provides conclusive evidence that paternally derived genes on human chromosome 1 are not imprinted.     

Complex rearrangements of chromosome 15 in two patients with mild/atypical Prader Willi syndrome

Multiple mechanisms are responsible for the development of Prader Willi syndrome (PWS), the most common genetic cause of obesity in childhood. Molecular findings are usually deletions and uniparental disomy (UPD) of the 15q11-13 region. Rarely, structural rearrangements of the pericentromeric region of chromosome 15 are also detected. Two cases with mild PWS phenotype and complex maternal UPD identified by microsatellite analysis are described: the first patient had uniparental iso and heterodisomy and the second displayed biallelic inheritance and uniparental isodisomy.     

Novel ENAM and LAMB3 Mutations in Chinese Families with Hypoplastic Amelogenesis Imperfecta

Amelogenesis imperfecta is a group of inherited diseases affecting the quality and quantity of dental enamel. To date, mutations in more than ten genes have been associated with non-syndromic amelogenesis imperfecta (AI). Among these, ENAM and LAMB3 mutations are known to be parts of the etiology of hypoplastic AI in human cases. When both alleles of LAMB3 are defective, it could cause junctional epidermolysis bullosa (JEB), while with only one mutant allele in the C-terminus of LAMB3, it could result in severe hypoplastic AI without skin fragility. We enrolled three Chinese families with hypoplastic autosomal-dominant AI. Despite the diagnosis falling into the same type, the characteristics of their enamel hypoplasia were different. Screening of ENAM and LAMB3 genes was performed by direct sequencing of genomic DNA from blood samples. Disease-causing mutations were identified and perfectly segregated with the enamel defects in three families: a 19-bp insertion mutation in the exon 7 of ENAM (c.406_407insTCAAAAAAGCCGACCACAA, p.K136Ifs*16) in Family 1, a single-base deletion mutation in the exon 5 of ENAM (c. 139delA, p. M47Cfs*11) in Family 2, and a LAMB3 nonsense mutation in the last exon (c.3466C>T, p.Q1156X) in Family 3. Our results suggest that heterozygous mutations in ENAM and LAMB3 genes can cause hypoplastic AI with markedly different phenotypes in Chinese patients. And these findings extend the mutation spectrum of both genes and can be used for mutation screening of AI in the Chinese population.     

Uniparental disomy of the entire X chromosome in a female with Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe, progressive, X-linked muscle-wasting disorder with an incidence of approximately 1/3,500 male births. Females are also affected, in rare instances. The manifestation of mild to severe symptoms in female carriers of dystrophin mutations is often the result of the preferential inactivation of the X chromosome carrying the normal dystrophin gene. The severity of the symptoms is dependent on the proportion of cells that have inactivated the normal X chromosome. A skewed pattern of X inactivation is also responsible for the clinical manifestation of DMD in females carrying X;autosome translocations, which disrupt the dystrophin gene. DMD may also be observed in females with Turner syndrome (45,X), if the remaining X chromosome carries a DMD mutation. We report here the case of a karyotypically normal female affected with DMD as a result of homozygosity for a deletion of exon 50 of the dystrophin gene. PCR analysis of microsatellite markers spanning the length of the X chromosome demonstrated that homozygosity for the dystrophin gene mutation was caused by maternal isodisomy for the entire X chromosome. This finding demonstrates that uniparental isodisomy of the X chromosome is an additional mechanism for the expression of X-linked recessive disorders. The proband's clinical presentation is consistent with the absence of imprinted genes (i.e., genes that are selectively expressed based on the parent of origin) on the X chromosome.     

A homozygous nonsense mutation in SOX9 in the dominant disorder campomelic dysplasia: a case of mitotic gene conversion

Campomelic dysplasia (CD; MIM 114290), an autosomal dominant skeletal malformation syndrome with XY sex reversal, is caused by heterozygous de novo mutations in and around the SOX9 gene on 17q. We report a patient with typical signs of CD, including sex reversal, who was, surprisingly, homozygous for the nonsense mutation Y440X. Since neither parent carried the Y440X mutation, possible mechanisms explaining the homozygous situation were a de novo mutation followed by uniparental isodisomy, somatic crossing over, or gene conversion. As the patient was heterozygous for six microsatellite markers flanking SOX9, uniparental isodisomy and somatic crossing over were excluded. Analysis of intragenic single-nucleotide polymorphisms suggested that the homozygous mutation arose by a mitotic gene conversion event involving exchange of at least 440 nucleotides and at most 2,208 nucleotides between a de novo mutant maternal allele and a wild-type paternal allele. Analysis of cloned alleles showed that homozygous mutant cells constituted about 80% of the leukocyte cell population of the patient, whereas about 20% were heterozygous mutant cells. Heterozygous Y440X mutations, previously described in three CD cases, have been identified in seven additional cases, thus constituting the most frequent recurrent mutations in SOX9. These patients frequently have a milder phenotype with longer survival, possibly because of the retention of some transactivation activity of the mutant protein on SOX9 target genes, as shown by cell transfection experiments. The fact that the patient survived for 3 months may thus be explained by homozygosity for a hypomorphic rather than a complete loss-of-function allele, in combination with somatic mosaicism. This is, to our knowledge, the first report of mitotic gene conversion of a wild-type allele by a de novo mutant allele in humans.     

Novel and recurrent mutations in the laminin-5 genes causing lethal junctional epidermolysis bullosa: molecular basis and clinical course of Herlitz disease

Herlitz disease (H-JEB), the lethal form of junctional epidermolysis bullosa, is a rare genodermatosis presenting from birth with widespread erosions and blistering of skin and mucosae because of tissue cleavage within the epidermal basement membrane. Mutations in any of the three genes encoding the 3, 3 and 2 chains of laminin-5 underlie this recessively inherited disorder. Here, we report the molecular basis and clinical course of H-JEB in 12 patients. Two novel nonsense mutations in the gene LAMA3 (E281X and K1299X) and a novel frame-shift mutation in the gene LAMB3 (1628insG) leading to a premature termination codon were identified by DNA sequencing and confirmed by restriction fragment length polymorphism analysis. In the four patients affected, neither the resulting truncated polypeptide chains nor assembled laminin-5 protein were detectable by immunofluorescence. Three patients were found to be heterozygous for the known hotspot mutation R635X and the recurrent mutations Q373X or 29insC in the gene LAMB3, whereas five others were homozygous for R635X. Significant variations in the disease progression and survival times between 1 and 30 months in this group of H-JEB patients emphasised the impact of modifying factors and the importance of immunostaining or mRNA assessment as parallel diagnostic methods. Interestingly, the only patients who survived for longer than 6 months were four females carrying the mutation R635X homozygously. In one of them, the clinical course may have been improved by treatment with artificial skin equivalents. These data may stimulate further investigation of genotype–phenotype correlations and facilitate mutation analysis and genetic counselling of affected families.     

Three Novel Homozygous Point Mutations and a New Polymorphism in the COL17A1 Gene: Relation to Biological and Clinical Phenotypes of Junctional Epidermolysis Bullosa

Junctional epidermolysis bullosa (JEB) is a clinically and biologically heterogeneous genodermatosis, characterized by trauma-induced blistering and healing without scarring but sometimes with skin atrophy. We investigated three unrelated patients with different JEB phenotypes. Patients 1 and 2 had generalized atrophic benign epidermolysis bullosa (GABEB), with features including skin atrophy and alopecia. Patient 3 had the localisata variant of JEB, with predominantly acral blistering and normal hair. All patients carried novel homozygous point mutations (Q1016X, R1226X, and R1303Q) in the COL17A1 gene encoding collagen XVII, a hemidesmosomal transmembrane component; and, therefore, not only GABEB but also the localisata JEB can be a collagen XVII disorder. The nonsense mutations led to drastically reduced collagen XVII mRNA and protein levels. In contrast, the missense mutation allowed expression of abnormal collagen XVII, and epidermal extracts from that patient contained polypeptides of normal size, as well as larger aggregates. The homozygous nonsense mutations in the COL17A1 gene were consistent with the absence of the collagen from the skin and with the GABEB phenotype, whereas homozygosity for the missense mutation resulted in expression of aberrant collagen XVII and, clinically, in localisata JEB.     

Normal phenotype with maternal isodisomy in a female with two isochromosomes: i(2p) and i(2q)

A 36-year-old normal healthy female was karyotyped because all of her five pregnancies had terminated in spontaneous abortions during the first 3 mo. Cytogenetic investigation disclosed a female karyotype with isochromosomes of 2p and 2q replacing the two normal chromosomes 2. Her husband and both of her parents had normal karyotypes. Molecular studies revealed maternal only inheritance for chromosome 2 markers. Reduction to homozygosity of all informative markers indicated that the isochromosomes derived from a single maternal chromosome 2. Except for the possibility of homozygosity for recessive mutations, maternal uniparental disomy 2 appears to have no adverse impact on the phenotype. Our data indicate that no maternally imprinted genes with major effect map to chromosome 2.     

Partial isodisomy for maternal chromosome 7 and short stature in an individual with a mutation at the COL1A2 locus.

Uniparental disomy for chromosome 7 has been described previously in two individuals with cystic fibrosis. Here, we describe a third case that was discovered because the proband was homozygous for a mutation in the COL1A2 gene for type I procollagen, although his mother was heterozygous and his father did not have the mutation. Phenotypically, the proband was similar to the two previously reported cases with uniparental disomy for chromosome 7, in that he was short in stature and growth retarded. Paternity was assessed with five polymorphic markers. Chromosome 7 inheritance in the proband was analyzed using 12 polymorphic markers distributed along the entire chromosome. Similar analysis of the proband's two brothers established the phase of the alleles at the various loci, assuming minimal recombination. The proband inherited only maternal alleles at five loci and was homozygous at all loci examined, except one. He was heterozygous for an RFLP at the IGBP-1 locus at 7p13-p12. The results suggest that the isodisomy was not complete because of a recombination event involving the proximal short arms of two maternal chromosomes. In addition, the phenotype of proportional dwarfism in the proband suggests imprinting of one or more growth-related genes on chromosome 7.     

Prader-Willi syndrome with an unusually large 15q deletion due to an unbalanced translocation t(4;15)

Prader-Willi syndrome (PWS) is a neurobehavioral disorder caused by deletions in the 15q11-q13 region, by maternal uniparental disomy of chromosome 15 or by imprinting defects. Structural rearrangements of chromosome 15 have been described in about 5% of the patients with typical or atypical PWS phenotype. An 8-year-old boy with a clinical diagnosis of PWS, severe neurodevelopmental delay, absence of speech and mental retardation was studied by cytogenetic and molecular techniques, and an unbalanced de novo karyotype 45,XY,der(4)t(4;15)(q35;q14),-15 was detected after GTG-banding. The patient was diagnosed by SNURF-SNRPN exon 1 methylation assay, and the extent of the deletions on chromosomes 4 and 15 was investigated by microsatellite analysis of markers located in 4qter and 15q13-q14 regions. The deletion of chromosome 4q was distal to D4S1652, and that of chromosome 15 was located between D15S1043 and D15S1010. Our patient's severely affected phenotype could be due to the extent of the deletion, larger than usually seen in PWS patients, although the unbalance of the derivative chromosome 4 cannot be ruled out as another possible cause. The breakpoint was located in the subtelomeric region, very close to the telomere, a region that has been described as having the lowest gene concentrations in the human genome.     

Characterization of the human laminin beta2 chain locus (LAMB2): linkage to a gene containing a nonprocessed, transcribed LAMB2-like pseudogene (LAMB2L) and to the gene encoding glutaminyl tRNA synthetase (QARS).

The laminin beta2 chain is an important constituent of certain kidney and muscle basement membranes. We have generated a detailed physical map of a 110-kb genomic DNA segment surrounding the human laminin beta2 chain gene (LAMB2) on chromosome 3p21.3-->p21.2, a region paralogous with the chromosome 7q22-->q31 region that contains the laminin beta1 chain gene locus (LAMB1). Several CpG islands and a novel polymorphic microsatellite marker (D3S4594) were identified. The 3' end of LAMB2 lies 16 kb from the 5' end of the glutaminyl tRNA synthetase gene (QARS). About 20 kb upstream of LAMB2 we found a gene encoding a transcribed, non-processed LAMB2-like pseudogene (LAMB2L). The sequence of 1.75 kb of genomic DNA at the 3' end of LAMB2L was similar to exons 8-12 of the laminin beta2 chain gene. The LAMB2L-LAMB2-QARS cluster lies telomeric to the gene encoding the laminin-binding protein dystroglycan (DAG1).     

Ornithine transcarbamylase (OTC) deficiency in a female patient with a de novo deletion of the paternal X chromosome

Summary A girl with ornithine transcarbamylase (OTC) deficiency was investigated for molecular and cytogenetic abnormalities that might explain this phenotype. Analysis with polymorphic DNA markers indicated that the patient did not inherit paternal alleles of the OTC locus, but that she did inherit the proximal locus DXS7 and the long arm of chromosome X. High-resolution cytogenetic analysis of the patient indicated a deletion of Xp11.4-p21, whereas both parents had normal karytoypes. Since the mother might be heterozygous according to biochemical tests, a second mutation within the maternal OTC gene cannot be excluded.     

Uniparental heterodisomy for chromosome 14 in a phenotypically abnormal familial balanced 13/14 Robertsonian translocation carrier.

A 9-year-old mentally retarded girl with multiple congenital anomalies was found to carry a balanced 13/14 Robertsonian translocation [45,XX,t(13q14q)] which was also present in her father. Her mother carried a balanced reciprocal translocation between chromosomes 1 and 14 [46,XX,t(1;14) (q32;q32)]. Both of her parents were phenotypically normal. Molecular studies were carried out to determine the parental origin of chromosomes 1, 13, and 14 in the patient. Using probes for D14S13 and D14S22, we could show that the patient inherited both chromosomes 14 from her father and none from her mother. Similar studies using probes for chromosomes 1 (D1S76) and 13 (D13S37) loci showed the presence of both maternal and paternal alleles in the patient. Our findings indicate that paternal uniparental heterodisomy for chromosome 14 most likely accounts for the phenotypic abnormalities observed in our patient. It is suggested that uniparental disomy may be the basis for abnormal development in at least some phenotypically abnormal familial balanced-translocation carriers.     

Cloning of the human type XVII collagen gene (COL17A1), and detection of novel mutations in generalized atrophic benign epidermolysis bullosa.

Generalized atrophic benign epidermolysis bullosa (GABEB) is a nonlethal variant of junctional epidermolysis bullosa (JEB). Previous findings have suggested that type XVII collagen is the candidate gene for mutations in this disease. We now have cloned the entire human type XVII collagen gene (COL17A1) and have elucidated its intron-exon organization. The gene comprises 56 distinct exons, which span approximately 52 kb of the genome, on the long arm of chromosome 10. It encodes a polypeptide, the alpha1(XVII) chain, consisting of an intracellular globular domain, a transmembrane segment, and an extracellular domain that contains 15 separate collagenous subdomains, the largest consisting of 242 amino acids. We also have developed a strategy to identify mutations in COL17A1 by use of PCR amplification of genomic DNA, using primers placed on the flanking introns. The PCR products are scanned for sequence variants by heteroduplex analysis using conformation-sensitive gel electrophoresis and then are subjected to direct automated sequencing. We have identified several intragenic polymorphisms in COL17A1, as well as mutations, in both alleles, in two Finnish families with GABEB. The probands in both families showed negative immunofluorescence staining with an anti-type XVII collagen antibody. In one family, the proband was homozygous for a 5-bp deletion, 2944del5, which resulted in frameshift and a premature termination codon of translation. The proband in the other family was a compound heterozygote, with one allele containing the 2944del5 mutation and the other containing a nonsense mutation, Q1023X. These results expand the mutation database in different variants of JEB, and they attest to the functional importance of type XVII collagen as a transmembrane component of the hemidesmosomes at the dermal/epidermal junction.     

Evidence consistent with human L1 retrotransposition in maternal meiosis I

We have used a unique polymorphic 3' transduction to show that a human L1, or LINE-1 (long interspersed nucleotide element-1), retrotransposition event most likely occurred in the maternal primary oocyte during meiosis I. We characterized a truncated L1 retrotransposon with a 3' transduction that was inserted, in a Dutch male patient, into the X-linked gene CYBB, thereby causing chronic granulomatous disease. We used the unique flanking sequence to localize the precursor L1 locus, LRE3, to chromosome 2q24.1. In a cell culture assay, the retrotransposition frequency of LRE3 is greater than that for any other element that has been tested to date. The patient's mother had two LRE3 alleles that differed slightly in the 3'-flanking genomic DNA. The patient had a single LRE3 allele that was identical to one of the maternal alleles; however, the patient's insertion matched the maternal LRE3 allele that he did not inherit. Other data indicate that there is only a small chance that the father (unavailable for analysis) carries the precursor LRE3 allele. In addition, paternal origin of the insertion would have required that an LRE3 mRNA transcribed before meiosis II be carried separately from its precursor LRE3 allele in the fertilizing sperm. Since the mother carries a potential precursor allele and the insertion was on the patient's maternal X chromosome, it is highly likely that the insertion originated during maternal meiosis I.     

Mutations in TRIOBP, which encodes a putative cytoskeletal-organizing protein, are associated with nonsyndromic recessive deafness

In seven families, six different mutant alleles of TRIOBP on chromosome 22q13 cosegregate with autosomal recessive nonsyndromic deafness. These alleles include four nonsense (Q297X, R788X, R1068X, and R1117X) and two frameshift (D1069fsX1082 and R1078fsX1083) mutations, all located in exon 6 of TRIOBP. There are several alternative splice isoforms of this gene, the longest of which, TRIOBP-6, comprises 23 exons. The linkage interval for the deafness segregating in these families includes DFNB28. Genetic heterogeneity at this locus is suggested by three additional families that show significant evidence of linkage of deafness to markers on chromosome 22q13 but that apparently have no mutations in the TRIOBP gene.     

First small supernumerary ring chromosome carrying 10q euchromatin in a patient with mild phenotype characterized by molecular cytogenetic techniques and review of the literature

We report the identification and characterization of the first supernumerary ring chromosome 10 containing a considerable proportion of 10q euchromatin by microdissection and reverse painting in a female patient presenting with short stature. Fluorescence in situ hybridization studies showed that the marker chromosome originates from chromosome 10 and includes the euchromatic bands p11.2 and q11.2. The supernumerary marker chromosome 10 was found in 14% of the peripheral blood lymphocytes analyzed. This constitutional mosaic could be confirmed in oral mucosa cells as a second cell system (16%) by interphase FISH using an alphoid centromeric probe for chromosome 10. Parental karyotypes were normal, uniparental disomy for the normal chromosomes 10 could be excluded by microsatellite analysis. The karyotype of the patient detected in peripheral blood cells can be described as mos 47,XX,+mar.rev ish r(10)(p11.2q11.2)(wcp10+,cep10+)/46,XX.