A dystrophin missense mutation showing persistence of dystrophin and dystrophin-associated proteins yet a severe phenotype

A muscle biopsy from an X-linked muscular dystrophy pedigree showed normal dystrophin and dystrophin-associated proteins. Linkage to multiple markers within the dystrophin gene (LOD=2.7, theta=0) indicated a primary dystrophinopathy. Sequencing of the entire dystrophin RNA revealed a single missense mutation (D3335H) in the unique carboxyl-terminal domain. This is the first report showing that a relatively severe dystrophinopathy can occur despite the correct localization of dystrophin and dystrophin-associated proteins.     

A novel missense mutation in patients from a retinoblastoma pedigree showing only mild expression of the tumor phenotype

We have used single strand conformation polymorphism analysis to study the 27 exons of the RB1 gene in individuals from a family showing 'mild' expression of the retinoblastoma phenotype. In this family affected individuals developed unilateral tumors and, as a result of linkage analysis, unaffected mutation carriers were also identified within the pedigree. A single band shift using SSCP was identified in exon 21 which resulted in a missense mutation converting a cys-->arg at nucleotide position 28 in the exon. The mutation destroyed an NdeI restriction enzyme site. Analysis of all family members demonstrated that the missense mutation co-segregated with patients with tumors or who, as a result of linkage analysis had been predicted to carry the predisposing mutation. These observations point to another region of the RB1 gene where mutations only modify the function of the gene and raise important questions for genetic counseling in families with these distinctive phenotypes.     

Mutation analysis of the MEN1 gene in multiple endocrine neoplasia type 1, familial acromegaly and familial isolated hyperparathyroidism

Multiple endocrine neoplasia type 1 (MEN 1) is an autosomal dominant disease characterized by neoplasia of the parathyroid glands, the endocrine pancreas, and the anterior pituitary gland. In addition, families with isolated endocrine neoplasia, notably familial isolated hyperparathyroidism (FIHP) and familial acromegaly, have also been reported. However, whether these families constitute MEN 1 variants or separate entities remains speculative as the genetic bases for these diseases are unclear. The gene for MEN 1 has recently been cloned and characterized. Using single strand conformation analysis (SSCA) and sequencing, we performed mutation analysis in: a) a total of 55 MEN 1 families from 7 countries, b) 13 isolated MEN 1 cases without family history of the disease, c) 8 acromegaly families, and d) 4 FIHP families. Mutations were identified in 27 MEN 1 families and 9 isolated cases. The 22 different mutations spread across most of the 9 translated exons and included frameshift (11), nonsense (6), splice (2), missense mutations (2), and in-frame deletions (1). Among the 19 Finnish MEN 1 probands, a 1466del12 mutation was identified in 6 families with identical 11q13 haplotypes and in 2 isolated cases indicating a common founder. One frameshift mutation caused by 359del4 (GTCT) was found in 1 isolated case and 4 kindreds of different origin and haplotypes; this mutation therefore represents a common "warm" spot in the MEN1 gene. By analyzing the DNA of the parents of an isolated case one mutation was confirmed to be de novo. No mutation was found in any of the acromegaly and small FIHP families, suggesting that genetic defects other than the MEN1 gene might be involved and that additional such families need to be analyzed.     

A missense mutation, Val62Ala, in the glucokinase gene in a Norwegian family with maturity-onset diabetes of the young

Hodgkin's disease primarily originating from the chest wall is very rare. A 48-year-old man was admitted to our hospital because of an abnormal shadow on a chest X-ray. Radiographic examinations suggested a neurogenic tumor located in the right second-intercostal space, and it was thus extirpated thoracoscopically. The tumor was thought to have arisen from the subpleural space, probably from a lymph node of the chest wall. The resected specimen measured 5.5 x 2.0 cm in size, and the pathological diagnosis was Hodgkin's disease of the diffuse lymphocyte predominant type. A clinical examination showed no other lesions in any other part of the body, including the bone marrow. Following surgery, adjuvant chemotherapy (COPP-ABVD) was given because of the possible scattering of malignant cells during surgery. To the best of our knowledge, this is the first report of Hodgkin's disease originating in the chest wall.     

Germline BRCA1 mutations and loss of the wild-type allele in tumors from families with early onset breast and ovarian cancer

The BRCA1 gene on human chromosome 17q21 is responsible for an autosomal dominant syndrome of inherited early onset breast/ovarian cancer. It is estimated that women harboring a germline BRCA1 mutation incur an 85% lifetime risk of breast cancer and a greatly elevated risk of ovarian cancer. The BRCA1 gene has recently been isolated and mutations have been found in the germline of affected individuals in linked families. Previous studies of loss of heterozygosity (LOH) in breast tumors have been carried out on sporadic tumors derived from individuals without known linkage to BRCA1 and on tumors from linked families. Loss of large regions of chromosome 17 has been observed, but these LOH events could not be unequivocally ascribed to BRCA1. We have studied 28 breast and 6 ovarian tumors from families with strong evidence for linkage between breast cancer and genetic markers flanking BRCA1. These tumors were examined for LOH using genetic markers flanking and within BRCA1, including THRA1, D17S856, EDH17B1, EDH17B2, and D17S183. Forty-six percent (16/34) of tumors exhibit LOH which includes BRCA1. In 8 of 16 tumors the parental origin of the deleted allele could be determined by evaluation of haplotypes of associated family members; in 100% of these cases, the wild-type allele was lost. In some of these families germline mutations in BRCA1 have been determined; analyses of tumors with LOH at BRCA1 have revealed that only the disease-related allele of BRCA1 was present. These data strongly support the hypothesis that BRCA1 is a tumor suppressor gene.     

Functional defects of Cx26 resulting from a heterozygous missense mutation in a family with dominant deaf-mutism and palmoplantar keratoderma

BACKGROUND: There is controversy about the impact on morbidity from delayed diagnoses of blunt hollow viscus injuries. A recent study suggested that the increased morbidity was primarily from delayed diagnosis of blunt duodenal injury (BDI). STUDY DESIGN: We studied the medical records from a 10-year period from June 1987 to June 1997 examining the data on 22,163 cases of blunt trauma. We assessed the incidence and consequences of delayed diagnoses of BDI, and identified preoperative factors associated with these delayed diagnoses. RESULTS: Thirty-five patients (0.2%) were identified in the retrospective study of the records from 22,163 blunt trauma patients to have sustained BDI. Of these, 25 patients (71%) were male. Ages ranged from 1 to 58 years (mean 18.8 years), and the predominant mechanism was motor vehicle accident in 18 patients (51%). Seven patients (20%) (group I) had a diagnostic delay of > 6 hours; 28 patients (80%) (group II) were diagnosed in < 6 hours. Six of the seven group I patients (86%) were evaluated initially with CT scans, and five (83%) showed findings suggestive of BDI. Among the 28 group II patients, 14 (50%) underwent initial diagnostic peritoneal lavage (DPL), and 14 (50%) had a CT scan. In seven of the group II patients (50%) who were initially evaluated by CT scan, there were findings suggestive of BDI. Diagnostic peritoneal lavage was initially equivocal (red blood cell count=5,000 to 100,000) in the remaining one group I patient compared with three of the group II patients who had DPL. Deterioration found on physical examinations prompted followup CT scans in 6 group I patients (86%), and the scans were diagnostic for BDI in all cases. CONCLUSIONS: Blunt duodenal injury is an uncommon entity. Despite the presence of suggestive CT and DPL findings, the diagnosis was delayed in 20% of the 35 patients whose records were examined in the study; this delayed diagnosis was associated with increased abdominal complications. Patients with persistent abdominal complaints and equivocal CT or DPL findings should undergo laparotomy or repeat CT scan evaluations.     

Modulation of disease severity of dystrophic epidermolysis bullosa by a splice site mutation in combination with a missense mutation in the COL7A1 gene

Dystrophic epidermolysis bullosa (EBD) is a clinically heterogeneous skin disorder, characterized by abnormal anchoring fibrils (AF) and loss of dermal-epidermal adherence. EBD has been linked to the COL7A1 gene at chromosome 3p21 which encodes collagen VII, the major component of the AF. Here we investigated two unrelated EBD families with different clinical phenotypes and novel combinations of recessive and dominant COL7A1 mutations. Both families shared the same recessive heterozygous 14 bp deletion at the exon-intron 115 boundary of the COL7A1 gene. The deletion caused in-frame skipping of exon 115 and the elimination of 29 amino acid residues from the pro-alpha1(VII) polypeptide chain. As a result, procollagen VII was not converted to collagen VII and the C-terminal NC-2 propeptide which is normally removed from the procollagen VII prior to formation of the anchoring fibrils was retained in the skin. All affected individuals also carried missense mutations in exon 73 of COL7A1 which lead to different glycine-to-arginine substitutions in the triple-helical domain of collagen VII. Combination of the deletion mutation with a G2009R substitution resulted in a mild phenotype. In contrast, combination of the deletion with a G2043R substitution led to a severe phenotype. The G2043R substitution was a de novo mutation which alone caused a mild phenotype. Thus, different combinations of dominant and recessive COL7A1 mutations can modulate disease activity of EBD and alter the clinical presentation of the patients.     

p53 functional loss in a colon cancer cell line with two missense mutations (218leu and 248trp) on separate alleles

We have sequenced p53 in three colon cancer cell lines capable of autonomous proliferation. SNU-C1 and SNU-C4 cells, whose autonomous growth is dependent upon autocrine stimulation of epidermal growth factor receptor (EGFR), had wildtype p53 sequence of exons 4-9. In contrast, an EGFR ligand-independent cell line, SNU-C5, had heterozygous missense mutations affecting codons 218 (valine to leucine) and 248 (arginine to tryptophan) of p53. Bacterial cloning of p53 from SNU-C5 cells showed that the 248trp and 218leu mutants were both expressed and on separate alleles. 248trp is a common 'hot spot' mutant of p53 with variable dominant negative activity depending on the celullar context. Valine 218, in contrast, is rarely affected by mutation in cancers and is located in a region of the hydrophobic core domain away from 'hot spot' DNA contact sights. However, valine 218 is completely conserved across species, prompting us to investigate the function of 218leu in SNU-C5 cells. SNU-C5 cells exhibited complete loss of normal p53 function as evidenced by over-expression of p53 protein and by failure to show induction of p53, waf-1, mdm-2 or G1/S arrest in response to the DNA damaging agent, bleomycin. In a yeast p53 functional assay (FASAY), 50% of the clones were unable to transactivate a p53-specific promoter required for yeast colony expansion at 25, 30 or 37 degrees C. Sequencing of the p53 insert from several randomly selected wild-type and mutant yeast clones revealed that 218leu-bearing clones retained their ability to transactivate the p53-specific promoter. As expected, the 248trp-bearing clones lost this function. These data indicate that although 218leu retains normal transactivation activity on a p53 promoter in yeast at physiological temperatures, it is not capable of normal p53 function in the presence of a 248trp allele in SNU-C5 cells. It remains unclear whether the strong dominant negative activity of 248trp in SNU-C5 cells is related to the cellular context or to an unresolved abnormality of 218leu function.     

A recurrent missense mutation in the KAL gene in patients with X-linked Kallmann's syndrome

Kallmann's syndrome (KS) is defined by the association of hypogonadotropic hypogonadism and anosmia or hyposmia. Segregation analysis in familial cases has demonstrated diverse inheritance patterns, suggesting the existence of several genes regulating GnRH secretion. Genetic defects have been demonstrated in the KAL gene, located on the Xp22.3 region, explaining the X-linked form of the disease. We report molecular findings regarding the KAL gene in 12 unrelated males with X-linked KS. PCR of the 14 exons of the KAL gene was performed on genomic DNA. PCR products of all exons were purified and sequenced. Genetic defects in the KAL gene were found in 7 patients. One exhibits a deletion from exon 3 to exon 5. Six individuals present a previously unidentified missense mutation in exon 11, consisting of a G to A substitution at codon 514 (GAA to AAA). In the remaining 5 individuals, no mutations were observed. We also found three different polymorphic changes. The first one, in exon 2, had not been reported previously. The other two were located at exons 11 and 12. The deletion described, comprises only part (exon 5) of the coding region of the first fibronectin type III-like repeat of the KAL protein. The rest of the deletion comprises part of the conserved cysteine-rich N-terminal region that corresponds to the whey acidic protein motif. The same missense mutation was found in 6 of the 12 patients, indicating the possibility that it derived from a common ancestor or suggesting the presence of a hot spot in this region of the gene.     

Identification of MEN1 gene mutations in sporadic carcinoid tumors of the lung

Lung carcinoids occur sporadically and rarely in association with multiple endocrine neoplasia type 1 (MEN1). There are no well defined genetic abnormalities known to occur in these tumors. We studied 11 sporadic lung carcinoids for loss of heterozygosity (LOH) at the locus of the MEN1 gene on chromosome 11q13, and for mutations of the MEN1 gene using dideoxy fingerprinting. Additionally, a lung carcinoid from a MEN1 patient was studied. In four of 11 (36%) sporadic tumors, both copies of the MEN1 gene were inactivated. All four tumors showed the presence of a MEN1 gene mutation and loss of the other allele. Observed mutations included a 1 bp insertion, a 1 bp deletion, a 13 bp deletion and a single nucleotide substitution affecting a donor splice site. Each mutation predicts truncation or potentially complete loss of menin. The remaining seven tumors showed neither the presence of a MEN1 gene mutation nor 11q13 LOH. The tumor from the MEN1 patient showed LOH at chromosome 11q13 and a complex germline MEN1 gene mutation. The data implicate the MEN1 gene in the pathogenesis of sporadic lung carcinoids, representing the first defined genetic alteration in these tumors.     

A missense mutation in the hepatocyte nuclear factor 4 alpha gene in a UK pedigree with maturity-onset diabetes of the young

Maturity-onset diabetes of the young (MODY) is a monogenic subgroup of non-insulin dependent diabetes mellitus (NIDDM) characterised bylan early age of onset (< 25 years) and an autosomal dominant mode of inheritance. MODY is genetically heterogeneous with three different genes identified to date; hepatocyte nuclear factor 4 alpha (HNF-4 alpha) [MODY1], glucokinase [MODY2] and hepatocyte nuclear factor 1 alpha (HNF-1 alpha) [MODY3]. A nonsense mutation in the HNF-4 alpha gene has recently been shown to cause MODY in a single large North American pedigree (RW). We screened a large UK Caucasian MODY family which showed weak evidence of linkage to the MODY1 locus on chromosome 20q (lod score for ADA 0.68 at theta = 0) for mutations in the coding region of the HNF-4 alpha gene by direct sequencing. A missense mutation resulting in the substitution of glutamine for glutamic acid was identified in exon 7 (E276Q). The mutation was present in all of the diabetic members of the pedigree plus two unaffected subjects and was not detected in 75 normal control subjects or 95 UK Caucasian subjects with late-onset NIDDM. This is the first missense mutation to be described in the HNF-4 alpha gene.     

Cure of hyperparathyroidism in pregnancy by sternotomy and removal of a mediastinal parathyroid adenoma

Primary hyperparathyroidism is rarely reported during pregnancy but can cause significant maternal and neonatal morbidity. We report a case of hyperparathyroidism during pregnancy requiring a median sternotomy for resection of a mediastinal parathyroid adenoma. Surgery resulted in normalisation of serum calcium, resolution of symptoms, and prevented neonatal hypocalcaemia.