Molecular basis of inherited factor XIII deficiency: identification of multiple mutations provides insights into protein function

Factor XIII (FXIII) is a zymogen essential for normal haemostasis. In inherited FXIII deficiency the majority of cases show absence of the FXIIIa subunit. Molecular analysis of PCR-amplified FXIIIa subunit exonic regions, and of RT-PCR amplified cDNA from six patients with FXIIIa subunit deficiency, from five unrelated families, has revealed 10 sequence changes: three mutations resulting in abnormal splicing of pre-mRNA, one nonsense mutation, one deletion/insertion change, three point mutations producing Val34Leu, Asn60Lys and Arg408Gln changes, and two silent mutations. In three families the patients are homozygous for a specific deficiency causing mutation, and patients from the remaining two families are compound heterozygotes. Understanding the molecular pathology of the disorder provides insights into the structure-function relationships of the various domains within the FXIII protein. From a clinical point of view, it enables direct diagnosis at the DNA level and may aid the development of FXIII analogues to promote wound healing.     

A molecular epidemiological study of porcine rotaviruses

15-20% of the CF mutation are expected to be rare and escape detection by systems designed to screen for common mutations. The highly polymorphic simple repeats would be particularly useful for genetic diagnosis in CF families where the mutations have not been identified. In this study, we used denaturing gradient gel electrophoresis with psoralen-modified oligonucleotide primers to study the GTnTm polymorphism previously identified at the intron 8 - exon 9 junction of the CFTR gene. Twelve characteristic patterns were identified. The most frequent genotype in CF alleles was GT10T9 and in non-CF alleles GT11T7. In this study, the heterozygous incidence is 70% in unrelated CF carriers. This polymorphism is full informative in 45% and half-informative in 50%. We conclude, that this polymorphism, easy to study by a relatively simple, rapid and cheap procedure, would be particularly useful in genetic counselling for CF and prenatal diagnosis in CF families in which mutations have not been yet identified.     

An investigation of a family tree with congenital deficiency in coagulation factor XIII]

In order to study the hereditary trait of a 20-year-old girl with congenital deficiency of coagulation factor XIII and her family tree, the following laboratory tests were done for the propositus and her family members: clot solubility test in 5 mol/L urea, estimation of factor XIII activity, amount of the subunits A and S of factor XIII with rocket electrophoresis. The results showed that the propositus had clinical history of bleeding in umbilical cord and its stump after birth. Her parents are not consanguineously related and have no history suggestive of hemorrhagic diathesis. The propositus has one brother and three sisters. One of her sisters died of bleeding of the umbilical cord after birth, another died at age of two, but the reason of her death is unclear and the remaining siblings are clinically healthy. The propositus had an abnormal urea clot lysis test, but the other family members had normal results. For the propositus factor XIII activity was 0%. A subunit 0% and S subunit 8.2%. Factor XIII activity of her father, her mother and her sister were 25%, 50%, 25% respectively. A subunit 52%, 58%, and 58% respectively and S subunit 66%, 68% and 66% respectively. The results showed that the propositus has a congenital deficiency in coagulation factor XIII, and her parents and sister are in fact carriers.     

First report of prenatal biochemical diagnosis of Lowe syndrome

The oculocerebrorenal syndrome of Lowe (OCRL) is a rare X-linked disorder with a severe phenotype characterized by congenital cataracts, renal tubular dysfunction and neurological deficits. The gene has been characterized and mutations have been identified in patients. Owing to the allelic heterogeneity exhibited by this gene, prenatal diagnosis by molecular analysis is limited to families in which the mutation is already known or in which linkage is informative. A more generally applicable diagnostic test would be valuable for families at risk for Lowe syndrome. Since ocrl1 is now known to encode a phosphatidylinositol 4,5-bisphosphate 5-phosphatase (Ptdlns(4,5)P2 phosphatase), we assessed whether biochemical testing could be used for prenatal diagnosis. We report here the first case of prenatal diagnosis for Lowe syndrome by measuring phosphatidylinositol 4,5-bisphosphate 5-phosphatase activity in cultured amniocytes.     

Affinity chromatography of human plasma and platelet factor XIII on organomercurial agarose

A method for affinity chromatography of plasma and platelet factor XIII has been developed, based on known structural characteristics of these molecules. Plasma factor XIII is composed of a and b subunits which are held together by noncovalent interactions; platelet factor XIII has only a subunits. a subunit contains free sulfhydryl groups, while in b subunit all the cystines form disulfide bonds. The affinity gel is an organomercurial agarose with p-chloromercuribenzoate as the reactive group. Both the zymogen and activated forms of a subunit reversibly bind to the ligand by forming covalent mercaptide bonds and are eluted by reducing agents. b subunit does not bind to the affinity gel and is held to it only through interaction with a subunit. Affinity chromatography can be used to purify plasma and platelet factor XIII and to study interactions of the subunits. Experiments on the affinity chromatography of purified plasma factor XIII in several stages of activation agree with earlier observations that activation is a two-step procedure in which b subunit is not quantitatively released from the complex until the final stage of activation by Ca2+.     

Relationship between parental trinucleotide GCT repeat length and severity of myotonic dystrophy in offspring

OBJECTIVE: To assess the relationship between the GCT repeat number in the myotonic dystrophy gene and the clinical phenotype and examine its predictive utility in prenatal testing. DESIGN: DNA from patients was examined for the length of the myotonic dystrophy GCT repeat region, using both Southern blot analysis and polymerase chain reaction. The results were compared with the clinical onset of disease, as well as with pregnancy outcomes. SETTING: Patient samples were referred to the Kleberg DNA Diagnostic Laboratory at the Baylor College of Medicine for DNA analysis by geneticists and genetic counselors (84%), neurologists (10%), and obstetricians and other specialists (6%). Clinical features including onset of disease and family pedigrees were determined by the referring centers. PATIENTS: A total of 241 patient samples from 118 families referred from primarily genetic or neurological centers for genetic linkage analysis or mutation analysis for myotonic dystrophy. This included 44 families referred for prenatal diagnosis. MAIN OUTCOME MEASURES: A relationship between myotonic dystrophy disease onset and length of the GCT repeat allele, parental origin of the disease allele, and results of prenatal diagnosis predictions of disease status were measured. RESULTS: There is a relationship between increasing repeat length and earlier clinical onset of disease. Essentially all (> 99%) myotonic mutations causing myotonic dystrophy are accounted for by GCT repeat amplification. Congenital myotonic dystrophy occurs with as few as 730 GCT repeats but only with alleles of maternal origin. Maternal GCT repeats were found as low as 75 (asymptomatic) that were amplified to result in a child with congenital myotonic dystrophy. Application of DNA diagnosis to 32 pregnancies provided an accurate method for identification of at-risk fetuses and allele enlargement. CONCLUSIONS: The GCT repeat in myotonic dystrophy is highly mutable. The triplet repeat amplification is highly specific for mutations involving the myotonin protein kinase gene accounting for myotonic dystrophy. The quantitation of triplet repeats can be more sensitive than physical, ophthalmologic, and electromyography examinations since the mutation can be detected in patients without evidence of myotonic dystrophy clinical findings. The length of the triplet expansion is influenced by the sex of the transmitting parent and is related to the clinical onset of disease features. Prenatal measurement of the GCT triplet repeat has utility for families with myotonic dystrophy risk since mutant and normal repeats are distinguishable and the length of mutant repeat alleles is associated with clinical severity. Thus, GCT triplet measurement provides a highly accurate means of detecting the myotonic dystrophy mutation in patients and offers a new reproductive option for families at risk for myotonic dystrophy.     

Nonisotopic single strand conformation analysis of the 5 alpha-reductase type 2 gene for the diagnosis of 5 alpha-reductase deficiency

5 alpha-Reductase deficiency is a rare autosomal recessive disorder of defective virilization in karyotypic males due to reduced conversion of testosterone to dihydrotestosterone. The gene encoding the affected 5 alpha-reductase type 2 enzyme has recently been cloned, and mutations within the coding region have been discovered as the cause of this disease. We address the possibility of a rapid nonradioactive molecular genetic screening technique for initial diagnosis and report different point mutations in this gene in eight unrelated patients with clinical features of 5 alpha-reductase deficiency. For molecular genetic analysis, DNA from peripheral blood leukocytes was studied. The coding region of the 5 alpha-reductase type 2 gene was characterized by exon-specific PCR amplification, nonradioactive single strand conformation analysis, and direct sequencing. In seven patients, homozygous point mutations were identified (Leu55-Gln, delta Met157, Gly196-Ser, Arg227-Gln, Ala228-Thr, and His231-Arg). One individual was a compound heterozygote carrier of two mutations (Ile112-Asn and Gln126-Arg). We conclude that molecular genetic characterization of point mutations in the 5 alpha-reductase type 2 gene may be used as an additional valuable procedure for the diagnosis of this disorder.     

Microsatellite analysis of Duchenne muscular dystrophy

Duchenne muscular dystrophy is X-linked recessive neuromuscular disorders caused by mutations in the dystrophin gene. Prenatal diagnosis and carrier detection are usually performed using diallelic RFLP-markers which are not always informative. Now 30 of microsatellite marker have reported, these microsatellite polymorphism can easily be amplified using PCR technique. If mutations are known to localize in this region of the dystrophin gene or if routine RFLP-analysis is uninformative, the analysis of microsatellite markers is the preferable technique in prenatal diagnosis and carrier detection.     

The Val34Leu polymorphism in the A subunit of coagulation factor XIII contributes to the large normal range in activity and demonstrates that the activation peptide plays a role in catalytic activity

There is a wide normal range of coagulation factor XIII activity that has never been adequately explained. A polymorphism substituting leucine for valine at position 34 in the activation peptide of the A subunit of factor XIII has recently been discovered in nondeficient individuals, and the present studies indicate that the leucine substitution results in a significant increase in transglutaminase activity. The frequency of the Leu34 allele in the Australian Caucasian population is 0.27, which is high enough to suggest that the inheritance of either the Val34 or Leu34 alleles may contribute to the wide normal range of activity. Although there has been structural evidence indicating that the activation peptide does not dissociate from the enzyme after thrombin cleavage, the discovery of elevated activity resulting from the Leu34 substitution is the first direct evidence that the activation peptide plays a continuing role in the function of factor XIII.     

Clinical and molecular heterogeneity in carbonic anhydrase II deficiency and prenatal diagnosis in an Italian family

Carbonic anhydrase (CA) II deficiency is characterized by osteopetrosis, renal tubular acidosis, cerebral calcification, and usually severe mental retardation. We describe an Italian boy with this disease whose mental retardation was relatively mild and whose renal tubular acidosis had only a distal component. A novel mutation of a gt-->tt change of splice donor site at the 5' end of intron 6 was demonstrated. Comparison of this patient with two previous Italian families with different mutations illustrates the clinical and molecular heterogeneity of this disease. The identification of the mutation in this family provided the opportunity for prenatal diagnosis in a subsequent pregnancy.     

The clinical and molecular genetic approach to Duchenne and Becker muscular dystrophy: an updated protocol

Detection of large rearrangements in the dystrophin gene in Duchenne and Becker muscular dystrophy is possible in about 65-70% of patients by Southern blotting or multiplex PCR. Subsequently, carrier detection is possible by assessing the intensity of relevant bands, but preferably by a non-quantitative test method. Detection of microlesions in Duchenne and Becker muscular dystrophy is currently under way. Single strand conformational analysis, heteroduplex analysis, and the protein truncation test are mostly used for this purpose. In this paper we review the available methods for detection of large and small mutations in patients and in carriers and propose a systematic approach for genetic analysis and genetic counselling of DMD and BMD families, including prenatal and preimplantation diagnosis.     

CDKN2A mutations in multiple primary melanomas

BACKGROUND: Germ-line mutations in the CDKN2A tumor-suppressor gene (also known as p16, p16INK4a, and MTS1) have been linked to the development of melanoma in some families with inherited melanoma. Whether mutations in CDKN2A confer a predisposition to sporadic (nonfamilial) melanoma is not known. In some patients with sporadic melanoma, one or more additional primary lesions develop, suggesting that some of these patients have an underlying genetic susceptibility to the cancer. We hypothesized that this predisposition might be due to germ-line CDKN2A mutations. METHODS: We used the polymerase chain reaction, single-strand conformation polymorphism analysis, and direct DNA sequencing to identify germ-line mutations in the CDKN2A gene in patients with multiple primary melanomas who did not have family histories of the disease. A quantitative yeast two-hybrid assay was used to evaluate the functional importance of the CDKN2A variants. RESULTS: Of 33 patients with multiple primary melanomas, 5 (15 percent; 95 percent confidence interval, 4 percent to 27 percent) had germ-line CDKN2A mutations. These included a 24-bp insertion at the 5' end of the coding sequence, three missense mutations (Arg24Pro, Met53Ile, and Ser56Ile), and a 2-bp deletion at position 307 to 308 (resulting in a truncated CDKN2A protein). In three families, CDKN2A mutations identical to those in the probands were found in other family members. In two families with mutations, we uncovered previously unknown evidence of family histories of melanoma. CONCLUSIONS: Some patients with multiple primary melanomas but without family histories of the disease have germ-line mutations of the CDKN2A gene. The presence of multiple primary melanomas may signal a genetic susceptibility to melanoma not only in the index patient but also in family members, who may benefit from melanoma-surveillance programs.     

Analysis of factor VIII gene polymorphisms in Brazilian blacks reveals further differences in the black population

We report here the analysis of four intragenic (BclI, HindIII, XbaI, and BglI) and two extragenic (MspI and TaqI/St14) polymorphisms associated with the factor VIII gene in the Brazilian Black population. No difference was observed for the allelic frequencies of the BclI, HindIII, and BglI polymorphisms when we compared our results with those reported for North American Blacks. For the first time, the XbaI and MspI polymorphisms were investigated in a Black population. Interestingly, the XbaI polymorphism is almost monomorphic for the Brazilian Black population, differing in this respect from all other populations studied thus far. The MspI polymorphism presents inverse allelic frequencies for the Brazilian Blacks when compared with Caucasians, as observed for the BclI and HindIII polymorphisms. Analysis of the TaqI/St14 multiallelic system revealed five novel alleles (ranging from 5.8 to 11.0 kb) which may represent unique alleles for the Black population. The results show that analysis of factor VIII gene polymorphisms and haplotypes may be successfully used for investigating interrelationships between human populations. The results also allow definition of a strategy for carrier detection and prenatal diagnosis of hemophilia A in this population based on analysis of factor VIII gene polymorphisms, which is considered an alternative approach for those families where identification of gene mutation is not feasible.     

Screening for tumour suppressor p16(CDKN2A) germline mutations in Israeli melanoma families

Approximately ten percent of patients with malignant melanoma have family histories of the disease, suggesting a genetic predisposition. Germline mutations in tumour suppressor p16 gene have been implicated as disease causing mutations in some of the melanoma families. The frequency of families with p16 germline mutations among melanoma prone families varies from eight to fifty percent. The range of the variability is influenced apparently by the number of melanoma affected individuals within the family, as well as by other, yet unidentified factors. Ethnic background is known to determine both the frequency and the nature of germline alterations. Recently, specific mutations in tumour suppressor genes involved in breast cancer and in colon cancer were found at elevated frequency among Ashkenazi Jews. This report describes results of a screening for p16 germline alterations in a collection of Israeli melanoma families. We have analyzed genomic DNA from thirty one Ashkenazi and non-Ashkenazi Jewish melanoma families, as well as from thirty melanoma patients without an apparent family history of the disease. The entire coding region of the p16 gene was screened by single strand conformation polymorphism analysis and direct DNA sequencing. We have detected a number of carriers with the Ala148 Thr polymorphism at the end of the second exon and several instances of 500(G=>C) substitution at the 3' untranslated portion of the gene.     

Roe v. Wade and the euthanasia debate

Epidermolysis bullosa (EB) is a group of heritable diseases which manifest with blistering and erosions of the skin and mucous membranes. Due of life-threatening complications and significant long-term morbidity associated with the severe, neonatal lethal (Herlitz) form of junctional EB (H-JEB), there has been a demand for prenatal diagnosis from families at risk for recurrence. Previously, the only reliable method of prenatal diagnosis of EB was a fetal skin biopsy performed at 16-20 weeks' gestation and analysed by electron microscopy. Recently, the genes LAMA3, LAMB3, and LAMC2, encoding the polypeptide subunits of laminin 5, an anchoring filament protein, have been shown to contain mutations in H-JEB. In this study, direct detection of pathogenetic mutations in the laminin 5 genes was used to perform polymerase chain reaction (PCR)-based prenatal testing. DNA was obtained by chorionic villus sampling (CVS) at 10-15 weeks or amniocentesis at 12-19 weeks' gestation in 15 families at risk for recurrence of JEB. In 13 cases, the fetus was predicted to be either genetically normal or a clinically unaffected carrier of a mutation in one allele. These predictions have been validated in all cases by the birth of a healthy child. In two cases, an affected fetus was predicted, and the diagnosis was confirmed by subsequent fetal skin biopsy. These results demonstrate that DNA-based prenatal testing offers an early, expedient, and accurate method of prenatal diagnosis or an exclusion of Herlitz JEB.     

Analysis of inversion in intron 22 of the factor F VIII:C gene in patients with hemophilia A in the Slovak population

Hemophilia A is the result of Factor F VIIIC (F8C) gene mutations. Predominating mutation is inversion, occurring in about 50% of patients with severe form of the disease. Inversion is the result of homologous recombination between gene A located on the 22. introne of the F8C gene and one of its telomeric copies located about 500 kb from 5'end of the factor F VIIIC gene. This study presents the results of this mutation screening in 84 nonrelated patients with hemophilia A. Inversion was identified in 22 (50%) of 44 patients with severe form and in 1 (from 13) with moderate form of the disease. Distal type of inversion was more frequent (82.6%) than proximal one. The identification of iversions enabled direct DNA diagnosis in 50% of patients with severe form of the disease and will be successfully used in the prenatal diagnosis and carrier testing, mainly in families with sporadic occurrence of the disease. (Tab. 1, Fig. 2, Ref. 18.)     

Prenatal diagnosis

The enormous progress witnessed in the field of prenatal diagnosis during the past two decades is likely to continue into the future. Improved imaging techniques are likely to enhance the resolution of noninvasively obtained fetal images considerably over their current excellent quality. Although this undoubtedly will be true for ultrasonography, the increased speed of magnetic resonance equipment may offer a new realm of imaging possibilities. Computerized image processing, analysis, and three-dimensional reconstructions all should make interpretation of fetal images easier and more understandable to the nonspecialist. Advances in molecular genetics will continue to accelerate, greatly expanding the range and accuracy of prenatal diagnosis. The alert pediatrician who is sensitive to genetic issues may, by early detection of pediatric disorders and careful family history assessment, be in a position to identify families at risk for serious genetic conditions and provide the opportunity to make informed decisions on reproductive options that avert a major tragedy. The pediatrician, working with obstetric colleagues, should be part of a team effort to support families going through prenatal testing. Familiarity with these rapidly changing technologies will make it far easier to support the family needing additional explanation about prenatal diagnosis issues.     

Mutation of RFXAP, a regulator of MHC class II genes, in primary MHC class II deficiency

BACKGROUND: Major-histocompatibility-complex (MHC) class II deficiency is an autosomal recessive primary immunodeficiency disease in which MHC class II molecules are absent. It is a genetically heterogeneous disease of gene regulation resulting from defects in several transactivating genes that regulate the expression of MHC class II genes. The mutations responsible for MHC class II deficiency are classified according to complementation group (a group in which the phenotype remains uncorrected in pairwise fusions of cells). There are three known complementation groups (A, B, and C). METHODS: To elucidate the genetic defect in patients with MHC class II deficiency that was not classified genetically, we performed direct complementation assays with the three genes known to regulate the expression of MHC class II genes, CIITA, RFX5, and RFXAP, and the relevant mutations were identified in each patient. RESULTS: Mutations in the RFXAP gene were found in three patients from unrelated families, and the resulting defect was classified as belonging to a novel complementation group (D). Transfection with the wild-type RFXAP gene restored the expression of MHC class II molecules in the patients' cells. CONCLUSIONS: Mutations in a novel MHC class II transactivating factor, RFXAP, can cause MHC class II deficiency. These mutations abolish the expression of MHC class II genes and lead to the same clinical picture of immunodeficiency as in patients with mutations in the other two MHC class II regulatory genes.     

Sensory testing of the hands in leprosy

A case of mucopolysaccharidosis type VII (MPS VII, beta glucuronidase deficiency) causing fatal hydrops fetalis in the third trimester is presented. The diagnosis was suspected on histopathological examination by the presence of foam cells in many of the viscera and foamy change in the placental Hofbauer cells. Electron microscopy showed empty cytoplasmic inclusion bodies within macrophages and in the Hofbauer cells. Enzyme assay of cultured fibroblasts showed markedly deficient beta glucuronidase activity, thus confirming the diagnosis. A detailed and thorough histopathological examination of hydrops fetalis cases is important to detect subtle features of inherited metabolic disorders. Use of a structured necropsy protocol is recommended for cases of non-immune hydrops. Electron microscopy is a useful adjunct to light microscopy in cases where an inherited metabolic disorder is suspected. Precise necropsy diagnosis is important as there are implications for genetic counselling and possible prenatal diagnosis in subsequent pregnancies.