Qualitative assessment of the emotional and behavioural responses of haemophilia A carriers to negative experiences in their medical care.

Previous discussions with haemophilia A (HA) carriers suggested that carriers may experience inappropriate care, resulting in poor relationships with healthcare providers (HCPs; principally physicians and nurses), and unfortunate and extreme emotional and behavioural responses. This was a qualitative study to explore medical experiences of HA carriers and their emotional and behavioural responses. Eleven HA carriers and five Haemophilia Treatment Centre nurses were interviewed. Themes were identified using QSR NVivo 8.0. Carriers and nurses reported HA-related bleeding symptoms in carriers, including life-threatening haemorrhage following injury or medical intervention. Menorrhagia was common and distressing. Negative carrier experiences were related in the determination of genotypic and phenotypic status, management, precautions and HCP attitude, including dismissing carriers' symptoms, concerns or requests for care. Carriers responded with mistrust, lost confidence, disappointment, fear, anxiety, doubt of self or child, discussing experiences, avoidance of healthcare and self-treatment. Dismissive HCP attitudes, ignorance about bleeding disorders in women and unique aspects of the carrier population appear to make errors more likely. This study indicates that carriers experience inappropriate care and encounter dismissive attitudes, and respond emotionally and behaviourally. Our model suggests that systematic medical errors aggravate a negative feedback loop leading to negative emotional and behavioural responses and worsening carrier care. Improved carrier care policies and increased awareness of women's bleeding disorders may improve this situation. Further research is needed to determine whether the themes identified in this study accurately reflect the experiences of carriers in general.

Familial essential thrombocythemia with spontaneous megakaryocyte colony formation and acquired JAK2 mutations.

Essential thrombocythemia (ET) is a chronic myeloproliferative disorder, characterized by increased proliferation of megakaryocytes and elevated platelet count that usually occurs sporadically. We report a family with seven affected individuals in three generations, including one individual with a phenotype resembling polycythemia vera, a related disorder. Megakaryocyte (CFU-MK) colony formation occurred in the absence of added cytokines in cultures of peripheral blood from affected family members. Some reports of familial ET have identified mutations in THPO and MPL, the genes for a cytokine (thrombopoietin, TPO) that regulates platelet production and its receptor (c-MPL), respectively. In this family, the MPL gene was excluded by linkage analysis. Although TPO levels were elevated in most affected family members and evidence for linkage was found between the disease and THPO (theta=0.0, Z(max)=3.0), a THPO mutation was not identified by DNA sequencing. The JAK2 V617F mutation that has been associated with 50% of sporadic cases of ET was identified as a somatic mutation, an acquired defect, in peripheral blood of the two most severely affected family members. These patients also had elevated TPO levels. Further study of familial myeloproliferative diseases will help elucidate the initiating genetic events underlying ET.

Concurrent acute lymphoblastic leukemia and juvenile pilocytic astrocytoma in a pediatric patient.

The concurrence of acute lymphoblastic leukemia (ALL) and an asymptomatic juvenile pilocytic astrocytoma is described. A 6-year-old boy without clinical evidence of neurofibromatosis had a juvenile pilocytic astrocytoma diagnosed on radiologic examination and before treatment of acute pre-B cell lymphoblastic leukemia. The patient has had a partial resection of the astrocytoma and is 9 months into treatment of his ALL, which is in complete remission. p53 gene mutation was not identified in this patient. The concurrent diagnosis before treatment of ALL and juvenile pilocytic astrocytoma, the latter normally an indolent tumor, suggests that some cases of astrocytoma previously ascribed to radiotherapy or other treatment may in fact be caused by other factors.

Familial myelodysplastic syndrome with early age of onset.

A family is described in which three members, the propositus, his brother, and son, developed a myelodysplastic syndrome (MDS) at the ages of 52, 35, and 25, respectively. A fourth member, the paternal uncle of the propositus, was diagnosed with chronic lymphocytic leukemia. Two of the three affected Individuals had megaloblastoid marrows with recognizable bone marrow cytogenetic abnormalities and progressive, nonleukemic bone marrow failure. The propositus was unresponsive to G-CSF and eventually died of sepsis. The second affected family member died of bone marrow transplant complications. The third affected family member underwent bone marrow transplantation and is showing signs of graft survival despite minor complications. The affected members of this pedigree appear to represent a continuum in severity of disease and, therefore, pathogenesis. The pattern of inheritance and clinical progression of the disease suggest a genetic defect which may predispose individuals to the development of MDS.

Mutations in NPC1 highlight a conserved NPC1-specific cysteine-rich domain.

Niemann-Pick type II disease is an autosomal recessive disorder characterized by a defect in intracellular trafficking of sterols. We have determined the intron/exon boundaries of eight exons from the conserved 3' portion of NPC1, the gene associated with most cases of the disease. SSCP analyses were designed for these exons and were used to identify the majority of mutations in 13 apparently unrelated families. Thirteen mutations were found, accounting for 19 of the 26 alleles. These mutations included eight different missense mutations (including one reported by Greer et al. [1998]), one 4-bp and two 2-bp deletions that generate premature stop codons, and two intronic mutations that are predicted to alter splicing. Two of the missense mutations were present in predicted transmembrane (TM) domains. Clustering of these and other reported NPC1 mutations in the carboxy-terminal third of the protein indicates that screening of these exons, by means of the SSCP analyses reported here, will detect most mutations. The carboxy-terminal half of the Npc1 protein shares amino acid similarity with the TM domains of the morphogen receptor Patched, with the largest stretch of unrelated sequence lying between two putative TM spans. Alignment of this portion of the human Npc1 protein sequence with Npc1-related sequences from mouse, yeast, nematode, and a plant, Arabidopsis, revealed conserved cysteine residues that may coordinate the structure of this domain. That 7 of a total of 13 NPC1 missense mutations are concentrated in this single Npc1-specific domain suggests that integrity of this region is particularly critical for normal functioning of the protein.

Linkage disequilibrium mapping of the Nova Scotia variant of Niemann-Pick disease.

Niemann-Pick type D (NPD) disease is a severe degenerative disorder of the nervous system characterized by the accumulation of tissue cholesterol and sphingomyelin. Because of a founder effect, it is unusually common in southwestern Nova Scotia, Canada. We have confirmed that almost all patients from 20 affected sibships descended on both sides from a small group of Acadians who settled in this region in about the year 1767. Previously using classic linkage analysis of this large kindred, we defined the critical gene region to a 13-cM chromosome segment between D18S869 and D18S66. Seven ESTs have been positioned within this interval. Carstea et al. (Niemann Pick C disease gene: homology to mediators of cholesterol homeostasis. Science 1997: 277: 232-235) recently demonstrated that one of these ESTs is the Niemann-Pick type C (NPCI) gene, the gene disrupted in most patients with NPC disease, and we have shown that a G3097-->T mutation in the NPC1 gene is also responsible for NPD. Here we report the development of five new polymorphic microsatellite markers and the testing for complete linkage disequilibrium in our single large NPD kindred that allowed us to reduce the NPD critical region to a 1-cM (1.3-1.6 Mb) interval between D18S1398 and D18S1108. In contrast, Carstea et al., using classic linkage analysis, required more than 18 unrelated NPC families to reduce the NPC1 critical region to a 5-cM interval. Our work supports the finding that NPD is an allelic variant of NPC1, and illustrates the power of large kindreds, which are common in Atlantic Canada and other relatively isolated areas, for gene mapping and identification.

FISH mapping and inter-Alu fingerprinting define the YAC contig map around the centromeric region of human chromosome 18.

Previous reports concerning the location of D18S44 with respect to the centromere have been ambiguous. Also, it has not been possible, based on formerly reported markers, to show that contigs WC18.0 and WC18.1 overlap. However, the data presented here definitively show, using FISH technology, that D18S44 (located on WC18.0) maps to proximal 18q. Furthermore, inter-Alu fingerprinting shows a clear overlap between WC18.0 and WC18.1, thereby establishing a complete contig between D18S44 and markers from WC18.1.

Genetic analysis of familial myelodysplastic syndrome: absence of linkage to chromosomes 5q31 and 7q22.

Myelodysplastic syndrome (MDS) is a hematological disorder that occurs primarily in the elderly as an acquired, sporadic disease. Familial cases of MDS are rare. We have identified a kindred with three affected individuals, with early age of onset, suggesting a possible inherited predisposition to this disease. Using a molecular genetic approach, we examined whether bands 5q31 or 7q22 or both, the chromosomal regions most frequently associated with sporadic MDS, are involved in familial expression of MDS in this pedigree. Linkage analysis using polymorphic microsatellite DNA markers demonstrated that neither 5q31 nor 7q22 cosegregated with MDS in this family. There was no history of common environmental or occupational exposure among family members with MDS. In addition, analysis of polymorphisms at two loci [glutathione S-transferase T1 and M1 (GSTT1 and GSTM1)] involved in carcinogen detoxification and associated with cancer susceptibility, including increased risk for MDS, showed no evidence for enhanced sensitivity to environmental carcinogens in affected family members. Taken together, our findings suggest that (1) there is an inherited predisposition to MDS in this kindred; and (2) genes at 5q31 and 7q22, the regions most commonly associated with sporadic MDS, are excluded from a causal role in this family's disease.

The Nova Scotia (type D) form of Niemann-Pick disease is caused by a G3097-->T transversion in NPC1.

Niemann-Pick type D (NPD) disease is a progressive neurodegenerative disorder characterized by the accumulation of tissue cholesterol and sphingomyelin. This disorder is relatively common in southwestern Nova Scotia, because of a founder effect. Our previous studies, using classic linkage analysis of this large extended kindred, defined the critical gene region to a 13-cM chromosome segment between D18S40 and D18S66. A recently isolated gene from this region, NPC1, is mutated in the majority of patients with Niemann-Pick type C disease. We have identified a point mutation within this gene (G3097-->T; Gly992-->Trp) that shows complete linkage disequilibrium with NPD, confirming that NPD is an allelic variant of NPC1.

Limiting the Niemann-Pick type C critical region to a 1-cM interval.

OBJECTIVE: To refine the position of and isolate the gene responsible for Niemann-Pick Type II (NP Type II) disease, an autosomal, recessive neurodegenerative disorder usually affecting children. The underlying biochemical defect results in an impairment in transport of intracellular cholesterol. This disease has been classified into two subtypes, NPC and NPD. NPD and the major complementation group of NPC both map to chromosome 18q11-12; therefore, they are likely allelic variants. The NP Type II gene was previously localized between microsatellite markers D18S44 and D18S1108. DESIGN: Linkage analysis. SETTING: Pathology department of a university-associated hospital. PATIENTS: An NPC family, including proband, parents and sister. OUTCOME MEASURES: NP Type II disease phenotype and biochemical phenotype (cholesterol esterification). RESULTS: DNA from the individuals in the NPC family was genotyped at 12 microsatellite loci from the critical region. The deduced haplotypes identify a meiotic recombinant that has allowed the distal limit of the critical region to be moved from D18S1108 to D18S1101. CONCLUSION: The NP Type II gene lies proximal to the microsatellite marker D18S1101, within the 1-cM interval between D18S1101 and D18S1398. This represents approximately 1.1 mb on the physical map.

Increased expression of caveolin-1 in heterozygous Niemann-Pick type II human fibroblasts.

Human Niemann-Pick type II fibroblasts, which encompass the panethnic type C (NPC) and Nova Scotia Acadian type D (NPD) variants, exhibit altered expression of caveolin-1 protein when examined by immunoblotting using an anti-caveolin-1 monoclonal antibody. Unexpectedly, caveolin-1 in heterozygous fibroblasts was significantly elevated as much as 10-fold compared to caveolin-1 in normal and homozygous affected fibroblasts. Homozygous NPC fibroblasts expressed caveolin-1 levels similar to those in normal fibroblasts, while the expression of caveolin-1 in homozygous NPD fibroblasts was slightly elevated. Northern analysis indicates that normal fibroblasts and NPC heterozygous fibroblasts have similar amounts of caveolin-1 mRNA, while NPC homozygous fibroblasts have significantly less caveolin-1 mRNA. In contrast, heterozygous and homozygous NPD fibroblasts exhibit increased levels of caveolin-1 mRNA. These novel findings suggest that caveolin-1 containing subcellular structures are involved in the pathophysiology of Niemann-Pick type II disease. Furthermore, altered caveolin-1 protein expression may serve as a useful marker for the diagnosis of carriers of NPC or NPD.

Linkage of Niemann-Pick disease type D to the same region of human chromosome 18 as Niemann-Pick disease type C.

Niemann-Pick type II disease is a severe disorder characterized by accumulation of tissue cholesterol and sphingomyelin and by progressive degeneration of the nervous system. This disease has two clinically similar subtypes, type C (NPC) and type D (NPD). NPC is clinically variable and has been identified in many ethnic groups. NPD, on the other hand, has been reported only in descendants of an Acadian couple who lived in Nova Scotia in the early 18th century and has a more homogeneous expression resembling that of less severely affected NPC patients. Despite biochemical differences, it has not been established whether NPC and NPD are allelic variants of the same disease. We report here that NPD is tightly linked (recombination fraction .00; maximum LOD score 4.50) to a microsatellite marker, D18S480, from the centromeric region of chromosome 18q. Carstea et al. have reported that the NPC gene maps to this same site; therefore we suggest that NPC and NPD likely result from mutations in the same gene.

Identification of WASP mutations, mutation hotspots and genotype-phenotype disparities in 24 patients with the Wiskott-Aldrich syndrome.

The Wiskott-Aldrich syndrome (WAS), an X-linked immunodeficiency disease caused by mutation in the recently isolated gene encoding WAS protein (WASP), is known to be associated with extensive clinical heterogeneity. Cumulative mutation data have revealed that WASP genotypes are also highly variable among WAS patients, but the relationship of phenotype with genotype in this disease remains unclear. To address this issue we characterized WASP mutations in 24 unrelated WAS patients, including 18 boys with severe classical WAS and 6 boys expressing mild forms of the disease, and then examined the degree of correlation of these as well as all previously published WASP mutations with disease severity. By analysis of these compiled mutation data, we demonstrated clustering of WASP mutations within the four most N-terminal exons of the gene and also identified several sites within this region as hotspots for WASP mutation. These characteristics were observed, however, in both severe and mild cases of the disease. Similarly, while the cumulative data revealed a predominance of missense mutations among the WASP gene lesions observed in boys with isolated thrombocytopenia, missense mutations were not exclusively associated with milder WAS phenotypes, but also comprised a substantial portion (38%) of the WASP gene defects found in patients with severe disease. These findings, as well as the detection of identical WASP mutations in patients with disparate phenotypes, reveal a lack of phenotype concordance with genotype in WAS and thus imply that phenotypic outcome in this disease cannot be reliably predicted solely on the basis of WASP genotypes.

A diagnostic assay for the Wiskott-Aldrich syndrome and its variant forms.

BACKGROUND: The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disease characterized by severe thrombocytopenia, eczema, and impaired immunity. While the diagnosis is usually straightforward, the syndrome may be expressed in an attenuated form, a phenotype which is difficult to distinguish from other types of congenital thrombocytopenia. Although a molecular-based assay for diagnosis of the spectrum of WAS patients has not been available, recent data indicate that WAS is associated with a specific profile of impaired mitogen responsiveness and suggest that detection of this abnormality may provide a diagnostic marker for all forms of the disease. To address this issue, we have studied patients with classical and atypical WAS for their lymphocyte proliferative responses to four T cell mitogenic stimuli and compared their response patterns to those detected in unaffected children. METHODS: Clinical histories and informed consent were obtained from 23 patients with either classical or putative (ie, atypical) WAS, 16 subjects with other disorders, and 12 healthy children. Peripheral blood mononuclear cells (PBMCs) collected from patients and controls were resuspended in culture medium, stimulated with the T cell mitogens phytohemagglutinin (PHA), concanavalin A (Con A), neuraminidase/galactose oxidase (NAGO), or periodate, and cultured for 60 h in 0.2 mL aliquots. Following a 20 h pulse with 3H-thymidine, cultures were harvested and the 3H-thymidine uptake was evaluated by liquid scintillation counting. RESULTS: The most striking observation involved response to periodate. While lymphocytes from all healthy control children proliferated in response to periodate treatment, cells from both classical as well as atypical WAS patients consistently failed to proliferate in response to this mitogen. By contrast, lymphocyte proliferative responses to PHA, Con A, and NAGO were detected in all patients and controls, although responses generally were lower in cells from classical WAS patients compared to other children. In two WAS patients, bone marrow transplantation and clinical improvement were associated with a change from no periodate response (pre-transplant) to periodate responsiveness (post-transplant). In contrast to the WAS patients, cells from patients with other hematologic and primary immune deficiency diseases responded uniformly to all four mitogens, including periodate. CONCLUSIONS: The data presented here indicate that T cells from patients with either classical or attenuated WAS fail to undergo proliferation in response to periodate, an agent that induced extensive T cell mitogenesis of cells from all healthy controls as well as patients with diseases other than WAS. As the WAS patients' cells did proliferate in response to treatment with other T cell mitogens, it appears that periodate induced T cell proliferation is selectively impaired in WAS and that detection of this defect may be of value in the distinction of both classical and attenuated WAS from other thrombocytopenic conditions.

Linkage of Wiskott-Aldrich syndrome with three marker loci, DXS426, SYP and TFE3, which map to the Xp11.3-p11.22 region.

Linkage analysis was performed in 19 families segregating for the Wiskott-Aldrich syndrome (WAS) and in 1 family with X-linked thrombocytopenia using nine polymorphic DNA markers spanning the interval DXS7-DXS14. The results confirm close linkage of WAS to the DXS7, TIMP, OATL1, DXS255, DXS146, and DXS14 loci and reveal three additional marker loci, DXS426, SYP, and TFE3, to be closely linked to WAS. The linkage data are also consistent with the localization of X-linked thrombocytopenia to the same chromosomal region as WAS and support localization of the WAS gene between the TIMP and DXS146 loci. However, the data were insufficient for positioning these disease genes with respect to the four marker loci that map within this latter interval. Analysis of recombination events between the marker loci place the TFE3 gene distal to DXS255 and favor the marker loci order Xpter-DXS7-(DXS426, TIMP)-(OATL1, SYP, TFE3)-DXS255-DXS146-DXS14.

Selective impairment of CD43-mediated T cell activation in the Wiskott-Aldrich syndrome.

The Wiskott-Aldrich syndrome (WAS) is associated with defective glycosylation and altered membrane expression of the leukocyte sialoglycoprotein CD43. To investigate whether such modifications of CD43 are relevant to T cell dysfunction in WAS, we have analyzed peripheral blood mononuclear cells from WAS patients for proliferative responses to both CD43-interacting and other T cell mitogenic stimuli. While patient lymphocytes proliferated in response to phytohaemagglutinin, concanavalin A, interleukin-2 and neuraminidase/galactose oxidase, no responses were elicited upon attempted triggering of the CD43 signalling pathway using periodate or anti-CD43 antibody. Cells from four of five patients with clinical profiles resembling, but not identical, to that of classic WAS also failed to respond to periodate or anti-CD43 antibody stimulation. These results indicate that the aberrant expression of CD43 on WAS lymphocytes is associated with selective impairment of CD43-induced T cell proliferation and that detection of this defect may be useful in the diagnosis of WAS and its variant forms.

The Wiskott-Aldrich syndrome: refinement of the localization on Xp and identification of another closely linked marker locus, OATL1.

The Wiskott-Aldrich syndrome (WAS) has previously been mapped to the proximal short arm of the X chromosome between the DXS14 and DXS7 loci. In this study, further segregation analysis has been performed using a newly identified WAS family as well as an additional marker probe, HOATL1. The results indicate close linkage between the WAS and OATL1 loci (Z = 6.08 at theta = 0.00) and localize the TIMP, OATL1, DXS255, and WAS loci distal to DXS146 and the OATL1 and WAS loci proximal to TIMP. These linkage data narrow the boundaries within which the WAS locus maps to the chromosomal region bracketed by TIMP and DXS146 and support the loci order Xpter-DXS7-TIMP-(OATL1, WAS, DXS255)-DXS146.

Hunter disease (mucopolysaccharidosis type II) associated with unbalanced inactivation of the X chromosomes in a karyotypically normal girl.

The mechanism of profound generalized iduronate sulfatase (IDS) deficiency in a developmentally delayed female with clinical Hunter syndrome was studied. Methylation-sensitive RFLP analysis of DNA from peripheral blood lymphocytes from the patient, using MspI/HpaII digestion and probing with M27 beta, showed that the paternal allele was resistant to HpaII digestion (i.e., was methylated) while the maternal allele was digested (i.e., was hypomethylated), indicating marked imbalance of X-chromosome inactivation in peripheral blood lymphocytes of the patient. Similar studies on DNA from maternal lymphocytes showed random X-chromosome inactivation. Among a total of 40 independent maternal fibroblast clones isolated by dilution plating and analyzed for IDS activity, no IDS- clone was found. Somatic cell hybrid clones containing at least one active human X chromosome were produced by fusion of patient fibroblasts with Hprt- hamster fibroblasts (RJK88) and grown in HAT-ouabain medium. Methylation-sensitive RFLP analysis of DNA from the hybrids showed that of the 22 clones that retained the DXS255 locus (M27 beta), all contained the paternal allele in the methylated (active) form. No clone was isolated containing only the maternal X chromosome, and in no case was the maternal allele hypermethylated. We postulate from these studies that the patient has MPS II as a result of a mutation resulting in both the disruption of the IDS locus on her paternal X chromosome and unbalanced inactivation of the nonmutant maternal X chromosome.

Linkage relationships of the Wiskott-Aldrich syndrome to 10 loci in the pericentromeric region of the human X chromosome.

Twelve families with Wiskott-Aldrich syndrome (WAS) were studied by linkage analysis using 10 polymorphic marker loci from the X-chromosome pericentromeric region. The results confirm close linkage of WAS to the DXS14, DXS7, TIMP, and DXZ1 loci and are consistent with previous data suggesting that WAS maps to the proximal Xp and is flanked by the DXS14 and DXS7 loci. The strongest linkage (Z = 10.19 at theta = 0.00) was found to be between WAS and the hypervariable DXS255 locus, a marker locus already mapped between DXS7 and DXS14 and which was informative for all meioses included in this analysis. Linkage of the WAS to two pericentromeric Xq loci, DXS1 and PGK1, was also established. On the basis of these results, accurate predictive testing should now be feasible in the majority of WAS families.