Characterization of steroid 21-hydroxylase gene mutations in a oligosymptomatic form of congenital adrenal hyperplasia: family study

BACKGROUND: 21-hydroxilase deficiency accounts for over 90% of all cases of congenital adrenal hyperplasia (CAH). There is a non-negligible incidence of both severe and nonclassical forms of this genetic disorder. Enzyme deficiency is due to mutations in the gene encoding adrenal 21-hydroxylase (named CYP 21B) and is inherited in an autosomical recesive way. Complete or partial impairment of enzyme activity has been correlated with the different clinical forms of the disease. PATIENTS AND METHODS: In the present paper CYP 21B gene analysis results obtained in a family with two kindred affected by a nonclassical form of the disease are shown. Clinical assessment of these two kindred showed a very mild form of the disease, whereas biochemical results suggested a late-onset partial 21-hydroxylase deficiency. Genotyping for deletions and 10 point mutations in the CYP 21B gene was performed by Southern blot analysis and polymerase chain reaction (PCR) allele-specific oligonucleotide (ASO) hybridation technique. RESULTS: Molecular genetic analysis performed in the two affected patients and two further relatives allowed us to detect the presence of different mutations in the two alleles of the CYP 21B gene. One of these mutations was severe (655G) and came from maternal line, whereas the other was mild (Val281Leu) and originated in paternal line. CONCLUSION: Molecular genetic analysis allows the possibility of finding severe (and non-expected) mutations in patients with clinically mild and late-onset forms of the 21-hydroxylase deficiency.     

Familial hyperaldosteronism type II: description of a large kindred and exclusion of the aldosterone synthase (CYP11B2) gene

Familial hyperaldosteronism type II (FH-II) is characterized by autosomal dominant inheritance and hypersecretion of aldosterone due to adrenocortical hyperplasia or an aldosterone-producing adenoma; unlike FH type I (FH-I), hyperaldosteronism in FH-II is not suppressible by dexamethasone. Of a total of 17 FH-II families with 44 affected members, we studied a large kindred with 7 affected members that was informative for linkage analysis. Family members were screened with the aldosterone/PRA ratio test; patients with aldosterone/PRA ratio greater than 25 underwent fludrocortisone/salt suppression testing for confirmation of autonomous aldosterone secretion. Postural testing, adrenal gland imaging, and adrenal venous sampling were also performed. Individuals affected by FH-II demonstrated lack of suppression of plasma A levels after 4 days of dexamethasone treatment (0.5 mg every 6 h). All patients had negative genetic testing for the defect associated with FH-I, the CYP11B1/CYP11B2 hybrid gene. Genetic linkage was then examined between FH-II and aldosterone synthase (the CYP11B2 gene) on chromosome 8q. A polyadenylase repeat within the 5'-region of the CYP11B2 gene and 9 other markers covering an approximately 80-centimorgan area on chromosome 8q21-8qtel were genotyped and analyzed for linkage. Two-point logarithm of odds scores were negative and ranged from -12.6 for the CYP11B2 polymorphic marker to -0.98 for the D8S527 marker at a recombination distance (theta) of 0. Multipoint logarithm of odds score analysis confirmed the exclusion of the chromosome 8q21-8qtel area as a region harboring the candidate gene for FH-II in this family. We conclude that FH-II shares autosomal dominant inheritance and hyperaldosteronism with FH-I, but, as demonstrated by the large kindred investigated in this report, it is clinically and genetically distinct. Linkage analysis demonstrated that the CYP11B2 gene is not responsible for FH-II in this family; furthermore, chromosome 8q21-8qtel most likely does not harbor the genetic defect in this kindred.     

Molecular diagnosis of salt wasting congenital adrenal hyperplasia, caused by deficit of 21-hydroxylase, in the Chilean population

BACKGROUND: The most frequent cause of congenital adrenal hyperplasia, manifested as virilization and salt wasting, is the deficit of 21-hydroxylase. This disease is originated by mutations of the gene CYP21 that codifies this enzyme, mostly recombination between this gene and its inactive pseudogene called CYP21P. AIM: To study the molecular origin of this enzyme deficiency in Chilean patients. PATIENTS AND METHODS: Twenty five patients with salt wasting congenital adrenal hyperplasia, that had 17-hydroxyprogesterone levels above 30 ng/ml, were studied. In all patients, a polymerase chain reaction (PCR) with selective primers was done with extracted genomic DNA, to amplify the active gene and specific primers for normal or mutated alleles (Allele-specific PCR). RESULTS: The affected allele was identified in 39 (78%) of the 50 chromosomes of the 25 patients. The higher frequency affected the ASIn2 in 26% of cases, followed by mutations Arg357Trp in 22% of cases and Gln319Stop in 12% and deletion in 12%. The identification of two affected alleles in a same patient was achieved in 17 cases (68%). The most frequent genotypes were homozygosity for ASIn2 (16%), homozygosity for Arg357Trp (12%) and the homozygote deletion of the gene in 12%. CONCLUSION: The most frequent mechanisms of genetic damage in this population of patients with salt wasting congenital adrenal hyperplasia due to deficiency of 21-hydroxylase were the mutations ASIn2 and Arg357Trp. This type of studies allows prenatal diagnosis and genetic counseling.     

Congenital adrenal hyperplasia (21-hydroxylase deficiency) without demonstrable genetic mutations

Congenital adrenal hyperplasia (CAH) owing to 21-hydroxylase deficiency (21-OHD) is the most common inherited defect of adrenal steroid biosynthesis. At least 36 mutations in the CYP21 gene, which is mapped to chromosome 6p21.3, have been described. We performed genetic analysis of the CYP21 gene in a patient with classic 21-OHD CAH and her family. The entire exonic coding regions and intronic regions, as well as the -1 kb 5' upstream promoter region, were thoroughly sequenced and analyzed. Despite extensive sequencing, no mutation was found in this 3.7 kb area. The 11beta-hydroxylase defect, closely mimicking the clinical and biochemical phenotype of classic 21-OHD, was excluded by directly sequencing 2.6 kb covering the entire coding of the CYP11B1 gene. Herein we describe a phenotypically and hormonally affected patient with classic simple virilizing 21-OHD CAH who lacks a mutation in the entire CYP21 gene and coding region of the CYP11B1 gene.     

High variability of CYP21 gene rearrangements in Spanish patients with classic form of congenital adrenal hyperplasia

Mutations that cause deficiency of the 21-hydroxylase (21-OH) appear as a result of recombinations between the CYP21B coding gene and the highly homologous CYP21A pseudogene, which are tandemly arranged with the C4A and C4B genes. We report a detailed analysis of a major chromosomic rearrangement by Southern blot using 21-OH and complement C4 cDNA probes, in a wide sample of classic Spanish congenital adrenal hyperplasia (CAH) patients. This study made it possible to observe that 50% of the patients carried at least one allele with gross abnormalities and that the frequencies of alleles with large deletions (16.66%) and gene conversions (14.16%) in the CYP21B gene were very similar. Moreover, our analysis revealed the existence of sixteen different restriction patterns of C4/CYP21 genes. Besides the detection of a new haplotype, which does not seem to appear from unequal crossing-over mechanisms, we observed the highest frequency on CYP21A duplications reported, as well as no duplications of the CYP21B gene. We also observed that although gross abnormalities of the CYP21A pseudogene did not affect 21-OH activity, alleles carrying deleterious point mutations had more rearrangements of the CYP21A gene than normal alleles. Even though the 21-OH deficiency is an autosomic trait, boys in our sample carried 2.6 times more deletions than girls. In contrast, conversion alleles were found equally frequently.     

Mucoepidermoid carcinoma arising in Warthin''s tumour of the parotid gland: report of two cases with histopathological, ultrastructural and immunohistochemical studies

OBJECTIVE: To study the molecular defects of congenital adrenal hyperplasia (CAH). STUDY DESIGN: Twenty Chinese patients, including 8 with salt-wasting (SW) type CAH, 11 with simple virilizing (SV) type CAH and 1 with nonclassical (NC) type CAH, were recruited. Two rounds of the polymerase chain reaction (PCR) were used to study the 21-hydroxylase gene (CYP21). The primary PCR amplified CYP21-specific DNA fragments, and the secondary PCR used products from the primary PCR for analysis of amplification-created restriction sites (ACRS) and direct DNA sequencing. In all patients, ACRS analysis was done at 12 possible mutation sites, and then direct DNA sequencing was performed to confirm or define the molecular defects. RESULTS: Ten different mutations, including nine point mutations and gross gene deletion or conversion, were found in this study. Of the nine point mutations, eight could be easily detected by ACRS analysis. The three most common mutations were codon (CD)172 t-->a (I172N), IVS-II 656 c/a-->g, and gross gene deletion or conversion, accounting for 27.5% (11/40 alleles), 25% (10/40) and 20% (8/40) of all identified mutations, respectively. All SW patients were compound heterozygotes of IVS-II 656, gross gene deletion or conversion, or other severe defects, including CDs236 (t-->a) (I236N)+ 237 (t-->a) (V237E)+ 239 (t-->a) (M239K), CD306 (+t), CD318 (c-->t) (Q318X) and CD356 (c-->t) (R356W) mutations. All SV patients had one allele with a CD172 (I172N) mutation. One allele of an NC patient had a CD183 (c-->g) (D183E) mutation, and the other allele was not defined. In the whole series, four alleles (10%) had more than one mutation. CONCLUSION: We found 10 different mutations in this study. The correlation between genotypes and phenotypes was compatible with the reported data. Two rounds of PCR and ACRS analysis may provide important information for genetic counseling, prenatal diagnosis and management of families at risk for CAH.     

Absence of steroid biosynthetic defects in heterozygote individuals for classic 11 beta-hydroxylase deficiency due to a R448H mutation in the CYP11B1 gene

Steroid 11 beta-hydroxylase deficiency (failure to convert 11-deoxy-cortisol to cortisol) is responsible for less than 5% of cases of classic congenital adrenal hyperplasia, but it is relatively frequent in Israel, among Jews of Moroccan origin. Affected individuals have a single base substitution in exon 8 of CYP11B1 gene, codon 448, from CGC (arginine) to CAC (histidine) (R448H), a mutation that abolishes enzyme activity completely. We studied the hormonal response to ACTH stimulation in individuals genotyped to have the R448H mutation in one allele only (heterozygotes), and who were therefore assumed to have 50% of 11 beta-hydroxylase activity. No demonstrable hormonal abnormalities were found in the 6 adults (3 mothers and 3 fathers) and 2 sons studied, suggesting that a quantitatively reduced 11 beta-hydroxylase is still enough for normal adrenal biosynthesis.     

Circulating endotoxin during initial antibiotic treatment of severe gram-negative bacteremic infections

The second most common cause of congenital adrenal hyperplasia is 11 beta-hydroxylase deficiency, an autosomal recessive disorder. We performed genetic analysis of CYP11B1, the gene encoding steroid 11 beta-hydroxylase, in three patients with classic 11 beta-hydroxylase deficiency. Herein we describe the first splice donor site mutation, a new nonsense mutation, and a new missense mutation in this disorder. An African-American patient was found to be a compound heterozygote for a codon 318 + 1G --> A substitution at the 5'-splice donor site of intron 5, in combination with Q356X, a nonsense mutation previously reported in an African-American patient. A Caucasian patient was found to be a compound heterozygote with a novel missense mutation, T318R, in combination with a previously reported 28-bp deletion in exon 2. A different mutation at codon 318 (T318M) has been described previously. A Caucasian patient was heterozygous for a novel nonsense mutation (Q19X) in exon 2. The second mutation was not identified in this patient. Multiple apparent polymorphisms were also observed. Two of these polymorphisms in CYP11B1 represent sequences from CYP11B2, suggesting that gene conversion may have occurred. In summary, we have identified three novel mutations and two previously reported mutations in CYP11B1 patients with 11 beta-hydroxylase deficiency. Our data suggest the presence of a mutational hot spot at codon 318 of CYP11B1, and the possibility of a founder effect in frequently identified mutations.     

CGH-detected DNA sequence copy number amplifications can be confirmed by interphase-FISH: new possiblities for prognostic approaches in oral squamous cell carcinomas

OBJECTIVE: Following the observation of two patients affected by true precocious puberty who went on to develop polycystic ovary syndrome (PCOS) and who were found to be heterozygotes (carriers) for 21-hydroxylase deficiency (21-OHD), we decided to evaluate the frequency of heterozygosity for adrenal 21-OHD in patients with true precocious puberty. STUDY DESIGN: We investigated 32 girls affected by true precocious puberty, by the single-dose ACTH stimulation test, HLA typing and the molecular analysis of the CYP21B gene encoding for the 21-OH enzyme, in order to detect gene deletions or point mutations. Twenty-eight cases were on LHRH analogue treatment and the remaining four, untreated owing to parental refusal, were investigated 0.5-1.5 years after spontaneous menarche. RESULTS: After ACTH testing, 13 out of the 32 (41%) cases displayed higher 17-hydroxyprogesterone (17-OHP) levels than normal but less than those found in patients affected by nonclassical adrenal hyperplasia (CAH); these levels were similar to those observed in obligate heterozygotes for CAH due to 21-hydroxylase deficiency (21-OHD). HLA typing showed a significantly increased frequency of the HLA alleles A28 and B14 which are peculiar to the HLA haplotypes of nonclassical CAH due to 21-OHD. Molecular analysis of the CYP21B gene showed that in four out of the 10 tested patients with an exaggerated 17-OHP response there were heterozygous point mutations of the CYP21B gene. In contrast, no CYP21B gene abnormalities were detected in the eight tested patients with normal 17-OHP. No differences were found between carriers and non-carriers of the 21-OHD with regard to age at onset of precocious puberty, clinical features, bone age acceleration and gonadal suppression induced by LH-RH analogue treatment. Two out of the four untreated patients who were investigated after menarche were found to be carriers of the 21-OHD; these girls showed signs of androgen excess, irregular menses and polycystic ovaries. CONCLUSIONS: A high frequency of heterozygosity for adrenal steroid 21-OHD was found in our patients with true precocious puberty. This adrenal defect does not seem to influence the pattern of central precocious puberty, but these patients require long-term follow-up as they might go on to develop polycystic ovary syndrome (PCOS). Whether or not heterozygosity of the 21-OHD may be related to the premature activation of the hypothalamo-pituitary-gonodal axis remains to be defined.     

Salt-wasting congenital adrenal hyperplasia: detection of mutations in CYP21B gene in a Chilean population

The steroid 21-hydroxylase deficiency (21OHD) is the most frequent cause of congenital adrenal hyperplasia. We have characterized the disease-causing mutations in the 21-hydroxylase genes of 63 patients with salt-wasting congenital adrenal hyperplasia from a Chilean population of Hispanic origin, a group that has been scarcely evaluated. Using allele-specific PCR, lesions were identified in 97 chromosomes out of 126 tested (77%). The most frequent findings were the gene deletion or large gene conversion (LGC) = 22.9%, I2 splice = 19%, R357W = 12.7%, and Q319X = 10.5%. We did not find alleles with the mutation F308insT and we found three alleles with the cluster E6. The frequency of the point mutation R357W was at least two times more frequent than the one found in Caucasians populations, but similar to that communicated in Asian populations; this finding may be explained by the Asian ancestry of our South-Amerindian population. The frequency of Q319X was also high, similar only to those patients studied in Italy and in a neighboring Argentinian population. In summary, this is a genetic characterization of 21OHD made in an almost pure Hispanic population in Latin America. The high frequency of deletion of CYP21B gene, I2 splice, R357W, and Q319X mutations probably reflects the European-Caucasian-Spanish influence of the conquerors, mixed with Amerindians of Asian ancestry and modulated by other European immigrations.     

Allele-dropout using PCR-based diagnosis for the splicing mutation in intron-2 of the CYP21B-gene: successful amplification with a Taq/Pwo-polymerase mixture

The splicing mutation in intron 2 (nucleotide 656) of the 21-hydroxylase gene (CYP21B) is the most common mutation causing congenital adrenal hyperplasia (CAH). Homozygosity for nt656G is associated with the classical phenotype of CAH. In several studies, a number of clinically asymptomatic relatives of CAH-patients were genotyped as nt656G homozygotes. We have proposed that the putative asymptomatic nt656G/G individuals are incorrectly typed due to dropout of one allele (in most cases nt656C) during PCR amplification. Here, we report the successful amplification of all alleles at nt656 with a Taq/Pwo DNA polymerase mixture in the primary PCR reaction. The results were independent from the type of polymerase used for sequencing reactions as the second step in mutation analysis.     

Uniparental disomy for chromosome 6 results in steroid 21-hydroxylase deficiency: evidence of different genetic mechanisms involved in the production of the disease

Congenital adrenal hyperplasia (CAH) is an inherited recessive disorder of adrenal steroidogenesis caused by mutations in the steroid 21-hydroxylase gene (CYP21) in more than 90% of affected patients. The CYP21 gene is located within the HLA complex locus on chromosome 6 (6p21.3). During a molecular characterisation study of a group of 47 Mexican families with 21-hydroxylase deficiency, we identified nine in which the mutation or mutations found in the patient did not appear to originate from one of the parents. Through DNA fingerprinting, paternity was established in all nine families with a probability of non-paternity in the range of 10(-19) to 10(-23). Among these families, we identified one patient with exclusive paternal inheritance of all eight markers tested on chromosome 6p, despite normal maternal and paternal contributions for eight additional markers on three different chromosomes. We did not identify duplication of paternal information for markers in the 6q region, consistent with lack of expression of transient neonatal diabetes owing to genomic imprinting in this patient. Our results substantiate evidence for the existence of different genetic mechanisms involved in the expression of this recessive condition in a substantial portion (approximately 19%) of affected Mexican families. In addition to the identification of a patient with paternal uniparental disomy, the occurrence of germline mutations may explain the unusual pattern of segregation in the majority of the remaining eight families.     

A novel helix termination mutation in keratin 10 in annular epidermolytic ichthyosis, a variant of bullous congenital ichthyosiform erythroderma

Annular epidermolytic ichthyosis is a distinct phenotypic variant of bullous congenital ichthyosiform erythroderma that has recently been described in two separate kindreds. Individuals with this variant present with bullous ichthyosis in early childhood and hyperkeratotic lichenified plaques in the flexural areas and extensor surfaces at later ages. Characteristically, they also develop intermittent bouts of annular and polycyclic, erythematous, scaly plaques on the trunk and proximal extremities. We now describe a third kindred with annular epidermolytic ichthyosis. Molecular analysis of this family revealed a novel mutation resulting in an isoleucine to threonine substitution at residue 107 (codon 446) within the highly conserved helix termination motif at the end of the rod domain of keratin 10.     

Comprehensive analytical strategy for mutation screening in 21-hydroxylase deficiency

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease with a wide range of clinical manifestations. It is most often caused by deficiency of steroid 21-hydroxylase, reflecting any of a wide range of mutations in the 21-hydroxylase (CYP21) gene. A major challenge in molecular diagnostics of CAH is the high homology between the CYP21 gene and the CYP21P pseudogene and the phenomenon of apparent gene conversion, which inactivates the functional gene. In this study we devised an improved stepwise diagnostic procedure involving nonradioactive Southern blotting and direct DNA sequencing. This strategy led to a successful elucidation of the molecular cause of the disease in 181 out of 182 unrelated alleles in a total of 91 clinically and biochemically characterized patients. We were able to identify all classical known disease-causing mutations of the 21-hydroxylase gene and a novel nonsense mutation (bp 670, A-->C, Y97X). Our method also allows the reliable, secure diagnosis of the heterozygous configuration and may therefore be used for pre-, peri-, and postnatal diagnosis of CAH, even when informative data of the index patient are lacking. Furthermore, it can be used to confirm the diagnosis of CAH in newborns detected in 17-hydroxyprogesterone screening programs.     

Germline mutations in the 3' part of APC exon 15 do not result in truncated proteins and are associated with attenuated adenomatous polyposis coli

Familial adenomatous polyposis (FAP) is an inherited predisposition to colorectal cancer characterized by the development of numerous adenomatous polyps predominantly in the colorectal region. Germline mutations in the adenomatous polyposis coli (APC) gene are responsible for most cases of FAP. Mutations at the 5' end of APC are known to be associated with a relatively mild form of the disease, called attenuated adenomatous polyposis coli (AAPC). We identified a frameshift mutation in the 3' part of exon 15, resulting in a stop codon at 1862, in a large Dutch kindred with AAPC. Western blot analysis of lymphoblastoid cell lines derived from affected family members from this kindred, as well as from a previously reported Swiss family carrying a frameshift mutation at codon 1987 and displaying a similar attenuated phenotype, showed only the wild-type APC protein. Our study indicates that chain-terminating mutations located in the 3' part of APC do not result in detectable truncated polypeptides and we hypothesize that this is likely to be the basis for the observed AAPC phenotype.     

CYP11B1 mutations causing non-classic adrenal hyperplasia due to 11 beta-hydroxylase deficiency

Steroid 11 beta-hydroxylase deficiency is the second most common cause of congenital adrenal hyperplasia, the inherited inability to synthesize cortisol. Severely affected patients carry mutations in the CYB11B1 gene that destroy enzymatic activity. Such patients have signs of androgen excess and usually have hypertension. Mild or non-classic 11 beta-hydroxylase deficiency has been reported previously but not studied genetically. In this study we report analysis of the CYP11B1 genes of three patients thought to suffer from non-classic 11 beta-hydroxylase deficiency. Mutations were detected in the CYP11B1 genes of two patients. One was a compound heterozygote for missense mutations N133H and T319M, whereas the other carried a nonsense mutation (Y423X) on one allele and a missense mutation (P42S) on the other. All three missense mutations affected enzymatic activity when expressed in vitro. No mutations were detected in the coding regions or intron-exon boundaries of the CYP11B1 genes of the other putative non-classic patient. In addition, we were unable to detect CYP11B1 mutations in two hirsute women with mildly elevated levels of 11 beta-hydroxylase precursors who had previously been identified in a screening study of patients in a reproductive endocrinology clinic. We conclude that nonclassic 11 beta-hydroxylase deficiency is a rare disorder. It is not a significant cause of hyperandrogenism in women and relatively stringent criteria should be used to prevent its misdiagnosis.     

Neuroscience nursing research: challenges for the next decade

A case of bilateral adrenal myelolipoma in association with congenital adrenal hyperplasia caused by 21-hydroxylase deficiency is presented. The clinical, radiologic, and endocrinologic features of this case are correlated with a review of the literature.     

The ACTH test in the diagnosis of hirsutism

The ACTH test has been used to confirm the diagnosis of adrenal insufficiency and the classic and the non-classic adrenal hyperplasia due to the 3-HSD, 21 OH e 110H deficiencies. This article reviews the historical aspects of the use of ACTH in the diagnosis of hirsutism and points out its mains indications. In spite of new biological molecular advances in the diagnosis of adrenal enzymatic deficiencies, the use of the ACTH test can help the physician to predict both genothipus and fenothipus in populations with hyperandrogenic manifestations due to non-classical or late-onset congenital adrenal hyperplasia.     

Novel beta-thalassemia mutation in patients of Jewish descent: [beta 30(B12)Arg-->Gly or IVS-I(-2)(A-->G)]

Classical congenital adrenal hyperplasia (CAH) results from an inherited enzymatic defect in cortisol synthesis and more than 90 per cent of cases are due to 21-hydroxylase deficiency. The androgen excess associated with this condition typically results in ambiguous external genitalia in affected females. It has been shown that prenatal treatment with dexamethasone is successful in preventing or reducing genital ambiguity in affected females. Rather than treating with dexamethasone, some couples choose to terminate the pregnancy when an affected fetus is prenatally diagnosed. We report a female with classical CAH who was born with normal external genitalia, although maternal treatment with dexamethasone did not begin until 16 weeks' gestation.     

A nonsense mutation in the erythrocyte band 3 gene associated with decreased mRNA accumulation in a kindred with dominant hereditary spherocytosis

We studied a French kindred with typical hereditary spherocytosis (HS). Studies of erythrocytes and erythrocyte membranes from HS individuals revealed abnormal erythrocyte membrane mechanical stability as well as 15-20% deficiency of band 3, the anion transporter. Anion transport studies of red cells from two affected individuals revealed decreased sulfate flux. Nucleotide sequence of cDNA encoding the distal third of the cytoplasmic domain and the entire transmembrane domain of band 3 obtained by RT-PCR of reticulocyte RNA of an affected family member was normal. Sequence analysis of genomic DNA from an HS individual identified a nonsense mutation of the band 3 gene, Q330X, near the end of the band 3 cytoplasmic domain. This mutation was present in genomic DNA of all HS family members and absent in DNA of unaffected family members. Using an RT-PCR-based assay, a marked quantitative decrease in accumulation of the mutant band 3 RNA was detected. Thus the codon 330 nonsense mutation is responsible for the decreased accumulation of mutant band 3 RNA and the deficiency of band 3 protein in this kindred. These results have important implications for the role of band 3 defects in the membrane pathobiology of HS as well as for the techniques used in detection of HS mutations.