Novel Mutations in Dax1 of X-Linked Adrenal Hypoplasia Congenita Over Several Generations in One Family

ObjectiveX-linked adrenal hypoplasia congenital (AHC) is a rare disorder caused by mutations in DAX1 gene. We report a case of X-linked AHC in a large family to analyze the pathogenesis of this rare disease and to add to our clinical knowledge of it.MethodsWe describe 3-year-old boy’s clinical features and laboratory test results, as well as the patient’s nuclear family members’ clinical symptoms, especially those with features of adrenal insufficiency. Genomic deoxyribonucleic acid (DNA) was extracted from the patient’s and the family members’ peripheral blood leukocytes, and the coding region and promoter region of DAX1 were directly sequenced.ResultsA 3-year-old boy who was diagnosed with X-linked AHC presented with atypical symptoms, and his laboratory test results revealed elevated serum adrenocorticotropic hormone levels (ACTH) and decreased serum cortisol levels. Three novel mutations were detected in the DAX1 coding sequence in this family: a missense mutation (c.376G>A, p.Val126Met), a synonymous mutation (c.498G>A, p.Arg166Arg), and a nonsense mutation (c.1225C>T, p.Gln409X).ConclusionsThis report describes the familial transmission of AHC over several generations and further expands the number of DAX1 mutations reported in the literature. Early diagnosis and prompt treatment of X-linked AHC are important and may provide a good prognosis. (Endocr. Pract. 2013;19:e105-e111)     

Identification of novel mutations of the DAX-1 gene in patients with X-linked adrenal hypoplasia congenita

OBJECTIVE: X-linked adrenal hypoplasia congenita (AHC) is a condition clinically featuring adrenal insufficiency and hypogonadotropic hypogonadism caused by mutations of DAX-1. This study was undertaken to characterize the molecular defects of DAX-1 in 3 unrelated Korean patients with AHC. PATIENTS AND METHODS: Patient 1 is a 6-year-old boy who presented with a salt-losing adrenal crisis in the neonatal period. Patient 2 is a 3-year-old boy who manifested aspiration pneumonia and adrenal insufficiency at the age of 1 month. Patient 3 is a 7-year-old boy who developed an adrenal crisis at the age of 3 days. In each of these patients, DAX-1 was analyzed by direct DNA sequencing after polymerase chain reaction amplification of the entire coding region. RESULTS: Direct sequencing of DAX-1 revealed two novel mutations, 1156_1157delCT in patient 1 and another novel nonsense mutation W105X in patient 2. Patient 3 had complete deletion of DAX-1. In patient 3, serum transaminases and creatine kinase levels were elevated while the glycerol kinase activity of leukocytes was decreased. Markedly elevated glycerol excretion was detected by urine organic acid analysis. Patient 3 was diagnosed as Xp21 contiguous gene syndrome associated with deletions of the entire IL1RAPL, GK genes and the C-terminal region of DMD gene. CONCLUSIONS: Two novel mutations of DAX-1 were detected in 2 unrelated patients with AHC, and complete deletion of DAX-1 in a patient with Xp21 contiguous gene syndrome who also presented with glycerol kinase deficiency, Duchenne muscular dystrophy, and AHC.     

A novel mutation in DAX1 gene causing different phenotypes in three siblings with adrenal hypoplasia congenita

Adrenal hypoplasia congenita (AHC) is a rare disease that can be caused by many abnormalities, including an X-linked form. Mutations in the DAX1 gene have been assigned as the genetic cause of AHC. We describe here three siblings with AHC, clinically presented at different ages, two in the neonatal period and one oligosymptomatic during infancy. Molecular analysis was able to detect a novel mutation in exon 1 of the DAX1 gene, consisting of a transition of C to T at position 359, determining a stop codon at position 359 (Q359X). The mutated gene encodes a truncated protein missing a large portion of the ligand-binding domain (C-terminal domain). The recognition of the disease in the index case suggested the diagnosis in the other siblings. Interestingly, the same mutation is presented with different phenotypes, suggesting that first-degree family members of patients with DAX1 mutations should be carefully evaluated routinely.     

Importance of genetic diagnosis of DAX-1 deficiency: example from a large, multigenerational family

BACKGROUND: Inactivating mutations of DAX-1 give rise to the X-linked form of adrenal hypoplasia congenita (AHC). Affected fetuses are at risk of early postnatal Addisonian crisis, but the variable phenotypic expression of DAX-1 insufficiency renders this diagnosis challenging. METHODS: We describe the familial transmission of AHC over several generations. The proband was diagnosed with adrenal insufficiency at age 3.5 years: molecular analysis revealed a novel, 373-bp deletion including the second exon of DAX-1. Given the familial history of several unexplained deaths in male infants related to the proband via his maternal great-grandmother, we hypothesized that all these boys had been affected with AHC. Another female member of the family being pregnant with a male fetus at the time, we performed DAX-1 analysis on the mother and the newborn. The mother was heterozygous for the deletion, and the newborn hemizygous: he presented an adrenal crisis at 10 days of life, and is now doing well on hormone replacement therapy. CONCLUSION: The unfortunate deaths of male infants at each generation of this family underlie the importance of early and precise diagnosis of this rare condition, stressing the value of genetic diagnosis in six potential female carriers of this family entering their reproductive years.     

Molecular pathogenesis of lipoid adrenal hyperplasia and adrenal hypoplasia congenita

Congenital lipoid adrenal hyperplasia (lipoid CAH) is the most severe form of CAH in which the synthesis of all gonadal and adrenal cortical steroids is markedly impaired. Lipoid CAH may be caused by the defect in either the steroidogenic acute regulatory (StAR) protein or the P450scc. More than 34 different mutations in StAR gene have been identified. Clinically, most of the patients manifest adrenal insufficiency from 1 day to 2 months of age, but some patient show delayed onset of adrenal insufficiency. Affected 46, XY subjects do not show pubertal development, whereas affected 46, XX subjects undergo spontaneous feminization, breast development and cyclical vaginal bleeding at the usual age of puberty.

X-linked adrenal hypoplasia congenital (AHC) is a rare congenital adrenal disorder characterized by severe adrenal insufficiency and hypogonadotropic hypogonadism. More than 80 different several intragenic mutations of DAX-1 have been identified. The failure of pubertal development may be caused by either abnormal hypothalamic or pituitary regulation of gonadotropin secretion. In addition, although the testicular steroidogenesis is largely intact, the functional maturity of Sertoli cells and also spermatogenesis are impaired. The type of mutation does not predict clinical phenotype. Thus, unified mechanism how DAX-1 gene defect gives rise to adrenal insufficiency, hypothalamic/pituitary hypogonadism and impaired spermatogenesis remains established.     

A novel missense mutation in exon 4 of the human coproporphyrinogen oxidase gene in two patients with hereditary coproporphyria

Hereditary coproporphyria (HCP) is an autosomal dominant disease characterized by a deficiency of coproporphyrinogen oxidase. To date, four mutations of the gene have been reported. We report here another mutation in two Japanese families with HCP, which was revealed by analysis of polymerase chain reaction (PCR)-amplified DNA fragments of the gene by a direct-sequencing method. A point mutation, G to A, was found in exon 4 of the gene at position 538 of the cDNA from the reported putative translation initiation codon ATG. This mutation results in a glycine to arginine substitution at amino acid 180. Two carriers in the family were successfully diagnosed by detecting the mutation using restriction analysis of the PCR products. Received: 23 April 1996 / Revised: 15 July 1996     

Five patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency (one with associated neuroblastoma) discovered in three generations of one family

BACKGROUND: Most patients with 21-hydroxylase deficiency (21-OHD) are compound heterozygous carriers. Their phenotype usually reflects a less severe allelic mutation, although discordance between the genotype and the phenotype has been observed. CASE REPORT: We present 5 patients with congenital adrenal hyperplasia (CAH) due to 21-OHD belonging to the 3 generations of the same family (grandmother, parents and their 2 children). As each patient carries at least one mild mutation of the CYP21 gene, their genotypes correspond to nonclassical CAH. The propositus is the older brother, who is compound heterozygous with a mild and severe CYP21 mutation (P30L/R356W). In spite of one mild CYP21 mutation, he presented with the clinical picture of a simple virilizing form of 21-OHD and required glucocorticoid replacement therapy from the age of 4. Both probands' parents are compound heterozygous carriers of different CYP21 gene mutations causing various degrees of enzymatic activity impairment, which explains the different genotypes and phenotypes in their offspring. The probands' mother, besides the nonclassical 21-OHD, also had neuroblastoma of the adrenal gland. CONCLUSION: The potential discordance between the genotype and the phenotype in some patients with CAH is emphasized. The existence of a mild CYP21 mutation P30L in a compound heterozygous with CAH might be associated with progressive virilization requiring glucocorticoid therapy from early childhood. The occurrence of neuroblastoma with 21-OHD may support the hypothesis that an impairment in the synthesis and secretion of glucocorticoids may play role in the development and functioning of the adrenal medulla.     

Physical mapping distal to the DMD locus

We report a new locus, designated JC-1, which maps between the gene responsible for adrenal hypoplasia (AHC) and the gene that encodes glycerol kinase (GK) in Xp21.2-21.3. The probe identifying this locus was obtained by cloning the distal sequence of a junction fragment from a Duchenne muscular dystrophy (DMD) patient with a large deletion. Pulsed-field gel electrophoresis analysis shows that a region of at least 4 Mb separates the 3' end of the dystrophin gene and the closest distal marker to AHC, DXS28. This region of the human genome contains few genes whose deletion results in a clinical phenotype. JC-1 is a useful probe from which to initiate strategies directed at cloning the AHC and GK loci.     

ATP1A3 Mutations and Genotype-Phenotype Correlation of Alternating Hemiplegia of Childhood in Chinese Patients

Alternating hemiplegia of childhood (AHC) is a rare and severe neurological disorder. ATP1A3 was recently identified as the causative gene. Here we report the first genetic study in Chinese AHC cohort. We performed whole-exome sequencing on three trios and three unrelated patients, and screened additional 41 typical cases and 100 controls by PCR-Sanger sequencing. ATP1A3 mutations were detected in 95.7% of typical AHC patients. At least 93.3% were de novo. Four late onset, atypical AHC patients were also mutation positive, suggesting the need for testing ATP1A3 mutations in atypical cases. Totally, 13 novel missense mutations (T370N, G706R, L770R, T771N, T771I, S772R, L802P, D805H, M806K, P808L, I810N, L839P and G893R) were identified in our study. By homology modeling of the mutant protein structures and calculation of an extensive list of molecular features, we identified two statistically significant molecular features, solvent accessibility and distance to metal ion, that distinguished disease-associated mutations from neutral variants. A logistic regression classifier achieved 92.9% accuracy by the average of 100 times of five-fold cross validations. Genotype-phenotype correlation analysis showed that patients with epilepsy were more likely to carry E815K mutation. In summary, ATP1A3 is the major pathogenic gene of AHC in Chinese patients; mutations have distinctive molecular features that discriminate them from neutral variants and are correlated with phenotypes.     

A new mutation of the ALAS2 gene in a large family with X-linked sideroblastic anemia

X-linked sideroblastic anemia is a genetic disorder characterized by a hypochromic microcytic anemia of variable intensity with the presence of ring sideroblasts in the bone marrow of the patients. Two different mutations have been reported in the ALAS2 gene in patients with this diseae. We have studied a large kindred with a pyridoxine-sensitive form of X-linked sideroblastic anemia. Sequencing amplified cDNA of the proband revealed a guanine-to-adenine change at nucleotide 871 of the coding sequence (exon 7 of the gene). This results in a glycine to serine substitution that is responsible for a marked decrease in the enzymatic activity of the mutated protein. A polymerase chain reaction assay demonstrated the presence of the same mutation in three affected males and two female carriers in the kindred. The carrier status was excluded in eight females at risk. Early detection of the mutant allele in family members may thus be important for the prevention of anemia in males and of iron overload both in affected males and carrier females.     

Angiotensinogen gene M235T variant and pre-eclampsia in Romanian pregnant women

Background. Association between the human angiotensinogen gene and essential hypertension has been confirmed in recent studies. Pre-eclampsia is a complication of pregnancy characterised by increased vascular resistance, high blood pressure, proteinuria and oedema, that appears in the second and third trimester of pregnancy. The aim of our study was the analysis of M235T mutation in the gene encoding angiotensinogen in Romanian women with different forms of hypertension during pregnancy. Methods. Fourteen women with obstetric complications were tested for M235T angiotensinogen gene mutation. Indications for testing were: severe or mild pre-eclampsia and pre-eclampsia associated with chronic hypertension. We also tested for control 6 healthy women. The M235T angiotensinogen gene mutation was analysed by polymerase chain reaction followed by enzymatic digestion with Tth 111I restriction endonuclease enzyme and agarose gel electrophoresis of the products. Results. Eleven (78.57%) of the 14 women with complications of pregnancy had M235T mutation: 9 (64.28%) were found to be heterozygous carriers of the M235T variant of the angiotensinogen gene and 2(14.28%) were found to be homozygous carriers. In the group of women with normal pregnancy, 3 (50%) of the 6 women had M235T mutation: 2 (33.33%) were found to be heterozygous carriers of the M235T variant of the angiotensinogen gene and 1 (16.66%) was found to be homozygous carrier. Conclusions. Our study shows that the M235T variant in the gene encoding angiotensinogen could be a risk factor in mild and severe pre-eclampsia.     

ATM gene mutations result in both recessive and dominant expression phenotypes of genes and microRNAs

The defining characteristic of recessive disorders is the absence of disease in heterozygous carriers of the mutant alleles. However, it has been recognized that recessive carriers may differ from noncarriers in some phenotypes. Here, we studied ataxia telangiectasia (AT), a classical recessive disorder caused by mutations in the ataxia telangiectasia mutated (ATM) gene. We compared the gene and microRNA expression phenotypes of noncarriers, AT carriers who have one copy of the ATM mutations, and AT patients with two copies of ATM mutations. We found that some phenotypes are more similar between noncarriers and AT carriers compared to AT patients, as expected for a recessive disorder. However, for some expression phenotypes, AT carriers are more similar to the patients than to the noncarriers. Analysis of one of these expression phenotypes, TNFSF4 level, allowed us to uncover a regulatory pathway where ATM regulates TNFSF4 expression through MIRN125B (also known as miR-125b or miR125b) [corrected] In AT carriers and AT patients, this pathway is disrupted. As a result, the level of MIRN125B is lower and the level of its target gene, TNFSF4, is higher than in noncarriers. A decreased level of MIRN125B is associated with breast cancer, and an elevated level of TNFSF4 is associated with atherosclerosis. Thus, our findings provide a mechanistic suggestion for the increased risk of breast cancer and heart disease in AT carriers. By integrating molecular and computational analyses of gene and microRNA expression, we show the complex consequences of a human gene mutation.     

Identification of an HD patient with a (CAG)180 repeat expansion and the propagation of highly expanded CAG repeats in lambda phage

The Huntington’s disease mutation has been identified as a CAG/polyglutamine repeat expansion in a large gene of unknown function. In order to develop the transgenic systems necessary to uncover the molecular pathology of this disorder, it is necessary to be able to manipulate highly expanded CAG repeats in a cloned form. We have identified a patient with an expanded allele of greater than 170 repeat units and have cloned the mutant allele in the lambda zap vector. The recovery of highly expanded repeats after clone propagation was more efficient when repeats were maintained as lambda phage clones rather than as the plasmid counterparts. Manipulation of the repeats as phage clones has enabled us to generate Huntington’s disease transgenic mice that contain highly expanded (CAG)₁₁₅–(CAG)₁₅₀ repeats and that develop a progressive neurological phenotype. Received: 7 October 1996 / Revised: 5 December 1996     

Novel insertion 496_497insG creating a stop codon D194X in a Chinese family with X-Linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (XALD, MIM 300100), the commonest inherited peroxisomal disorder, is characterized by central nervous system demyelination, primary adrenal failure and the systemic accumulation of saturated very long chain fatty acids (VLCFAs). The defective gene ABCD1 encodes an ATP-binding cassette (ABC) transport protein named ALDP, which functions as a crucial transporter of VLCFAs into the peroxisomes for beta-oxidation. Here, we report a Chinese man with adrenomyeloneuropathy characterized by Addison's disease and spastic paraparesis. His plasma VLCFA levels, ratios of C24:0/C22:0 and C26:0/C22:0 were all significantly elevated. We performed mutation analysis of the ABCD1 gene in the proband and the family members using direct DNA sequencing and restriction analysis. A novel insertion 496_497insG in exon 1 causing a frame shift and a premature stop codon at amino acid position 194 (D194X) was identified (GenBank accession No. NM_000033). The insertional mutation abolishes an HhaI restriction site. The same mutation was found in his mother and the eldest sister even though their clinical and biochemical abnormalities were milder. Diagnosis of XALD often relies upon the detection of elevated VLCFA levels and ratios of C26:0/C22:0 and C24:0/C22:0 in fasting blood, however, 5-15% of the obligate heterozygotes would give normal values. DNA-based testing thus remains the most reliable tool for heterozygote detection when the disease-causing mutations are known. Using restriction fragment length polymorphism with HhaI, we have devised a rapid method for the identification of the carriers among the proband's family members and possibly for the screening of the mutations in other XALD patients.     

Exon Redefinition by a Point Mutation within Exon 5 of the Glucose-6-Phosphatase Gene is the Major Cause of Glycogen Storage Disease Type 1a in Japan

Glycogen storage disease (GSD) type 1a (von Gierke disease) is an autosomal recessive disorder caused by a deficiency in microsomal glucose-6-phosphatase (G6Pase). We have identified a novel mutation in the G6Pase gene of a individual with GSD type 1a. The cDNA from the patient's liver revealed a 91-nt deletion in exon 5. The genomic DNA from the patient's white blood cells revealed no deletion or mutation at the splicing junction of intron 4 and exon 5. The 3' splicing occurred 91 bp from the 5' site of exon 5 (at position 732 in the coding region), causing a substitution of a single nucleotide (G to T) at position 727 in the coding region. Further confirmation of the missplicing was obtained by transient expression of allelic minigene constructs into animal cells. Another eight unrelated families of nine Japanese patients were all found to have this mutation. This mutation is a new type of splicing mutation in the G6Pase gene, and 91% of patients and carriers suffering from GSD1a in Japan are detectable with this splicing mutation.     

Fluorescence-assisted mismatch analysis (FAMA) for exhaustive screening of the α-galactosidase A gene and detection of carriers in Fabry disease

We used the fluorescence-assisted mismatch analysis (FAMA) method to screen rapidly the α-galactosidase A gene in patients with Fabry disease in order to identify unknown mutations and help define genotype-phenotype correlations in this X-linked lysosomal storage disorder. Chemical cleavage at mismatches on heteroduplex DNA end-labeled with strand-specific fluorescent dyes, reliably detects sequence changes in DNA fragments of up to 1.5 kb and locates them precisely. Exhaustive scanning of the α-galactosidase gene was accomplished on four polymerase chain reaction-generated amplicons, covering all seven exons, the exon-intron boundaries, and 700 bp of 5′-flanking sequence. Mutations were identified in each of the 15 patients studied from nine unrelated kindreds. Among the seven previously undescribed sequence changes, three are obviously pathogenic because they lead to premature protein termination. The other four, a splice-site mutation and three missense mutations, were the only changes found upon complete scanning of the gene and its promoter. In addition, FAMA also detects female heterozygous carriers more dependably than direct sequencing, and thus provides a valuable diagnostic test. In Fabry disease, this molecular criterion is especially important for genetic counseling since heterozygotes can be asymptomatic and their enzymatic values within the normal range. Received: 9 April 1996 / Revised: 8 July 1996     

Detection of New Paternal Dystrophin Gene Mutations in Isolated Cases of Dystrophinopathy in Females

Duchenne muscular dystrophy is one of the most common lethal monogenic disorders and is caused by dystrophin deficiency. The disease is transmitted as an X-linked recessive trait; however, recent biochemical and clinical studies have shown that many girls and women with a primary myopathy have an underlying dystrophinopathy, despite a negative family history for Duchenne dystrophy. These isolated female dystrophinopathy patients carried ambiguous diagnoses with presumed autosomal recessive inheritance (limbgirdle muscular dystrophy) prior to biochemical detection of dystrophin abnormalities in their muscle biopsy. It has been assumed that these female dystrophinopathy patients are heterozygous carriers who show preferential inactivation of the X chromosome harboring the normal dystrophin gene, although this has been shown for only a few X:autosome translocations and for two cases of discordant monozygotic twin female carriers. Here we study X-inactivation patterns of 13 female dystrophinopathy patients—10 isolated cases and 3 cases with a positive family history for Duchenne dystrophy in males. We show that all cases have skewed X-inactivation patterns in peripheral blood DNA. Of the nine isolated cases informative in our assay, eight showed inheritance of the dystrophin gene mutation from the paternal germ line. Only a single case showed maternal inheritance. The 10-fold higher incidence of paternal transmission of dystrophin gene mutations in these cases is at 30-fold variance with Bayesian predictions and gene mutation rates. Thus, our results suggest some mechanistic interaction between new dystrophin gene mutations, paternal inheritance, and skewed X inactivation. Our results provide both empirical risk data and a molecular diagnostic test method, which permit genetic counseling and prenatal diagnosis of this new category of patients.     

Recurrent and founder mutations in the Netherlands

Background. Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is an inherited cardiac disease with reduced penetrance and a highly variable expression. Mutations in the gene encoding the plakophilin-2 gene (PKP2) are detected in about 50% of ARVC/D patients. The p.Arg79X mutation in PKP2 has been identified in Europe and North America and has been functionally characterised. We evaluated the prevalence of the p.Arg79X mutation in PKP2 in the Dutch population.Methods. Twelve index patients and 41 family members were evaluated in three university hospitals in the Netherlands. The diagnosis of ARVC/D was established according to the recently revised Task Force Criteria. Segregation of the p.Arg79X mutation was studied and haplotypes were reconstructed to determine whether the p.Arg79X mutation was a recurrent or a founder mutation.Results. The p.Arg79X mutation in PKP2 was identified in 12 index patients. Haplotype analysis revealed a shared haplotype among Dutch p.Arg79X mutation carriers, indicating a common founder. Six index patients (50%) had a first- or second-degree relative who had died of sudden cardiac death below 40 years of age. At age 60, only 60% of the mutation carriers had experienced any symptoms. There was no significant difference in symptom-free survival and event-free survival between men and women.Conclusion. We have identified the largest series of patients with the same desmosome gene mutation in ARVC/D reported to date. This p.Arg79X mutation in PKP2 is a founder mutation in the Dutch population. The phenotypes of PKP2 p.Arg79X mutation carriers illustrate the clinical variability and reduced penetrance often seen in ARVC/D. (Neth Heart J 2010;18:583–91.)     

Non-Invasive Prenatal Diagnosis of Multiple Endocrine Neoplasia Type 2A Using COLD-PCR Combined with HRM Genotyping Analysis from Maternal Serum

The multiple endocrine neoplasia type 2A (MEN2A) is a monogenic disorder characterized by an autosomal dominant pattern of inheritance which is characterized by high risk of medullary thyroid carcinoma in all mutation carriers. Although this disorder is classified as a rare disease, the patients affected have a low life quality and a very expensive and continuous treatment. At present, MEN2A is diagnosed by gene sequencing after birth, thus trying to start an early treatment and by reduction of morbidity and mortality. We first evaluated the presence of MEN2A mutation (C634Y) in serum of 25 patients, previously diagnosed by sequencing in peripheral blood leucocytes, using HRM genotyping analysis. In a second step, we used a COLD-PCR approach followed by HRM genotyping analysis for non-invasive prenatal diagnosis of a pregnant woman carrying a fetus with a C634Y mutation. HRM analysis revealed differences in melting curve shapes that correlated with patients diagnosed for MEN2A by gene sequencing analysis with 100% accuracy. Moreover, the pregnant woman carrying the fetus with the C634Y mutation revealed a melting curve shape in agreement with the positive controls in the COLD-PCR study. The mutation was confirmed by sequencing of the COLD-PCR amplification product. In conclusion, we have established a HRM analysis in serum samples as a new primary diagnosis method suitable for the detection of C634Y mutations in MEN2A patients. Simultaneously, we have applied the increase of sensitivity of COLD-PCR assay approach combined with HRM analysis for the non-invasive prenatal diagnosis of C634Y fetal mutations using pregnant women serum.