Recent Advances in Imprinting Disorders.

Imprinting disorders (ImpDis) are a group of currently 12 congenital diseases with common underlying (epi)genetic etiologies and overlapping clinical features affecting growth, development and metabolism. In the last years it has emerged that ImpDis are characterized by the same types of mutations and epimutations, i.e. uniparental disomies, copy number variations, epimutations, and point mutations. Each ImpDis is associated with a specific imprinted locus, but the same imprinted region can be involved in different ImpDis. Additionally, even the same aberrant methylation patterns are observed in different phenotypes. As some ImpDis share clinical features, clinical diagnosis is difficult in some cases. The advances in molecular and clinical diagnosis of ImpDis help to circumvent these issues, and they are accompanied by an increasing understanding of the pathomechanism behind them. As these mechanisms have important roles for the etiology of other common conditions, the results in ImpDis research have a wider effect beyond the borders of ImpDis. For patients and their families, the growing knowledge contributes to a more directed genetic counseling of the families and personalized therapeutic approaches.

A homozygous mutation in a consanguineous family consolidates the role of ALDH1A3 in autosomal recessive microphthalmia.

Anomalies of eye development can lead to the rare eye malformations microphthalmia and anophthalmia (small or absent ocular globes), which are genetically very heterogeneous. Several genes have been associated with microphthalmia and anophthalmia, and exome sequencing has contributed to the identification of new genes. Very recently, homozygous variations within ALDH1A3 have been associated with autosomal recessive microphthalmia with or without cysts or coloboma, and with variable subphenotypes of developmental delay/autism spectrum disorder in eight families. In a consanguineous family where three of the five siblings were affected with microphthalmia/coloboma, we identified a novel homozygous missense mutation in ALDH1A3 using exome sequencing. Of the three affected siblings, one had intellectual disability and one had intellectual disability and autism, while the last one presented with normal development. This study contributes further to the description of the clinical spectrum associated with ALDH1A3 mutations, and illustrates the interfamilial clinical variation observed in individuals with ALDH1A3 mutations.

A novel founder BBS1 mutation explains a unique high prevalence of Bardet-Biedl syndrome in the Faroe Islands.

BACKGROUND/AIM: Bardet-Biedl syndrome is a multiorgan disease presenting with retinitis pigmentosa leading to blindness. The aim of the study was to investigate the genetic background of Bardet-Biedl syndrome in the Faroe Island. It was hypothesised that a common genetic background for the syndrome would be found. METHODS: Patients were identified from the files of the Retinitis Pigmentosa Register at the National Eye Clinic, Denmark. The diagnosis of Bardet-Biedl syndrome was verified from medical files. Mutational screening of BBS1, BBS2, BBS4, MKKS and BBS10 was done by denaturing high-performance liquid chromatography. RESULTS: Out of 13 prevalent cases in the Faroe Islands, 10 patients from nine families were included. A novel splice site mutation in BBS1, c.1091+3G>C, was identified, and this was predicted to affect protein function by skipping 16 amino acids. Nine patients were homozygous for this mutation, while one patient was compound heterozygous with a recurrent BBS1 mutation, p.Met390Arg. The patients presented with severe ophthalmic phenotypes, while the systemic manifestations of the disease were apparently milder. CONCLUSION: A novel BBS1 mutation was identified, most probably a founder mutation, further confirming the Faroe Islands as a genetic isolate. The phenotypic expression of the Faroese patients suggests that different mutations in BBS1 affect various organs differently.

The A1555G mtDNA mutation in Danish hearing-impaired patients: frequency and clinical signs.

The A1555G mutation of the mtDNA is associated with both aminoglycoside-induced and non-syndromic hearing loss. The A1555G is relatively frequent in the Spanish and some Asian populations, but has only been reported rarely in other populations, possibly because of ascertainment bias. We studied 85 Danish patients with varying degrees of hearing impairment and found two patients with the A1555G mutation (2.4%). Neither had received aminoglycosides. Our study indicates that the mutation might not be uncommon in Danish patients with hearing impairment.

Is there a relationship between U-shaped audiograms and mutations in connexin 26?

The knowledge about gene mutations causing permanent hearing impairment (HI) is rapidly increasing, offering clinicians the possibility of analysing different gene mutations in relation to various phenotypes. This study examines a possible relationship between U-shaped audiograms and mutations in the GJB2-gene, coding for Connexin 26 (Cx 26). Thirty-eight subjects at a median age of 42 years, range 18-60 years with symmetric U-shaped audiograms classified as sensorineural were included in the genetic investigation. The gender distribution was 13 males and 25 females. No subjects had any indication of syndromic HI, and any possible exogenous factor that might cause HI was excluded. Three subjects had self-reported prelingual HI and 34 subjects had self-reported postlingual HI. Thirty-five subjects had one or more family members with HI. In 19 subjects the entire Cx 26 gene was examined, whereas 19 subjects were investigated for the 35delG mutation only. One female with mild HI and postlingual onset of the HI was heterogeneous for the L9OP-mutation in the Cx 26 gene. In all other subjects no mutations in the Cx 26 gene could be identified. Mutations of the Cx 26 gene are very rare among subjects exhibiting a U-shaped phenotype of the audiogram. However the majority of the investigated subjects (35/38) had a family history of HI and it seems therefore reasonable to ascribe U-shaped hearing deficit to genetic factors which has to be searched for in alternative gene mutations.

Population-based risk estimates of Wilms tumor in sporadic aniridia. A comprehensive mutation screening procedure of PAX6 identifies 80% of mutations in aniridia.

Aniridia is a severe eye disease characterized by iris hypoplasia; both sporadic cases and familial cases with an autosomal dominant inheritance exist. Mutations in the PAX6 gene have been shown to be the genetic cause of the disease. Some of the sporadic cases are caused by large chromosomal deletions, some of which also include the Wilms tumor gene (WAGR syndrome), resulting in an increased risk of developing Wilms tumor. Based on the unique registration of both cancer and aniridia cases in Denmark, we have made the most accurate risk estimate to date for Wilms tumor in sporadic aniridia. We have found that patients with sporadic aniridia have a relative risk of 67 (confidence interval: 8.1-241) of developing Wilms tumor. Among patients investigated for mutations, Wilms tumor developed in only two patients out of 5 with the Wilms tumor gene (WT1) deleted. None of the patients with smaller chromosomal deletions or intragenic mutations were found to develop Wilms tumor. Our observations suggest a smaller risk for Wilms tumor than previous estimates, and that tumor development requires deletion of WT1. We report a strategy for the mutational analysis of aniridia cases resulting in the detection of mutations in 68% of sporadic cases and 89% of familial cases. We also report four novel mutations in PAX6, and furthermore, we have discovered a new alternatively spliced form of PAX6.

Mutation analysis of the GJB2 (connexin 26) gene by DGGE in Greek patients with sensorineural deafness.

The GJB2 (connexin 26) gene, one of the major genes responsible for autosomal recessive deafness, has been investigated previously by a variety of techniques, including PCR-SSCP and sequencing of the entire gene for screening of unknown mutations, and allele-specific PCR, ASO, and PCR-mediated site-directed mutagenesis for the detection of the common mutation 35delG. Here, we present the development of a DGGE method for the characterization of the full spectrum of mutations in the GJB2 gene. The GJB2 cDNA and flanking sequences were amplified in three overlapping segments. We screened 26 Greek patients with prelingual, sensorineural deafness, where syndromic forms and environmental causes of deafness had been excluded. The 35delG mutation was detected in 28 chromosomes (53.8%), while another three sequence variations accounted for 7.6% of the alleles. The sequence variation R127H, previously described in a few Spanish and Balkan patients, was detected in two patients as the sole mutation. A novel sequence variation, K224Q, was identified as the sole mutation in one patient. Use of this approach may contribute to the full description of mutations in this important deafness gene.

Haplotype and AGG-interspersion analysis of FMR1 (CGG)(n) alleles in the Danish population: implications for multiple mutational pathways towards fragile X alleles.

The AGG interspersion pattern and flanking microsatellite markers and their association with instability of the FMR1 (CGG)(n) repeat, involved in the fragile X syndrome, were analyzed in DNA from filter-paper blood spots randomly collected from the Danish newborn population. Comparison of DXS548-FRAXAC1 haplotype frequencies in the normal population and among fragile X patients suggested strong linkage disequilibrium between normal alleles and haplotype 7-3 and between fragile X alleles and haplotype 2-1 and 6-4. Comparison of the AGG interspersion pattern in 143 alleles, ranging in size from 34-62 CGG, and their associated haplotypes indicates the existence of at least three mutational pathways from normal alleles toward fragile X alleles in the Danish population. Two subgroups of normal alleles, with internal sequences of (CGG)(10)AGG(CGG)(19) and (CGG)(9)AGG(CGG)(12) AGG(CGG)(9), possibly predisposed for expansion, were identified in the data set. When alleles larger than 34 CGG were investigated, comparing the length of 3' uninterrupted CGG triplets (uCGG), we found that alleles associated with haplotype 2-1 and 6-4 contain significantly longer stretches of uCGG than alleles associated with haplotype 7-3. Thus, the data support that (CGG)(n) instability is correlated to the length of uCGG.

Analysis of FMR1 (CGG)n alleles and FRAXA microsatellite haplotypes in the population of Greenland: implications for the population of the New World from Asia.

The fragile X syndrome is caused by the expansion of a polymorphic (CGG)n tract in the promoter region of the FMR1 gene. Apparently the incidence of fragile X syndrome is rare in the population of Greenland. In order to examine population-related factors involved in stability of the (CGG)n sequence, DNA samples obtained randomly from the Greenlandic population were analysed for size and AGG interspersion pattern of the FMR1 (CGG)n region and associated DXS548-FRAXAC1 haplotypes. In addition a large Greenland family with unstable transmission in the premutation range was analysed. The (CGG)n allele sizes in the Greenland population showed a narrow distribution similar to that reported for Asian populations. DNA sequencing of alleles with 36 CGG repeats revealed an AGG(CGG)6 insertion previously reported exclusively in Asian populations and a high frequency of alleles with a (CGG)10AGG(CGG)9AGG(CGG)9 or (CGG)9AGG(CGG)9AGG(CGG)6AGG(CGG)9 sequence pattern was found. Thus the data confirm the Asian origin of the Greenlandic (Eskimo) population and indicates that some (CGG)n alleles have remained stable for 15-30,000 years, since the population of the New World arrived from Asia via the Bering Strait.

Mutational analysis of PAX6: 16 novel mutations including 5 missense mutations with a mild aniridia phenotype.

Mutations in the developmental control gene PAX6 have been shown to be the genetic cause of aniridia, which is a severe panocular eye disease characterised by iris hypoplasia. The inheritance is autosomal dominant with high penetrance but variable expressivity. Here we describe a mutational analysis of 27 Danish patients using a dideoxy fingerprinting method, which identified PAX6 mutations in 18 individuals with aniridia. A thorough phenotype description was made for the 18 patients. A total of 19 mutations, of which 16 were novel, are described. Among these were five missense mutations which tended to be associated with a milder aniridia phenotype, and in fact one of them seemed to be non-penetrant. Four of the five missense mutations were located in the paired domain. We also describe a third alternative spliced PAX6 isoform in which two of the four missense mutations would be spliced out. Our observations support the concept of dosage effects of PAX6 mutations as well as presenting evidence for variable expressivity and gonadal mosaicism.

[Fragile X syndrome. Diagnosis, genetics and clinical findings]. / Fragilt X-syndrom. Diagnostik, genetik og klinik.

The most common heritable form of mental retardation is the fragile X syndrome. It is X-linked and affects 1:1500-1:4000 boys. In Denmark 230 affected individuals are known, thereby rendering it underdiagnosed. Only minor dysmorphic traits are associated with the syndrome, more pronounced in boys, namely a long face with large, prominent ears, and macroorchidism postpubertally. The psychological manifestations are autistic features, hyperactivity and deviant behavior. Today no medical curative treatment is available but much can be gained from social and educational intervention. The syndrome is caused by a dynamic mutation on the X chromosome and displays a remarkable pattern of inheritance. Carrier diagnosis and prenatal diagnosis are feasible. This article describes the clinical, diagnostic and genetic aspects of fragile X syndrome.

Mutational analysis of the FMR1 gene in 118 mentally retarded males suspected of fragile X syndrome: absence of prevalent mutations.

Fragile X syndrome is usually caused by expansion of a trinucleotide (CGG) repeat in the 5'-untranslated region of the FMR1 gene. However, both deletions and point mutations in FMR1 have been identified as rare causes of the fragile X syndrome. We have screened the FMR1 gene for mutations by single-stranded conformational polymorphism analysis in 118 mentally retarded males who were referred to us for fragile X testing, and who had a CGG repeat number in the normal size range. We found one patient with a 2-bp deletion in intron 1 and two unrelated patients with identical silent mutations in exon 1. Neither of these mutations were found in 83 controls. Further investigation of the exon 1 silent mutation by Western blot analysis showed normal expression of FMRP in lymphoblastoid cells and reverse-transcription-polymerase chain reaction analysis showed that intron 1 and 2 were spliced out as in the normal control. Furthermore, we found two common polymorphisms, one in intron 1 and one in exon 5. However, no pathogenic FMR1 mutation was found.

High-throughput analysis of fragile X (CGG)n alleles in the normal and premutation range by PCR amplification and automated capillary electrophoresis.

Fragile X syndrome is caused by expansion of a (CGG)n trinucleotide repeat within the 5' untranslated region of the FMR1 gene transcript. The disease is reliably diagnosed by Southern blotting, but this method constitutes a significant workload and requires large samples. Therefore, for large research or screening projects in which a large majority of the samples will be normal, a more rapid and less expensive method is needed. We present a method for accurate, high-throughput analysis of the FRAXA (CGG)n region in the normal and premutation range. The method is based on polymerase chain reaction (PCR) amplification of DNA extracted from whole blood or eluted from dried blood spots on filter-paper, followed by automated capillary electrophoresis and detection by multicolour fluorescence. This method allows a throughput of 144 samples in 48 h, with an intra-assay accuracy in size determination of 0.2-1.8 bp. We performed a blind reanalysis of samples from 30 patients, previously analysed by Southern blotting or PCR with radioactive labelling. In this study normal and premutation alleles, ranging from 28-121 (CGG)n repeats, were correctly determined with respect to number of (CGG)n repeats. All full-mutation alleles and one large premutation allele in a sample of a heterozygote failed to amplify. The method was used to determine the distribution of FRAXA (CGG)n repeats in the Danish population.

Deletion of all CGG repeats plus flanking sequences in FMR1 does not abolish gene expression.

The fragile X syndrome is due to the new class of dynamic mutations. It is associated with an expansion of a trinucleotide repeat (CGG) in exon 1 of the fragile X mental retardation gene 1 gene (FMR1). Here we present a fragile X family with an unique female patient who was rendered hemizygous for the FRAXA locus due to a large deletion of one X chromosome. In addition, the other X had a microdeletion in FMR1. PCR and sequence analysis revealed that the microdeletion included all CGG repeats plus 97 bp of flanking sequences, leaving transcription start site and translation start site intact. Despite this total lack of CGG repeats in the FMR1 gene, Western blot analysis showed expression of FMRP, and the patient's phenotype was essentially normal. X-inactivation studies of the androgen-receptor (AR) locus and haplotype determination of microsatellite markers gave evidence that the deletion probably originated from regression of a fully mutated FMR1 gene. Although the minimal number of CGG repeats hitherto reported in FRAXA is six, and at least four other genes associated with CGG repeats are known, suggesting an as yet unknown function of these repeats, our study clearly demonstrates that the absence of CGG repeats does not abolish expression of the FMR1 gene in lymphoblastoid cells.

Role of the time factor in signaling specificity: application to mitogenic and metabolic signaling by the insulin and insulin-like growth factor-I receptor tyrosine kinases.

The signal transduction pathways activated by hormones, growth factors, and cytokines show an extraordinary degree of cross-talk and redundancy. This review addresses the question of how the specificity conferred at the binding step is maintained through the signaling network despite the convergence of multiple signals on common efferent pathways such as mitogen-activated protein (MAP) kinase. The mechanism of receptor activation by ligand-induced dimerization provides a signaling device with both a switch and a timer. The role of the time factor, ie, of signaling kinetics, as a determinant of selectivity is discussed with emphasis on the receptor tyrosine kinases and cytokine receptors, and especially mitogenic versus metabolic signaling by insulin and insulin-like growth factor-I (IGF-I).

The insulin-like growth factor-I receptor. Structure, ligand-binding mechanism and signal transduction.

The nonclassical binding kinetics of IGF-I and insulin to their respective receptors, suggestive of negative cooperativity, can be readily explained by our recently proposed novel binding mechanism whereby the bivalent ligand bridges the two receptor alpha-subunits alternatively at opposite sites in a symmetrical receptor structure. The bivalent binding mechanism also explains bell-shaped bioactivity curves. The possible role of different binding modes versus differences in downstream signaling by insulin and IGF-I in producing specific mitogenic or metabolic responses is discussed.

Expression of insulin receptor spliced variants and their functional correlates in muscle from patients with non-insulin-dependent diabetes mellitus.

Due to alternative splicing of exon 11 of the receptor gene, the human insulin receptor exists in two forms, that have distinct tissue-specific expression and are functionally different. Needle biopsies obtained from vastus lateralis muscle from 20 patients with noninsulin-dependent diabetes mellitus (NIDDM) and 20 normal control subjects were analyzed for the relative expression of insulin receptor mRNA variants in a novel assay using fluorescence-labeled primers and subsequent analysis on an automated DNA sequencer. In subgroups of patients and control subjects, insulin binding and tyrosine kinase activity were examined in wheat germ agglutinin-purified insulin receptors isolated from muscle biopsies. Moreover, insulin-stimulated glucose disposal was studied by means of the euglycemic hyperinsulinemic clamp technique. No difference in the relative expression of spliced variants of the insulin receptor mRNA was observed (control subjects, 71.4 +/- 1.3% insulin receptor mRNA with exon 11; NIDDM patients, 71.5 +/- 1.3% insulin receptor mRNA with exon 11). No significant interrelationships were demonstrated among the relative expression of insulin receptor mRNA variants, insulin binding, and tyrosine kinase activity toward the exogenous substrate poly(Glu-Tyr(4:1)). Furthermore, no significant relationship was demonstrated between the glucose disposal rate and the relative expression of insulin receptor splice variants. In conclusion, in skeletal muscle from both normal control subjects and NIDDM patients, the proportion of insulin receptor mRNA with exon 11 is about 70%. In addition, no significant correlations exist among insulin binding, insulin receptor tyrosine kinase activity, glucose disposal rate, and expression of alternative spliced insulin receptors in human skeletal muscle.

Mutation of arginine 86 to proline in the insulin receptor alpha subunit causes lack of transport of the receptor to the plasma membrane, loss of binding affinity and a constitutively activated tyrosine kinase in transfected cells.

We have investigated the role of Ser 85 and Arg 86 of the human insulin receptor (HIR) in insulin binding and tyrosine kinase activity by mutational analysis. Four mutant cDNAs were created (R86P, R86N, S85T+R86N, S85W+R86K) and stably transfected into BHK cells. R86P-HIR was also transiently expressed in 293 cells. Only the R86P receptor had substantially altered properties: lack of transport to the plasma membrane, loss of insulin binding, a constitutively activated autophosphorylation and tyrosine kinase, and an incomplete processing. Some of these alterations mimic those reported for the insulin receptor of the leprechaun Atl, which has a homozygous R86P mutation (Longo, N., et al, Biochem. Biophys. Res. Commun., 167, 1229, 1990; Clin. Res., 40, 2, 329, 1992).