Search

Background: Jaffe-Campanacci syndrome is characterized by multiple non-ossifying fibromas, café-au-lait macules and giant cell granulomas of the jaw. Even if the association between all these peculiar features and neurofibromatosis type 1 have been described, it has not yet been clarified whether Jaffe-Campanacci syndrome represents a distinct entity or it can be regarded as a neurofibromatosis type 1 subtype., Case Presentation: The patient here described is a young boy, who fulfilled the clinical diagnostic criteria for both syndromes. He had a complex clinical history with café-au-lait macules, axillary and inguinal freckling, multiple non-ossifying fibromas, giant-cell granuloma of the jaw, neurofibromas, plexiform fibroma, ocular Lisch nodules, optic chiasmatic- hypothalamic glioma, pseudarthrosis, scoliosis, short stature, vascular anomalies, seizures. Molecular analysis of the NF1 gene both on blood cells and non-ossifying fibroma's biopsy tissue allowed the detection of a novel variant within the coding region, NM_000267.3:c.2789_2791delATC(p.Tyr930_Pro931delinsSer), with loss of heterozygosity (second hit mutation) in the non-ossifying fibroma., Conclusion: This result indicates that every patient with clinical features of Jaffe-Campanacci syndrome should be further evaluated to detect features related to neurofibromatosis type 1 and genetically investigated for mutations in the NF1 gene, since this could lead to a definite diagnosis, but also could clarify and quantify the real genotype-phenotype overlap between neurofibromatosis type 1 and Jaffe-Campanacci syndrome.

Background/aim: The McCune-Albright syndrome (MAS) is a potentially severe disorder hallmarked by fibrous bone dysplasia, café-au-lait skin spots, and endocrine hyperfunction. It is caused by postzygotic activating mutations at the R201 codon of the GNAS gene, leading to a state of somatic mosaicism. Our aim was to improve the mutation detection rate and to quantify the presence of R201 GNAS mutations in different DNA samples from MAS patients., Methods: Real-time COLD- and MAMA-PCR TaqMan techniques were combined to search for R201 mutations in the DNA of blood or affected tissues from 16 previously molecularly characterized MAS patients, from a further 84 subjects with MAS signs who were R201 negative at RFLP analysis, and from 36 controls. The ability of this new method to provide quantitative data was tested in the serial dilution of wild-type, R201H, or R201C cloned plasmid DNA samples; the mutant abundance was measured by spectrophotometry., Results: A linear correlation between the true and the relative mutant abundance was observed until 2.5%, indicating a reliable quantification of R201 mutations. The assay's sensitivity was 0.05%, similar to that of previously described molecular methods., Conclusion: The real-time COLD-MAMA-PCR approach is a rapid, efficient, and inexpensive molecular technique for the identification of mutant alleles poorly represented in DNA samples.
., (© 2017 S. Karger AG, Basel.)

Context: Congenital hypothyroidism (CH) is a common endocrine disorder with an incidence of 1:3000- 4000 newborns. In 80-85% of cases, CH is caused by defects in thyroid organogenesis, resulting in absent, ectopically located, and/or severely reduced gland, all conditions indicated as "thyroid dysgenesis" (TD). A higher prevalence of congenital heart diseases has been documented in children with CH compared to the general population. This association suggests a possible pathogenic role of genes involved in both heart and thyroid development. Among these, it can be included Isl1, a transcription factor containing a LIM homeodomain that is expressed in both thyroid and heart during morphogenesis., Objective: In the present study, we investigate the role of ISL1 in the pathogenesis of TD., Settings and Patients: By single stranded conformational polymorphism, we screened for mutations the entire ISL1 coding sequence in 96 patients with TD and in 96 normal controls., Results: No mutations have been found in patients and controls., Conclusion: Our data indicate that, despite the relevant role of ISL1 in thyroid and heart morphogenesis, mutations in its coding region are not associated with TD in our group of patients.

Context: Pseudohypoaldosteronism type 1 (PHA1) is a rare salt-wasting syndrome. Mutations in the NR3C2 gene coding for the mineralocorticoid receptor (MR) cause autosomal dominant PHA1., Objective: Our objective was to reveal the cause of renal salt loss in six PHA1 patients and analyze the mutants' functional impact on MR function., Design: Our study included the following: clinical and hormonal characterization of the patients' phenotype, analysis of the NR3C2 gene, determination of receptor affinities to aldosterone and the transcriptional activation abilities of the MR mutants, investigation of subcellular translocation using fluorescence-labeled MR, and studying changes in mutant receptor conformation with proteolysis experiments and three-dimensional modeling., Results: Six heterozygous NR3C2 mutations were detected. One frameshift mutation (c.1131dupT) has been reported previously. The second frameshift mutation (c.2871dupC), which has only recently been reported by our group, showed no aldosterone binding and no transactivation because of a major change in receptor conformation. Two novel nonsense mutations generate a truncated receptor protein. Two missense mutations differently affect MR function. S818L was reported recently without complete in vitro data. S818L does not bind aldosterone or activate transcription or translocate into the nucleus. A major displacement of several residues involved in aldosterone binding was PHA1 causing. The novel E972G mutation showed a significantly lower ligand-binding affinity and only 9% of wild-type transcriptional activity caused by major changes in receptor conformation., Conclusions: Our data on six mutations extend the spectrum of PHA1-causing NR3C2 gene mutations. Studying naturally occurring mutants helps to clarify their pathogenicity and to identify crucial residues for MR structure and function.

Heterozygous, de novo, loss-of-function mutations in SOX2 have been shown to cause bilateral anophthalmia. Here we provide a detailed description of the clinical features associated with SOX2 mutations in the five individuals with reported mutations and four newly identified cases (including the first reported SOX2 missense mutation). The SOX2-associated ocular malformations are variable in type, but most often bilateral and severe. Of the nine patients, six had bilateral anophthalmia and two had anophthalmia with contralateral microphthalmia with sclerocornea. The remaining case had anophthalmia with contralateral microphthalmia, posterior cortical cataract and a dysplastic optic disc, and was the only patient to have measurable visual acuity. The relatively consistent extraocular phenotype observed includes: learning disability, seizures, brain malformation, specific motor abnormalities, male genital tract malformations, mild facial dysmorphism, and postnatal growth failure. Identifying SOX2 mutations from large cohorts of patients with structural eye defects has delineated a new, clinically-recognizable, multisystem disorder and has provided important insight into the developmental pathways critical for morphogenesis of the eye, brain, and male genital tract., ((c) 2005 Wiley-Liss, Inc.)

Objective: To clarify the genotype-phenotype correlation and elucidate the role of digenic inheritance in cystinuria., Methods: 164 probands from the International Cystinuria Consortium were screened for mutations in SLC3A1 (type A) and SLC7A9 (type B) and classified on the basis of urine excretion of cystine and dibasic amino acids by obligate heterozygotes into 37 type I (silent heterozygotes), 46 type non-I (hyperexcretor heterozygotes), 14 mixed, and 67 untyped probands., Results: Mutations were identified in 97% of the probands, representing 282 alleles (86.8%). Forty new mutations were identified: 24 in SLC3A1 and 16 in SLC7A9. Type A heterozygotes showed phenotype I, but mutation DupE5-E9 showed phenotype non-I in some heterozygotes. Type B heterozygotes showed phenotype non-I, with the exception of 10 type B mutations which showed phenotype I in some heterozygotes. Thus most type I probands carried type A mutations and all type non-I probands carried type B mutations. Types B and A mutations contributed to mixed type, BB being the most representative genotype. Two mixed cystinuria families transmitted mutations in both genes: double compound heterozygotes (type AB) had greater aminoaciduria than single heterozygotes in their family., Conclusions: Digenic inheritance is an exception (two of 164 families), with a limited contribution to the aminoaciduria values (partial phenotype) in cystinuria. Further mutational analysis could focus on one of the two genes (SLC3A1 preferentially for type I and SLC7A9 for type non-I probands), while for mixed probands analysis of both genes might be required, with priority given to SLC7A9.

The PAX8 gene, mapped on 2q12-q14, encodes for a transcription factor involved in thyroid cell proliferation and differentiation. Five mutations in PAX8 have been so far described in both sporadic and rare familial forms of thyroid dysgenesis with proposed autosomal dominant inheritance, all associated with thyroid hypoplasia and/or dysfunction. Fifty-four subjects with congenital hypothyroidism detected during neonatal screening and associated with an ultrasound or scintiscan picture of thyroid dysgenesis were investigated for PAX8 mutations. The entire PAX8 coding region with exon-intron boundaries was amplified from genomic DNA, and a mutational screening was performed by denaturing HPLC followed by direct sequencing when denaturing HPLC elution abnormalities appeared. A new heterozygous deletion (c.989_992delACCC) in exon 7 causing a frameshift with premature stop codon after codon 277 was identified in a subject with thyroid hypoplasia. This mutation is the only one so far identified that lies outside the paired domain. The predicted mutant protein completely lacks the C-terminal region but contains the paired box, octapeptide, and homeodomain. It retains the ability to bind a paired-domain sequence in vitro but is transcriptionally inactive. These results provide evidence that the C-terminal region is essential for transcriptional activity. The new mutation has been inherited from the completely euthyroid mother. It was also present in a brother with slightly elevated TSH only. Thus, it is associated with thyroid dysgenesis in the proband and both euthyroidism and compensated hypothyroidism in her family. This suggests that other factors/genes may modulate phenotypic expression.

Brachydactyly, classically described as shortening of III, IV, and V metacarpals and I distal phalanx, is the typical and most specific sign of Albright's hereditary osteodystrophy, a peculiar phenotype reported in subjects with pseudohypoparathyroidism type Ia (PHP-Ia) caused by mutations in the GNAS gene, which encodes for the alpha-subunit of the stimulatory G protein (Gsalpha). It has been reported in 70% of PHP subjects from routine radiological examinations, but there are no specific data for hand alterations in genetically characterized PHP-Ia subjects. We evaluated the metacarpophalangeal pattern profile in 14 GNAS-mutated PHP-Ia subjects and determined the prevalence and patterns of left hand bone shortening. To search for genotype/phenotype correlations, we compared metacarpophalangeal pattern profiles in subjects with identical mutations. Shortening below -2 SD score (SDS) was present in at least one bone in each subject, with a prevalence of 100%; however, great variability existed between subjects and between hand bone segments. Between subjects, shortening ranged from -2 to -10.4 SDS and involved 1-19 hand bones (5.3-100%). Between segments, III-IV metacarpals were the most compromised (-10.4 and -10.0 SDS, respectively); V metacarpals and I-IV distal phalanges were the most frequently shortened (85.7%). Overall, bone length median values revealed shortening below -2 SDS in all metacarpals and all distal phalanges, i.e. brachymetacarpia and brachytelephalangy, that cluster together. These segments were shortened in 64.3-85.7% of patients, significantly differing from proximal and middle phalanges, which were shortened in 21.4-50%. Even if these hand alterations were a constant and typical finding in our PHP-Ia population, cluster analysis in subjects with the same genotypes did not generally show a genotype/phenotype correlation. Variability between subjects may be the result of complex interactions between GNAS defects and other genetic or epigenetic factors. In conclusion, hand shortening analysis in 14 genetically characterized patients showed typical brachymetacarpia and brachytelephalangy. Further studies in PHP-Ia subjects without GNAS mutations and in other brachydactyly syndromes will determine whether the pattern described is also specific.

McCune-Albright syndrome (MAS) is a rare disease caused by somatic postzygotic mutations at Arg201 in the GNAS1 gene that encodes for the Gsalpha protein. Arg201 mutations are gain-of-function mutations in affected tissues (including bone, skin, endocrine glands and other tissues) that result in the activation of cAMP. We used a polymerase chain reaction(PCR)-based technique for the selective enrichment and analysis of the Arg201 mutant allele in 27 different tissues from 24 Italian patients with one or more signs of MAS. Arg201 mutations were identified in 13 different tissues (48.1%) from 11 patients (45.8%). Mutation detection rates differed across the various types of tissue samples, and the mutation was not always found in every tissue sample from the same patient. To overcome problems in the analysis of mutations in somatic mosaicism, as occurs in MAS, a highly sensitive molecular technique should be applied, the most appropriate tissue source selected, and various affected tissues from the same patient analyzed.

Dihydropteridine reductase (DHPR) is an enzyme involved in recycling of tetrahydrobiopterin (BH4), the cofactor of the aromatic amino acid hydroxylases. Its deficiency is characterized by hyperphenylalaninemia due to the secondary defect of phenylalanine hydroxylase and depletion of the neurotransmitters dopamine and serotonin, whose syntheses are controlled by tryptophan and tyrosine hydroxylases. The DHPR cDNA has been cloned and mapped on 4p15.3. In the present study we report the genomic structure of the DHPR gene (QDPR). This gene includes seven exons within a range of 84-564 bp; the corresponding introns are flanked by canonic splice junctions. We also present a panel of PCR primers complementary to intronic sequences that greatly facilitates amplification of the gene and provides a genomic DNA approach for mutation detection. We have used this approach to study six patients with DHPR deficiency. Four known mutations (G23D, H158Y, IVS5G+ 1A, R221X) and two new mutations (Y150C and G218ins9bp) were found. The Y150C mutation was found in compound heterozygosity with G23D, a mutation always associated with a severe phenotype in homozygous patients. This patient has an intermediate phenotype (good response to monotherapy with BH4). The mutant enzyme for Y150C was expressed in an E. coli system. Comparison of its kinetic parameters with those of the G23D mutant enzyme showed that it is not as effective as the wild-type enzyme, but is more active than the G23D mutant. This patient's intermediate phenotype is thus due to the mild DHPR mutation Y150C. Correlations between genotypes and phenotypes were also found for the other mutations.

Ultraviolet (UV)-induced repair and mutational spectra were analyzed in an inducible marker gene, the metallothionein-l/guamine-xanthine phosphoribosyl transferase (gpt) fusion gene, carried by an Epstein-Barr virus-derived shuttle vector episomically maintained in human cells. The repair rate of UV photodimers from the shuttle-vector molecules was typical of transcriptionally active sequences, 70% of the dimers being removed within 8 h after irradiation. The spectrum obtained under basal gene transcription was compared with that obtained under induced transcription. In both cases, base substitutions at dipyrimidine sequences predominated. Multiple mutations and deletions probably due to recombinational events induced by UV damage were also observed. Most of the UV-mutated dipyrimidine sites were located in the transcribed strand and were independent of the transcriptional activity of the target gene. In contrast, the distribution of mutations throughout the coding region of the gpt gene was affected by transcription, with a preferential clustering of mutations occurring in the 3' half of the gene after transcription induction. The strand bias observed in the UV spectra most likely reflects selection for nonfunctional gpt protein.