A novel mutation in collagen gene COL1A2 associated with transient regional osteoporosis.

A young man was diagnosed with transient regional osteoporosis (TRO). The genetic analysis revealed a novel de novo likely pathogenic variant in COL1A2 gene. Our hypothesis is that TRO may be a possible clinical manifestation of osteogenesis imperfecta due to a reduced bone mass and an impaired trabecular mechanical competence. INTRODUCTION: Transient regional osteoporosis (TRO) is a disease characterized by episodes of pain in the lower limbs involving the hip, knee, ankle or foot. Here, we present a clinical case of a Caucasian 25-year-old man exhibiting TRO. Based on few mild clinical findings suggestive of osteogenesis imperfecta (OI), but without a history of fragility fractures, we performed a genetic assessment to investigate this hypothesis. METHODS: Medical history was obtained from the patient and family members, including biochemical, RMI and DXA assessments. Next-generation sequencing of COL1A1, COL1A2, COL2A1, CASR, CYP19A1, CUL7, CRTAP, KAL1, LEPRE1, LRP5, PPIB and SLC9A3R1, genes involved in juvenile osteoporosis, was performed. RESULTS: We identified a novel de novo heterozygous missense variant, c.488G > A, in exon 11 of the COL1A2 gene (NM_000089.3), resulting in the putative p.Gly163Asp substitution in the N-terminal part of the helical domain of type I collagen. The variant was predicted to be damaging by the in silico prediction tools and the mutation was therefore classified as likely pathogenic. This mutation can affect skeletal health impairing bone mass and trabecular mechanical competence, inducing a disease whose features strictly evoke a TRO. CONCLUSION: The present study describes a novel de novo heterozygous missense variant in COL1A2 gene, possibly inducing a propensity to trabecular microfractures. The recurrent symptomatic bone marrow oedema episodes could be the clinical picture consistent with the hypothesis of an inherited connective tissue disorder giving bone fragility.

A balanced reciprocal translocation t(10;15)(q22.3;q26.1) interrupting ACAN gene in a family with proportionate short stature.

PURPOSE: Few examples of the involvement of a single gene in idiopathic short stature have been described until now. Our aim was to identify the causative gene of proportionate short stature in a large family showing co-segregation of the phenotype with the reciprocal translocation t(10;15)(q22;q24). METHODS: FISH mapping was carried out with BACs and long-range PCR probes to identify the smallest genomic regions harboring the translocation breakpoints. Real-Time RT-PCR was performed in blood after pre-amplification of target genes cDNA. RESULT: The affected family members presented with a final height of between - 2.41 and - 4.18 SDS and very mild skeletal dysmorphisms. Growth rates of the proband and of her cousin, whose childhood and pre-pubertal bone age corresponded to the chronological age, showed a poor growth spurt during treatment with rhGH. However, their adult height was greater than that of their untreated mothers, suggesting efficacy of GH therapy. Breakpoint mapping revealed that the translocation t(10;15)(q22.3;q26.1) disrupts, on 15q, the ACAN gene at intron 1, decreasing its transcriptional expression. CONCLUSIONS: This is the first description of a chromosome rearrangement disrupting ACAN and leading to its haploinsufficiency. ACAN loss of function should be considered a potential underpinning of short patients who display a poor growth spurt and belong to families with autosomal dominant segregation of proportionate short stature. Besides this core phenotype, literature review suggests that advanced bone age, early onset osteochondritis dissecans, osteoarthritis, intervertebral disc disease as well as craniofacial dysmorphisms can be important suggestive phenotypes in affected families.

Identification of a New Mutation (L46P) in the Human NOG Gene in an Italian Patient with Symphalangism Syndrome.

Proximal symphalangism (SYM1) is a joint morphogenesis disorder characterized by stapes ankylosis, proximal interphalangeal joint fusion, skeletal anomalies and conductive hearing loss. Noggin is a bone morphogenetic protein (BMP) antagonist essential for normal bone and joint development in humans and mice. Autosomal dominant mutations have been described in the NOG gene, encoding the noggin protein. We analyzed an Italian sporadic patient with SYM1 due to a novel NOG mutation (L46P) based on a c.137T>C transition. A different pathogenic mutation in the same codon (L46D) has been previously described in an in vivo chicken model. An in silico model shows a decreased binding affinity between noggin and BMP7 for both L46D and L46P compared to the wild type. Therefore, this codon should play an important role in BMP7 binding activity of the noggin protein and consequently to the joint morphogenesis.

Novel mutations in the CDKL5 gene, predicted effects and associated phenotypes.

It has been found that CDKL5 gene mutations are responsible for early-onset epilepsy and drug resistance. We screened a population of 92 patients with classic/atypical Rett syndrome, 17 Angelman/Angelman-like patients and six idiopathic autistic patients for CDKL5 mutations and exon deletions and identified seven novel mutations: six in the Rett subset and one in an Angelman patient. This last, an insertion in exon 11, c.903_904 dupGA, p.Leu302Aspfx49X, is associated with a relatively mild clinical presentation as the patient is the only one capable of sitting and walking alone. Of the six mutations, two are de novo missense changes affecting highly conserved aminoacid residues, c.215 T > C p.Ile72Thr and c.380A > G p.His127Arg (present in a mosaic condition) found in two girls with the most severe clinical presentation, while the remaining are the splicing c.145 + 2 T > C and c.2376 + 5G > A, the c.1648C > T p.Arg550X and the MPLA-identified c.162_99del261 mutation. RNA characterisation of four mutations revealed the aberrant transcript of the missense allele (case 2) and not the stop mutation (case 3), but also allowed the splicing mutation (case 1) and the c.-162_99del261 (case 4) to be categorised as truncating. The obtained data reinforce the view that a more severe phenotype is due more to an altered protein than haploinsufficiency. Furthermore, the mutational repertoire of the CDKL5 gene is shown to be expanded by testing patients with phenotypical overlap to Rett syndrome and applying multiplex ligation-dependent probe amplification.

Disruption of friend of GATA 2 gene (FOG-2) by a de novo t(8;10) chromosomal translocation is associated with heart defects and gonadal dysgenesis.

FOG-2 (Friend of GATA 2) is a transcriptional cofactor able to differentially regulate the expression of GATA-target genes in different promoter contexts. Mouse models evidenced that FOG-2 plays a role in congenital heart disease and normal testis development. In human, while FOG-2 mutations have been identified in sporadic cases of tetralogy of Fallot, no mutations are described to be associated with impaired gonadal function. We here describe a young boy with a balanced t(8;10)(q23.1;q21.1) translocation who was born with congenital secundum-type atrial septal defect and gonadal dysgenesis. Fluorescence in situ hybridization mapped the chromosome 8 translocation breakpoint (bkp) to within the IVS4 of the FOG-2 gene, whereas the chromosome 10 bkp was found to lie in a desert gene region. Quantitative analysis of FOG-2 expression revealed the presence of a truncated transcript but there was no detectable change in the expression of the genes flanking the 10q bkp, thus making it possible to assign the observed clinical phenotype to altered FOG-2 expression. Genetic and clinical analyses provide insights into the signaling pathways by which FOG-2 affects not only cardiac development but also gonadal function and its preservation.

Molecular and genomic characterisation of cryptic chromosomal alterations leading to paternal duplication of the 11p15.5 Beckwith-Wiedemann region.

BACKGROUND: Beckwith-Wiedemann syndrome (BWS) is an overgrowth disorder with increased risk of paediatric tumours. The aetiology involves epigenetic and genetic alterations affecting the 11p15 region, methylation of the differentially methylated DMR2 region being the most common defect, while less frequent aetiologies include mosaic paternal 11p uniparental disomy (11patUPD), maternally inherited mutations of the CDKN1C gene, and hypermethylation of DMR1. A few patients have cytogenetic abnormalities involving 11p15.5. METHODS: Screening of 70 trios of BWS probands for 11p mosaic paternal UPD and for cryptic cytogenetic rearrangements using microsatellite segregation analysis identified a profile compatible with paternal 11p15 duplication in two patients. RESULTS: Fluorescence in situ hybridisation analysis revealed in one case the unbalanced translocation der(21)t(11;21)(p15.4;q22.3) originated from missegregation of a cryptic paternal balanced translocation. The second patient, trisomic for D11S1318, carried a small de novo dup(11)(p15.5p15.5), resulting from unequal recombination at paternal meiosis I. The duplicated region involves only IC1 and spares IC2/LIT1, as shown by fluorescent in situ hybridisation (FISH) mapping of the proximal duplication breakpoint within the amino-terminal part of KvLQT1. CONCLUSIONS: An additional patient with Wolf-Hirschorn syndrome was shown by FISH studies to carry a der(4)t(4;11)(p16.3;p15.4), contributed by a balanced translocation father. Interestingly, refined breakpoint mapping on 11p and the critical regions on the partner 21q and 4p chromosomal regions suggested that both translocations affecting 11p15.4 are mediated by segmental duplications. These findings of chromosomal rearrangements affecting 11p15.5-15.4 provide a tool to further dissect the genomics of the BWS region and the pathogenesis of this imprinting disorder.

Characterization of the genomic structure of the human neuronal nicotinic acetylcholine receptor CHRNA5/A3/B4 gene cluster and identification of novel intragenic polymorphisms.

Genes coding for the alpha5, alpha3, and beta4 subunits (CHRNA5, CHRNA3, and CHRNB4) of the neuronal nicotinic acetylcholine receptors (nAChRs) are clustered on chromosome 15q24. Linkage of this chromosomal region to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), an idiopathic partial epilepsy, was reported in one family. Moreover, mutations in other neuronal nAChR subunit genes coding for the alpha4 (CHRNA4) and the beta2 (CHRNB2) subunits were associated with ADNFLE. Apart from the exon-intron structure of CHRNA3, the genomic organization of this gene cluster was unknown, making comprehensive mutational analyses impossible. The genomic structure of CHRNA5 and CHRNB4 is here reported. Moreover, two hitherto unknown introns were identified within the 3' untranslated region of CHRNA3, causing a partial tail-to-tail overlap with CHRNA5. Four novel intragenic polymorphisms were identified and characterized in the cluster.

A new biallelic polymorphism in intron 1 of the CHRNA4 gene may cause erroneous genotyping of a closely linked CA repeat marker.

A new polymorphism in intron 1 of the neuronal nicotinic acetylcholine receptor alpha 4 subunit gene (CHRNA4) was identified. It consists of a G to T substitution located in the downstream flanking region of a previously reported CA repeat marker. This polymorphism whose frequency is about six percent in a control population occurs near the 3' end of the reverse primer generally used to type the CA repeat marker. Data are presented showing that the newly identified polymorphism causes erroneous genotyping of the CA repeat marker which can alter the results of linkage analysis for CHRNA4. The use of a different reverse primer located 34 nt downstream of the published sequence overcame errors in genotyping and identified two novel alleles of the CA repeat marker. Re-typing of the marker with the new proposed primer pair in a Caucasian control population of 107 unrelated individuals was also performed

Refined mapping of CHRNA3/A5/B4 gene cluster and its implications in ADNFLE.

The chromosome 15q24 region, containing the CHRNA3/A5/B4 gene cluster, coding for the alpha3, alpha5 and beta4 subunits of neuronal nicotinic acetylcholine receptors, has been reported to be linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) in one family. However, nor the gene nor the mutation involved have been identified. We report the refined mapping of CHRNA3/A5/B4 cluster. Segregation analyses of CHRNA3/A5/B4 polymorphisms in families showing recombinations for 15q24 G¿en¿ethon STR markers allowed to position the cluster in a 0.6 cM interval, between STRs D15S1027 and D15S1005. This location is external to the 15q24-ADNFLE-linked region, therefore excluding the involvement of this cluster in the pathogenesis of ADNFLE in the 15q24-linked family. Moreover, these data provide more precise information for further linkage studies.

SER252PHE and 776INS3 mutations in the CHRNA4 gene are rare in the Italian ADNFLE population.

41 patients (19 sporadic and 22 familial) affected by autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) were analyzed for the presence of two mutations (Ser252Phe, 776ins3) in the CHRNA4 gene, reported to be associated with this disease. Electroclinical findings of sporadic forms were indistinguishable from familial ones. In none of the patients, these mutations were found by dot blot analysis with allele specific oligonucleotides. These data, obtained on the largest group so far studied, suggest the rarity of the reported mutations.

Autosomal dominant nocturnal frontal lobe epilepsy. A video-polysomnographic and genetic appraisal of 40 patients and delineation of the epileptic syndrome.

A number of clinical and aetiological studies have been performed, during the last 30 years, on patients with abnormal nocturnal motor and behavioural phenomena. The aetiological conclusions of these studies were often conflicting, suggesting either an epileptic or a non-epileptic origin. Among the clinical characteristics of these patients, the familial clustering was one thoroughly accepted. A nocturnal familial form of frontal lobe epilepsy (autosomal dominant nocturnal frontal lobe epilepsy, ADNFLE), often misdiagnosed as parasomnia, has been recently described in some families. In one large Australian kindred, a missense mutation in the second transmembrane domain of the neuronal nicotinic acetylcholine receptor alpha 4 subunit (CHRNA4) gene, located on chromosome 20 q13.2-13.3, has been reported to be associated with nocturnal frontal lobe epilepsy. We performed an extensive clinical and video-polysomnographic study in 40 patients complaining of repeated abnormal nocturnal motor and/or behavioural phenomena, from 30 unrelated Italian families. Thirty-eight patients had an electroclinical picture strongly suggesting the diagnosis of ADNFLE. They had a wide clinical spectrum, ranging from nocturnal enuresis to sleep-related violent behaviour, thus including all the main features of the so-called 'typical' parasomnias. The video-polysomnographic recording confirmed the wide spectrum of abnormal manifestations, including sudden awakenings with dystonic/ dyskinetic movements (in 42.1% of patients), complex behaviours (13.2%) and sleep-related violent behaviour (5.3%). The EEG findings showed ictal epileptiform abnormalities predominantly over frontal areas in 31.6% of patients. In another 47.4% of patients the EEG showed ictal rhythmic slow activity over anterior areas. Only 18.4% of the patients had already received a correct diagnosis of epilepsy. In 73.3% of the patients treated with anti-epileptic drugs the seizures were readily controlled. Pedigree analysis on 28 of the families was consistent with autosomal dominant transmission with reduced penetrance (81%). DNAs from 20 representative affected individuals were sequenced in order to check for the presence of the missense mutation in the CHRNA4 gene found in the Australian kindred affected by ADNFLE. Nucleotide sequence analysis did not reveal the presence of this mutation, but it did confirm the presence of two other base substitutions, not leading to amino acid changes. These two intragenic polymorphisms, together with a closely linked restriction fragment length polymorphism at the D20S20 locus, have been used for linkage analysis of ADNFLE to the terminal region of the long arm of chromosome 20 in five compliant families. The results allowed us to exclude linkage of ADNFLE to this chromosomal region in these families, thus confirming the locus heterogeneity of the disorder. Large and full video-polysomnographical studies are of the utmost importance in order to clarify the real prevalence of both nocturnal frontal lobe epilepsy and parasomnias, and to provide a correct therapy.