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Deleterious variants of DYNC2H1 gene are associated with a wide spectrum of skeletal ciliopathies (SC). We used targeted parallel sequencing to analyze 25 molecularly unsolved families with different SCs. Deleterious DYNC2H1 variants were found in six sporadic patients and two monozygotic (MZ) twins. Clinical diagnoses included short rib-polydactyly type 3 in two cases, and asphyxiating thoracic dystrophy (ATD) in one case. Remarkably, clinical diagnosis fitted with EvC, mixed ATD/EvC and short rib-polydactyly/EvC phenotypes in three sporadic patients and the MZ twins. EvC/EvC-like features always occurred in compound heterozygotes sharing a previously unreported splice site change (c.6140-5A>G) or compound heterozygotes for two missense variants. These results expand the DYNC2H1 mutational repertoire and its clinical spectrum, suggesting that EvC may be occasionally caused by DYNC2H1 variants presumably acting as hypomorphic alleles.

Clinical expression of Ellis‐van Creveld syndrome (EvC) is variable and mild phenotypes have been described, including patients with mostly cardiac and limb involvement. Whether these cases are part of the EvC phenotypic spectrum or separate conditions is disputed. Herein, we describe a family with vertical transmission of atrioventricular canal defect (AVCD), common atrium, and postaxial polydactyly. Targeted sequencing of EVC, EVC2, WDR35, DYNC2LI1, and DYNC2H1 identified different compound heterozygosity in EVC genotypes in the two affected members, consisting of a nonsense (p.Arg622Ter) and a missense (p.Arg663Pro) variant in the father, and the same nonsense variant and a noncanonical splice‐site in‐frame change (c.1316–7A>G) in the daughter. Complementary DNA sequencing, immunoblot, and immunofluorescence experiments using patient‐derived fibroblasts and Evc–/– mouse embryonic fibroblasts showed that p.Arg622Ter is a loss‐of‐function mutation, whereas p.Arg663Pro and the splice‐site change c.1316–7A>G are hypomorphic variants resulting in proteins that retain, in part, the ability to complex with EVC2. Our molecular and functional data demonstrate that at least in some cases the condition characterized as “common atrium/AVCD with postaxial polydactyly” is a mild form of EvC due to hypomorphic EVC mutations, further supporting the occurrence of genotype‐phenotype correlations in this syndrome., This study was supported by funding from the Italian Ministry of Health (RC‐2019) to Alessandro De Luca, Fondazione Bambino Gesù (Vite Coraggiose) to Marco Tartaglia, and the Spanish Ministry of Science, Innovation and Universities to Victor L. Ruiz‐Perez (SAF2016‐75434‐R (AEI/FEDER, UE) and PID2019‐105620RB‐I00/AEI/10.13039/501100011033).

PRKACA and PRKACB code for two catalytic subunits (Cα and Cβ) of cAMP-dependent protein kinase (PKA), a pleiotropic holoenzyme that regulates numerous fundamental biological processes such as metabolism, development, memory, and immune response. We report seven unrelated individuals presenting with a multiple congenital malformation syndrome in whom we identified heterozygous germline or mosaic missense variants in PRKACA or PRKACB. Three affected individuals were found with the same PRKACA variant, and the other four had different PRKACB mutations. In most cases, the mutations arose de novo, and two individuals had offspring with the same condition. Nearly all affected individuals and their affected offspring shared an atrioventricular septal defect or a common atrium along with postaxial polydactyly. Additional features included skeletal abnormalities and ectodermal defects of variable severity in five individuals, cognitive deficit in two individuals, and various unusual tumors in one individual. We investigated the structural and functional consequences of the variants identified in PRKACA and PRKACB through the use of several computational and experimental approaches, and we found that they lead to PKA holoenzymes which are more sensitive to activation by cAMP than are the wild-type proteins. Furthermore, expression of PRKACA or PRKACB variants detected in the affected individuals inhibited hedgehog signaling in NIH 3T3 fibroblasts, thereby providing an underlying mechanism for the developmental defects observed in these cases. Our findings highlight the importance of both Cα and Cβ subunits of PKA during human development., This work was partially supported by funding from the Spanish Ministry of Science, Innovation and Universities (SAF2016-75434-R [AEI/FEDER, UE] and PID2019-105620RB-I00/AEI/10.13039/501100011033) to V.L.R.-P. S.S.T. was supported by NIH grant R03TR002947, E.M.F.M. by Kassel graduate school “Clocks”, and A.D.L. by the Italian Ministry of Health (RC-2019). The University of Antwerp supported G.M. and W.V.H. with Methusalem funding (FFB190208) and S.P. with a predoctoral grant. E.B. was supported by The Research Foundation Flanders with a postdoctoral grant (12A3814N). The study was also funded by a National Health and Medical Research Council Program Grant (1091593) to I.E.S., a Practitioner Fellowship (1006110) to I.E.S., a Senior Research Fellowship (1102971) to M.B., and an R.D. Wright Career Development Fellowship (1063799) to M.S.H. B.S.S. and G.L. were supported by Throne Holst Foundation UiO (2019-2021) and Strategic PhD fund by UiO/IMB.

DNA methylation in the human genome is largely programmed and shaped by transcription factor binding and interaction between DNA methyltransferases and histone marks during gamete and embryo development. Normal methylation profiles can be modified at single or multiple loci, more frequently as consequences of genetic variants acting in cis or in trans, or in some cases stochastically or through interaction with environmental factors. For many developmental disorders, specific methylation patterns or signatures can be detected in blood DNA. The recent use of high-throughput assays investigating the whole genome has largely increased the number of diseases for which DNA methylation analysis provides information for their diagnosis. Here, we review the methylation abnormalities that have been associated with mono/oligogenic diseases, their relationship with genotype and phenotype and relevance for diagnosis, as well as the limitations in their use and interpretation of results.

Ellis-van Creveld syndrome (EvC) is a chondral and ectodermal dysplasia caused by biallelic mutations in the EVC, EVC2 and WDR35 genes. A proportion of cases with clinical diagnosis of EvC, however, do not carry mutations in these genes. To identify the genetic cause of EvC in a cohort of mutation-negative patients, exome sequencing was undertaken in a family with 3 affected members, and mutation scanning of a panel of clinically and functionally relevant genes was performed in 24 additional subjects with features fitting/overlapping EvC. Compound heterozygosity for the c.2T>C (p.Met1?) and c.662C>T (p.Thr221Ile) variants in DYNC2LI1, which encodes a component of the intraflagellar transport-related dynein-2 complex previously found mutated in other short-rib thoracic dysplasias, was identified in the 3 affected members of the first family. Targeted resequencing detected compound heterozygosity for the same missense variant and a truncating change (p.Val141*) in 2 siblings with EvC from a second family, while a newborn with a more severe phenotype carried 2 DYNC2LI1 truncating variants. Our findings indicate that DYNC2LI1 mutations are associated with a wider clinical spectrum than previously appreciated, including EvC, with the severity of the phenotype likely depending on the extent of defective DYNC2LI1 function.

Previous reports summarized the seizure types occurring in patients with idic(15) syndrome. To better define this issue, we retrospectively analyzed the evolution of electroencephalogram findings and seizures in 35 patients with confirmed idic(15). Epilepsy occurred in 28 patients (80%), with a median age of onset of 3 years 3 months. The initial seizures were infantile spasms associated with a hypsarrhythmic electroencephalogram (nine patients), focal/generalized tonic (seven patients), or atypical absences (eight patients). High doses of oral steroids were given in all nine children with infantile spasms, with remission of seizures and resolution of electroencephalogram abnormalities. Among them, three were seizure free at the time of evaluation, but six later developed Lennox-Gastaut syndrome or Lennox-Gastaut-like syndrome. The eight patients with atypical absences developed Lennox-Gastaut syndrome or Lennox-Gastaut-like syndrome. Epilepsy was well controlled in 32% of the patients; satisfactorily controlled (seizures reduced >75%) in 21.4%; partially controlled (seizures reduced

We report the detailed clinical presentation and molecular features of a spinal neurofibromatosis familial case where a 40-year-old woman, presenting with multiple bilateral spinal neurofibromas and no other clinical feature of neurofibromatosis type 1 (NF1), inherited a paternal large multiexonic deletion (c.5944-?_7126+?del) which resulted in NF1 gene haploinsufficiency at the RNA level. In the clinically unaffected 73-year-old father, spinal cord MRI disclosed bilateral and symmetrical hypertrophy of spinal lumbosacral roots. Our study widens the phenotypic and mutational spectrum of NF1 and illustrates the difficulties of counseling patients with border-line or atypical presentation of this disorder.

Context: Type 2 deiodinase (D2) converts T4 in T3 in several human tissues, including hypothalamus and pituitary, and, therefore, plays a pivotal role in the negative feedback regulation of TSH secretion. A common variant of the gene, threonine (Thr) 92 alanine (Ala), has been identified and associated with decreased D2 enzymatic activity. Objective: Our objective was to investigate whether this polymorphism predicts the T4 dosage needed to obtain target TSH levels in thyroidectomized patients. Setting: Ambulatory patients were included in the study. Patients: A total of 191 consecutive thyroid cancer patients, previously treated by near total thyroidectomy and radioiodine ablation, were studied. They were on stable T4 dose treatment aimed at obtaining either suppressed (supp) (n = 117, < 0.1 mU/liter) or near-supp (n = 74, ≥ 0.1 < 0.5 mU/liter) serum TSH levels. Main Outcome Measures: DNA genotyping for D2 Thr92Ala variant and evaluation of T4 dose (μg/kg) needed to obtain target TSH levels were determined. Results: Ala/Ala homozygous patients needed a higher T4 dose as compared with patients carrying the Thr92 variant (X/Thr patients) according to a recessive genetic model (2.08 ± 0.43 vs. 1.90 ± 0.35 μg/kg; P < 0.05). This difference was observable in the near-supp group (P = 0.002), but not in the supp group (P = 0.4). Conclusions: D2 Thr92Ala polymorphism seems to predict the need for higher T4 intake in thyroidectomized patients. If this finding is confirmed in additional studies, it may predict the T4 requirement to suppress TSH on the basis of the individual genetic background.

Analysis of 786 NF1 mutation-positive subjects with clinical diagnosis of neurofibromatosis type 1 (NF1) allowed to identify the heterozygous c.5425C>T missense variant (p.Arg1809Cys) in six (0.7%) unrelated probands (three familial and three sporadic cases), all exhibiting a mild form of disease. Detailed clinical characterization of these subjects and other eight affected relatives showed that all individuals had multiple cafe-au-lait spots, frequently associated with skinfold freckling, but absence of discrete cutaneous or plexiform neurofibromas, Lisch nodules, typical NF1 osseous lesions or symptomatic optic gliomas. Facial features in half of the individuals were suggestive of Noonan syndrome. Our finding and revision of the literature consistently indicate that the c.5425C>T change is associated with a distinctive, mild form of NF1, providing new data with direct impact on genetic counseling and patient management.

Familial spinal neurofibromatosis (FSNF) is a rare form of neurofibromatosis type 1 (NF1) characterized by multiple, histologically proven neurofibromas of the spinal roots leaving no intact segments and associated neurofibromas of major peripheral nerves. It is sometimes associated with other NF1 stigmata. Most patients have NF1 gene mutations. We describe a patient who fulfilled the diagnostic criteria for spinal neurofibromatosis and belonged to a family in which other affected members exhibited classical NF1 stigmata. A novel missense (c.7109 T > A; p.Val2370Asp) mutation in exon 39 of the NF1 gene was present in the affected family members. The family displayed extreme phenotypic variability in the spectrum of NF1. To our knowledge, this is the first patient with spinal neurofibromatosis in the context of classical NF1 with an NF1 gene mutation. The term FSNF is inaccurate as this condition simply reflects the typical autosomal dominant pattern of NF1 inheritance with phenotypoc variability and does not encompass patients with sporadic disease or those in the context of a classical NF1 phenotype as reported in the present family. The term could be replaced by “spinal neurofibromatosis”.

X-linked Retinitis Pigmentosa (XLRP) shows a huge genetic heterogeneity with almost five distinct loci on the X chromosome. So far, only two XLRP genes have been identified, RPGR (or RP3) and RP2, being mutated in approximately 70% and 10% of the XLRP patients. Clinically there is no clearly significative difference between RP3 and RP2 phenotypes. In the attempt to assess the degree of involvement of the RP2 gene, we performed a complete mutation analysis in a cohort of patients and we identified five novel mutations in five different XLRP families. These mutations include three missense mutations, a splice site mutation, and a single base insertion, which, because of frameshift, anticipates a stop codon. Four mutations fall in RP2 exon 2 and one in exon 3. Evidence that such mutations are different from the 21 RP2 mutations described thus far suggests that a high mutation rate occurs at the RP2 locus, and that most mutations arise independently, without a founder effect. Our mutation analysis confirms the percentage of RP2 mutations detected so far in populations of different ethnic origin. In addition to novel mutations, we report here that a deeper sequence analysis of the RP2 product predicts, in addition to cofactor C homology domain, further putative functional domains, and that some novel mutations identify RP2 amino acid residues which are evolutionary conserved, hence possibly crucial to the RP2 function.

We have examined the relationship between the common dopamine D4 receptor (DRD4) exon III repeat polymorphism and infants' behavior measured with the Italian version of the Early and Revised Infancy Temperament Questionnaires (EITQ/RITQ) in 122 Italian neonates at 1 and 5 months of life, when the genetic contribution to the behavior can be more clearly assessed. Two-way (genotype x age) analysis of variance revealed a significant correlation with the temperamental subscale of adaptability [F(1, 120) = 5.26, P0.02]. At 1 month of life (early assessment), infants with long (L) DRD4 alleles presented significantly low scores (L 2.61 +/- 0.073; S 2.84 + 0.79; Newman-Keuls P = 0.03) in comparison with the high scores of infants with short (S) alleles (L 2.4 +/- 0.059; S 2.25 +/- 0.57). These differences were not detected at 5 months of life (late assessment), denoting a strong environmental effect at this age on the genetic background. These results confirm and extend the genetic influence of the DRD4 gene in human temperament at birth.

SummaryPatella aplasia-hypoplasia (PTLAH) is a rare genetic defect characterized by congenital absence or marked reduction of the patella. PTLAH can occur either as an isolated defect or in association with other malformations, and it characteristically occurs in the nail-patella syndrome and in some chromosome imbalances. We report the first evidence of linkage for isolated PTLAH in an extended Venezuelan family. After exclusion of the candidate chromosome regions where disorders associated with PTLAH have been mapped, a genomewide scan was performed that supported mapping of the disease locus within a region of 12 cM on chromosome 17q22. Two marker loci (D17S787 and D17S1604) typed from this region gave maximum LOD scores >3. Accordingly, multipoint analysis gave a maximum LOD score of 3.39, with a most likely location for the disease gene between D17S787 and D17S1604. Sequencing of the noggin gene, a candidate mapping between these markers, failed to reveal any mutation in affected subjects.

Mutations or exon deletions of the epsilon-sarcoglycan (SGCE) gene cause myoclonus-dystonia (M-D), but a subset of M-D patients are mutation-negative and the sensitivity and specificity of current genetic testing criteria are unknown. We screened 46 newly enrolled M-D patients for SGCE mutations and deletions; moreover, 24 subjects previously testing negative for SGCE mutations underwent gene dosage analysis. In our combined cohorts, we calculated sensitivity, specificity, positive and negative predictive values, and area under the curve of 2 published sets of M-D diagnostic criteria. A stepwise logistic regression was used to assess which patients' characteristics best discriminated mutation carriers and to calculate a new mutation predictive score ("new score"), which we validated in previously published cohorts. Nine of 46 (19.5%) patients of the new cohort carried SCGE mutations, including 5 novel point mutations and 1 whole-gene deletion; in the old cohort, 1 patient with a complex phenotype carried a 5.9-Mb deletion encompassing SGCE. Current diagnostic criteria had a poor ability to discriminate SGCE-positive from SGCE-negative patients in our cohort; conversely, age of onset, especially if associated with psychiatric features (as included in the new score), showed the best discriminatory power to individuate SGCE mutation carriers, both in our cohort and in the validation cohort. Our results suggest that young age at onset of motor symptoms, especially in association with psychiatric disturbance, are strongly predictive for SGCE positivity. We suggest performing gene dosage analysis by multiple ligation-dependent probe amplification (MLPA) to individuate large SGCE deletions that can be responsible for complex phenotypes.

PubMed ID: 23924873, [No abstract available]

Ellis van Creveld syndrome and Weyers acrofacial dysostosis are allelic disorders caused by mutations in EVC or EVC2 genes. We illustrate the results of direct analysis of whole EVC and EVC2 genes' coding regions in 32 unrelated families with clinical diagnosis of Ellis van Creveld syndrome and in 2 families with Weyers acrofacial dysostosis. We identified mutations in 27/32 (84%) cases with Ellis van Creveld syndrome and 2/2 cases with Weyers acrofacial dysostosis. Of the Ellis van Creveld syndrome cases, 20/27 (74%) had a mutation in EVC and 7/27 (26%) in EVC2 genes. The two subjects with Weyers acrofacial dysostosis had a heterozygous mutation in the last exon of EVC2. In total, we detected 25 independent EVC and 11 independent EVC2 mutations. Nineteen EVC mutations (19/25, 76%) and 4 EVC2 mutations (4/11, 36%) were novel. Also one EVC2 gene mutation found in Weyers acrofacial dysostosis was novel. In 5 unrelated cases with a clinical diagnosis of Ellis van Creveld syndrome, we did not find any mutation in either EVC or EVC2 genes. Current findings expand the Ellis van Creveld syndrome and Weyers acrofacial dysostosis mutation spectra, and provide further evidence that the last exon of EVC2 gene is a hot spot for Weyers acrofacial dysostosis mutations. Accordingly, EVC2 exon 22 should be analyzed with priority by mutation screening in individuals with a suspected diagnosis of Weyers acrofacial dysostosis.

Given that TP53 alterations predict prognosis and response to therapy in chronic lymphocytic leukemia (CLL), screening for TP53 mutations has an increasing role in patient management. TP53 direct sequencing is a time-consuming method, while the AmpliChip p53 Research Test is a novel non time-consuming microarray-based resequencing assay and queries Exons 2–11. We evaluated the impact of TP53 mutations on clinical outcome by analyzing 98 untreated CLL using the AmpliChip p53 Research Test and direct sequencing and performed microarrays analysis on TP53 mutated and/or deleted cases. The AmpliChip p53 Research Test detected 17 mutations in 14 patients (17.3%); a significant association between TP53 mutations and del(17p) was recorded. From a clinical standpoint, a higher percentage of mutation was found in CLL with unfavorable outcome (17.2% vs. 7.1% in progressive vs. stable cases). Detection of TP53 mutations by the AmpliChip p53 Research Test was associated with a significantly worse survival (P = 0.0002). Comparison of the array and direct sequencing tests showed that the p53 Research Test detected more mutations, although it failed to identify two microdeletions. Finally, microarrays analysis showed a more distinctive signature associated with del(17p) than with TP53 mutations, likely due to a concomitant gene dosage effect. The AmpliChip p53 Research Test is a straightforward method that bears prognostic value. This study confirms a high percentage of TP53 mutations in CLL with unfavorable outcome and a significant association between TP53 aberrations and del(17p). Finally, specific gene expression profiles are recognized for TP53 alterations. © 2011 Wiley-Liss, Inc.

Transitory depression of the compound muscle action potential during repetitive nerve stimulation is a well-documented neurophysiologic finding in recessive myotonia congenita. It represents the neurophysiologic counterpart of the transitory weakness often impairing patients at the beginning of a movement after rest, and it is usually better induced using high-rate nerve stimulations. The authors examined 30 patients with recessive myotonia congenita and carried out a 3 Hz nerve stimulation study to ascertain to what extent this protocol was able to detect the occurrence of transitory depression. Their findings were compared with the results obtained by 12 patients affected by dominant myotonia congenita and 12 patients affected by nondystrophic myotonia due to SCN4A mutations. Molecular genetic analysis of the CLCN1 and SCN4A genes was also performed. The 3 Hz nerve stimulation protocol was well tolerated and showed high sensitivity, resulting positive in 66% of recessive case and good reproducibility, if performed after an adequate period of rest. All dominant cases and all patients affected by myotonia due to SCN4A mutations showed negative results. Molecular studies identified 26 different CLCN1 mutations, 16 of which were novel. Transitory depression confirmed to vary in accordance to CLCN1 mutations. The 3 Hz protocol was well tolerated and showed good sensitivity and reproducibility. Furthermore, this test might be suitable for genotype-phenotype correlation studies.

Anecdotal cases of polymicrogyria (PMG; a malformation of cortical development consisting of an excessive number of small gyri with abnormal lamination) in patients with neurofibromatosis type 1 (NF1) have been described; however, the cases were unilateral and had negative NF1 genetic testing. We describe an 11-year-old girl with NF1 manifesting as a complex epileptic syndrome, including partial seizures secondarily generalized and status epilepticus, who had in association, bilateral, asymmetrical (opercular and paracentral lobular) PMG. She had a 1-bp deletion (c.1862delC) in exon 12b of the NF1 gene. It is notable that, given the key role played by the NF1 gene product, neurofibromin, in normal brain development, and the relatively high frequency of other brain findings in NF1, there are not more NF1 cases with brain malformations manifesting as PMG. © 2011 Wiley-Liss, Inc.

Congenital bilateral absence of vas deferens (CBAVD) is a manifestation of the mildest form of cystic fibrosis (CF) and is characterized by obstructive azoospermia in otherwise healthy patients. Owing to the availability of assisted reproductive technology, CBAVD patients can father children. These fathers are at risk of transmitting a mutated allele of the CF transmembrane conductance regulator (CFTR) gene, responsible for CF, to their offspring. The identification of mutations in both CFTR alleles in CBAVD patients is a crucial requirement for calculating the risk of producing a child with full-blown CF if the female partner is a healthy CF carrier. However, in the majority of CBAVD patients, conventional mutation screening is not able to detect mutations in both CFTR alleles, and this difficulty hampers the execution of correct genetic counselling. To obtain information about the most represented CFTR mutations in CBAVD patients, we analysed 23 CBAVD patients, 15 of whom had a single CFTR mutation after screening for 36 mutations and the 5T allele. The search for the second CFTR mutation in these cases was performed by using a triplex approach: (i) first, a reverse dot-blot analysis was performed to detect mutations with regional impact; (ii) next, multiple ligation-dependent probe amplification assays were conducted to search for large rearrangements; and (iii) finally, denaturing high-performance liquid chromatography was used to search for point mutations in the entire coding region. Using these approaches, the second CFTR mutation was detected in six patients, which increased the final detection rate to 60.8%.

We report on the clinical and molecular features of a family in which neurofibromatosis type 1 (NF1) occurred in two of three siblings born to unaffected parents and in one granddaughter. Linkage analysis showed that the two affected siblings and the daughter of one of them shared the same paternal allele, whereas they had inherited different maternal alleles. We detected a disease-causing deletion (c.4773-3622-?_5749+?del) encompassing three NF1 gene exons in affected individuals. This mutation occurred on the paternally derived allele, arguing for a germline mosaicism in the probands' father. Real-time PCR showed that the mutation was present in about 10-17% of the paternal sperms. Current results confirm that germline mosaicism can explain the recurrence of NF1 in offspring of unaffected parents.

The aim of this study was to collect the practices of cytogenetic and molecular genetic testing and genetic counseling activities in Italy in the year 2007 and provide guidance to the national and regional health systems to improve the organization of genetic services.A web-based survey was carried out to assess the total number and the type of analyses, the number and type of genetic counseling sessions, and the personnel attending these activities. The quality management system of the responding structures, in terms of certification and accreditation standards, was also investigated. The appropriateness of requests for genetic testing was evaluated for six disorders.Data were collected from 278 responding centers, half of which were located in the northern regions of the country. Twenty-eight percent of the total were certified according to quality standards. A total of 217 molecular genetic and 171 cytogenetic laboratories, and 102 clinical genetic services were surveyed. About 560,000 genetic tests, including 311,069 cytogenetic and 248,691 molecular genetic analyses of 556 genes, were recorded. The fetal karyotype was examined on either trophoblast or amniocytes in about one of every 4.4 pregnancies. Only 11.5% of cytogenetic analyses and 13.5% of molecular tests were accompanied by genetic counseling. Concerning the appropriateness of a request for genetic testing, a low congruity was found between the clinical diagnosis and the laboratory results.This study highlights the need for reorganizing the genetic structure network in Italy, which at present is oversized, improving the quality management systems, expanding the availability of testing for rare disease genes, and improving access to pretest and posttest genetic counseling.

Summary We screened ATM gene mutations in 104 Italian Ataxia-Telangiectasia patients from 91 unrelated families (detection rate 90%) and found 21 recurrent mutations in 63 families. The majority (67%) of patients were compound heterozygotes, while 33% were homozygotes. To determine the existence of common haplotypes and potential founder effects, we analyzed five microsatellite markers within and flanking the ATM gene. Haplotype analysis was carried out in 48/63 families harbouring 16 of the 21 recurrent mutations. Forty different haplotypes were detected in the 48 A-T families studied. We found that the majority of patients with the same recurrent mutation originated from the same geographical area. All but one recurrent mutation analyzed displayed a common haplotype suggesting a single origin that then spread to different geographical areas. The high number of different haplotypes does not allow the screening of ATM mutations by haplotype analysis alone in the Italian population. The finding of recurrent public mutations without founder effect suggests the existence of ‘mild’ hot spots of mutation located along the sequence of the ATM gene.

Background Neurofibromatosis type 1 is one of the most common autosomal dominant disorders, affecting about 1:3,500 individuals. NF1 exon 7 displays weakly defined exon-intron boundaries, and is particularly prone to missplicing. Methods In this study we investigated the expression of exon 7 transcripts using bioinformatic identification of splicing regulatory sequences, and functional minigene analysis of four sequence changes [c.910C>T (R304X), c.945G>A/c.946C>A (Q315Q/L316M), c.1005T>C (N335N)] identified in exon 7 of three different NF1 patients. Results Our results detected the presence of three exonic splicing enhancers (ESEs) and one putative exonic splicing silencer (ESS) element. The wild type minigene assay resulted in three alternative isoforms, including a transcript lacking NF1 exon 7 (NF1ΔE7). Both the wild type and the mutated constructs shared NF1ΔE7 in addition to the complete messenger, but displayed a different ratio between the two transcripts. In the presence of R304X and Q315Q/L316M mutations, the relative proportion between the different isoforms is shifted toward the expression of NF1ΔE7, while in the presence of N335N variant, the NF1ΔE7 expression is abolished. Conclusion In conclusion, it appears mandatory to investigate the role of each nucleotide change within the NF1 coding sequence, since a significant proportion of NF1 exon 7 mutations affects pre-mRNA splicing, by disrupting exonic splicing motifs and modifying the delicate balance between aberrantly and correctly spliced transcripts.

Genetic counselling of patients with small supernumerary ring chromosomes (sSRCs) can be difficult, especially in prenatal testing, due to the complexity in establishing a karyotype-phenotype correlation. In fact, it has been estimated that about 10% of extra ring(1) chromosomes are associated with an unremarkable phenotype. We report on five new cases of extra ring chromosomes(1) manifesting different clinical outcome. One case was familial, segregating from a mother with mosaic karyotype, while the others were de novo. Ring chromosomes were characterised by FISH. In three subjects the involvement of the same euchromatic 1p region was demonstrated. Present observations corroborate previous results and provide some insight into the identification of the harmless ring(1) structures.

Noonan and LEOPARD syndromes are developmental disorders with overlapping features, including cardiac abnormalities, short stature and facial dysmorphia. Increased RAS signaling owing to PTPN11, SOS1 and KRAS mutations causes approximately 60% of Noonan syndrome cases, and PTPN11 mutations cause 90% of LEOPARD syndrome cases. Here, we report that 18 of 231 individuals with Noonan syndrome without known mutations (corresponding to 3% of all affected individuals) and two of six individuals with LEOPARD syndrome without PTPN11 mutations have missense mutations in RAF1, which encodes a serine-threonine kinase that activates MEK1 and MEK2. Most mutations altered a motif flanking Ser259, a residue critical for autoinhibition of RAF1 through 14-3-3 binding. Of 19 subjects with a RAF1 mutation in two hotspots, 18 (or 95%) showed hypertrophic cardiomyopathy (HCM), compared with the 18% prevalence of HCM among individuals with Noonan syndrome in general. Ectopically expressed RAF1 mutants from the two HCM hotspots had increased kinase activity and enhanced ERK activation, whereas non-HCM-associated mutants were kinase impaired. Our findings further implicate increased RAS signaling in pathological cardiomyocyte hypertrophy.

To estimate the contribution of single and multi-exon NF1 gene copy-number changes to the NF1 mutation spectrum, we analysed a series of 201 Italian patients with neurofibromatosis type 1 (NF1). Of these, 138 had previously been found, using denaturing high-performance liquid chromatography or protein truncation test, to be heterozygous for intragenic NF1 point mutations/deletions/insertions, and were excluded from this analysis. The remaining 63 patients were analysed using multiplex ligation-dependent probe amplification (MLPA), which allows detection of deletions or duplications encompassingor=1 NF1 exons, as well as entire gene deletions. MLPA results were validated using real-time quantitative PCR (qPCR) or fluorescent in situ hybridisation. MLPA screening followed by real-time qPCR detected a total of 23 deletions. Of these deletions, six were single exon, eight were multi-exon, and nine were of the entire NF1 gene. In our series, deletions encompassingor=1 NF1 exons accounted for approximately 7% (14/201) of the NF1 gene mutation spectrum, suggesting that screening for these should now be systematically included in genetic testing of patients with NF1.

Type 2 iodothyronine deiodinase (DIO2) converts thyroid prohormone tetraiodothyronine into the biologically active triiodothyronine hormone, which increases insulin sensitivity at the skeletal muscle level. The DIO2 T92A polymorphism modulates deiodinase activity and has been inconsistently associated with insulin resistance. Also, the P121A polymorphism of the peroxisome proliferator-activated receptor (PPAR) gamma2 gene, which encodes a transcription factor involved in insulin signaling, has been inconsistently associated with insulin resistance. This study was aimed at evaluating the combined effect of DIO2 T92A and PPARgamma2 P12A polymorphisms on insulin resistance-related features in 590 non-diabetic whites. A significant gene-gene interaction was observed in the modulation of systolic (p = 0.01) and diastolic (p = 0.02) blood pressure and metabolic syndrome (p = 0.02), with carriers of both DIO2 A92 and PPARgamma2 A12 variants showing the worst phenotype. This latter interaction was also shown by multifactor dimensionality reduction analysis (p = 0.0045). A peroxisome proliferator response element in the DIO2 promoter was identified by in silico analysis and confirmed by in vitro gel shift mobility assay, thus providing a biological plausibility for the observed gene-gene interaction. If confirmed in other populations, comprising several thousand individuals, these data may help identify individuals at risk for insulin resistance-related abnormalities.

The gene for ataxia-telangiectasia (A-T:MIM:#208900), ATM, spans about 150~kb of genomic DNA and is composed of 62 coding exons. ATM mutations are found along the entire coding sequence of the gene, without evidence of mutational hot spots. Using DNA as the starting material, we used denaturing high performance liquid chromatography (DHPLC) technique to search for ATM gene mutations. Initially, DHPLC was validated in a retrospective study of 16 positive control samples that included 19 known mutations; 100% of mutations were detected. Subsequently, DHPLC was used to screen for mutations a cohort of 22 patients with the classical form of A-T. A total of 27 different mutations were identified on 38 of the 44 alleles, corresponding to a 86% detection rate. Fourteen of the mutations were novel. In addition, 15 different variants and polymorphisms of unknown functional significance were found. The high incidence of new and individual A-T mutations in our cohort of patients demonstrates marked mutational heterogeneity of A-T in Italy and corroborate the efficiency of DHPLC as a method for the mutation screening of A-T patients.

Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by degeneration of the anterior horn cells of the spinal cord, causing symmetric proximal muscle weakness. SMA is classified in three clinical types, SMA I, SMA II, and SMA III, based on the severity of the symptoms and the age of onset. About 95% of SMA cases are caused by homozygous deletion of the survival motor neuron 1 (SMN1) gene (5q13), or its conversion to SMN2. The molecular diagnosis of this disease is usually carried out by a polymerase chain reaction-restriction fragment length polymorphism approach able to evidence the absence of both SMN1 copies. However, this approach is not able to identify heterozygous healthy carriers, which show a very high frequency in general population (1:50). We used the multiple ligation-dependent probe amplification (MLPA) approach for the molecular diagnosis of SMA in 19 affected patient and in 57 individuals at risk to become healthy carriers. This analysis detected the absence of the homozygous SMN1 in all the investigated cases, and allowed to discriminate between SMN1 deletion and conversion to SMN2 on the basis of the size showed by the peaks specific for the different genes mapped within the SMA critical region. Moreover, MLPA analysis evidenced a condition of the absence of the heterozygous SMN1 in 33 out of the 57 relatives of the affected patients, demonstrating the usefulness of this approach in the identification of healthy carriers. Thus, the MLPA technique represents an easy, low cost, and high throughput system in the molecular diagnosis of SMA, both in affected patients and in healthy carriers.

A comprehensive and long-range monitoring of genetic testing is ongoing in Italy starting from 1987. The data collected by the last survey of year 2004, on behalf of the Italian Society of Human Genetics, included the activities of 88 clinical centres and 160 cytogenetic and 183 molecular genetic laboratories, hosted by 256 structures. Only 42% of them fulfilled the requirements of current Italian legislation. Genetic tests included 283,601 cytogenetic analyses. There have been 120,238 invasive prenatal samplings, 84% of which were amniocenteses. A significant north-to-south decreasing gradient was evident for all activities. This study has also surveyed 190,610 molecular genetic tests. CFTR gene analysis accounted for 23% of prenatal and 29% of postnatal molecular tests. In total, 420 different genes have been investigated, 10 of which comprised three-quarters of the whole activity. More than 10% of molecular tests were performed on fetal samples, the analysis of CFTR, DMD, FMR1, FMR2, and GJB2 genes accounting for 83% of all prenatal tests. In years 1997-2004, the demand of cytogenetic tests has increased two-fold and that of molecular tests has increased four-fold. Only 16% of cytogenetic and 12.5% of molecular tests have been followed by genetic counselling. This survey highlights the need for a major basic intervention in the general organisation of genetic structures in Italy, which should be rationalised in accordance with the national guidelines, and the necessity of constant training of general practitioners and education of consumers to the appropriate use of genetic testing.

Marfan Syndrome (MFS) is an autosomal dominant disorder of the connective tissue due to mutations of Fibrillin-1 gene (FBN1) in more than 90% of cases and Transforming Growth Factor-Beta-Receptor2 gene (TGFB2R) in a minority of cases. Genotyping is relevant for diagnosis and genotype-phenotype correlations. We describe the FBN1 genotypes and related phenotypes of 81 patients who were referred to our attention for MFS or Marfan-like phenotypes. Patients underwent multidisciplinary pertinent evaluation in the adult or paediatric setting, according to their age. The diagnosis relied on Ghent criteria. To optimise DHPLC analysis of the FBN1 gene, all coding regions of the gene were directly sequenced in 19 cases and 10 controls: heterozygous amplicons were used as true positives. DHPLC sensitivity was 100%. Then, DHPLC was used to screen 62 other cases. We identified 74 FBN1 mutations in 81 patients: 64 were novel and 17 known. Of the 81 mutations, 41 were missense (50.6%), 27, either nonsense or frameshift mutations and predicted a premature termination codon (PTC) (33%), 11 affected splice sites (13.6%), and two predicted in-frame deletions (2.5%). Most mutations (67.9%) occurred in cbEGF-like modules. Genotype was clinically relevant for early diagnosis and conclusion of the diagnostic work-up in patients with incomplete or atypical phenotypes.

Genetic testing of the cystic fibrosis transmembrane conductance (CFTR) gene is currently performed in couples undergoing assisted reproduction techniques (ART), because of the high prevalence of healthy carriers in the population and the pathogenic relationship with congenital bilateral absence of vas deferens (CBAVD). However, discordant data have been reported concerning the usefulness of this genetic test in couples with no family history of cystic fibrosis (CF). In this study, we report the results of CFTR molecular screening in 1195 couples entering ART. Genetic testing was initially carried out in a single partner of each couple. CFTR mutations were detected in 55 subjects (4.6%), a percentage that overlaps with the one reported in the general population. However, significantly higher frequencies of were found in CBAVD individuals (37.5%) and in males with nonobstructive azoospermia (6.6%). The 5T allele was found in 78 patients (6.5%). This figure was again significantly different in males with nonobstructive-azoospermia (9.9%) and in those with CBAVD (100%). All together, 139 subjects (11.6%) had either a CFTR mutation or the 5T allele. Subsequent molecular analysis of their partners disclosed a CFTR mutation or 5T allele in nine cases (6.5%). However, none of these couples had CFTR alterations in both members, a CFTR mutation being invariably present in one partner and the 5T allele in the other. In order to improve genetic counselling of these couples, the TG-M470V-5T association was analyzed, and a statistically significant relationship between 12TG-V470 and CBAVD was detected.

Our objective was to evaluate the similarities and differences in bone mass and structure between pairs of monozygotic twins as measured by means of the quantitative ultrasound (QUS) technique.A cohort of monozygotic twins was measured by QUS of the hand phalanges using the DBM sonic bone profiler (IGEA, Carpi, Italy). The parameters studied were amplitude-dependent speed of sound (AD-SoS), ultrasound bone profile index (UBPI), signal dynamics (SDy) and bone transmission time (BTT). Linear correlation coefficients, multivariate linear analysis and the ANOVA test were used to assess intrapair associations between variables and to determine which factors influence the intrapair differences in QUS variables.One hundred and six pairs of monozygotic twins were enrolled in the study, 68 females and 38 males in the age range 5 to 71 years.Significant intrapair correlations were obtained in the whole population and separately for males and females, regarding height ( r =0.98-0.99, p0.0001), weight ( r =0.95-0.96, p0.0001), AD-SoS ( r =0.90-0.92, p0.0001), BTT ( r =0.94-0.95, p0.0001) and other QUS parameters ( r0.74, p0.0001). Multivariate analysis revealed that intrapair differences between AD-SoS, SDy, UBPI and BTT are significantly influenced by age in the whole population and in the female population. Furthermore, the ANOVA test showed, for the female group, a significant increase in the intrapair differences in SDy and UBPI above 40 years.A relative contribution of genetic factors to skeletal status could be observed by phalangeal QUS measurement in monozygotic twins. A significant increase in the intrapair difference in QUS parameters with increasing age and onset of menopause also suggests the importance of environmental factors in the female twin population.

X-linked retinitis pigmentosa (XLRP) results from mutations in a number of loci, including RP2 at Xp11.3, and RP3 at Xp21.1. RP2 and RP3 genes have been identified by positional cloning. RP2 mutations are found in about 10% of XLRP patients. We performed a mutational screening of RP2 gene inpatients belonging to seven unrelated families in linkage with the RP2 locus. SSCP analysis detected three conformation variants, within exon 2 and 3. Direct sequencing of exon 2, disclosed a G-->A transition at nucleotide 449 (W150X), and a G-->T transversion in position 547 (E183X). Sequence analysis of exon 3 variant revealed an insertion (853/854insG), leading to a frameshift. In this patient, we detected an additional sequence alteration (A-->G at nucleotide 848, E283G). Each mutation was co-segregating with the disease in the affected family members available for the study. These mutations are expected to introduce a stop codon within the RP2 coding sequence probably resulting in a truncated or unstable protein.

A polymorphic site has been found in the human bone morphogenetic protein-4 (BMP4) gene. To our knowledge, it is the first defined polymorphism in this gene, being present with similar frequencies in Caucasian, Hispanic, and African populations.

The RPGR (retinitis pigmentosa GTPase regulator) gene has been shown to be mutated in 10-20% of patients with X-linked retinitis pigmentosa (XLRP), a severe form of inherited progressive retinal degeneration. A total of 29 different RPGR mutations have been identified in northern European and United States patients. We have performed mutation analysis of the RPGR gene in a cohort of 49 southern European males affected with XLRP. By multiplex SSCA and automatic direct sequencing of all 19 RPGR exons, seven different and novel mutations were identified in eight of the 49 families; these include three splice site mutations, two microdeletions, and two missense mutations. RNA analysis showed that the three splice site defects resulted in the generation of aberrant RPGR transcripts. Six of these mutations were detected in the conserved amino-terminal region of RPGR protein, containing tandem repeats homologous to the RCC1 protein, a guanine nucleotide-exchange factor for Ran-GTPase. Several exonic and intronic sequence variations were also detected. None of the RPGR mutations reported in other populations were identified in our series. Our results are consistent with the notions of heterogeneity and minority causation of XLRP by mutations in RPGR in Caucasian populations.

We report a personal experience of first-trimester prenatal diagnosis of Ellis-van Creveld (EvC) syndrome based on typing of microsatellite markers flanking the EvC locus. An heterozygous fetus was diagnosed with a diagnostic accuracy of 96 per cent. The DNA prediction was confirmed by ultrasound at 22 weeks of gestation and by clinical evaluation at birth.