Clinical variability in DYNC2H1-related skeletal ciliopathies includes Ellis-van Creveld syndrome.

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.

Mosaic genome-wide paternal uniparental disomy after discordant results from primary fetal samples and cultured cells.

Mosaic genome-wide paternal uniparental disomy (GWpUPD) is a rare condition in which two euploid cell lines coexist in the same individual, one with biparental content and one with genome-wide paternal isodisomy. We report a complex prenatal diagnosis with discordant results from cultured and uncultured samples. A pregnant woman was referred for placental mesenchymal dysplasia and fetal omphalocele. Karyotype, array-CGH and Beckwith-Wiedemann Syndrome (BWS) testing (methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) of 11p15) performed on amniocytes were negative. After intrauterine fetal demise, the clinical suspicion persisted and BWS MS-MLPA was repeated on cultured cells from umbilical cord and amniotic fluid, revealing a mosaicism for KvH19 hypermethylation/KCNQ1OT1:TSS:DMR hypomethylation. These results, along with microsatellite analysis of the BWS region, were consistent with mosaic paternal 11p15 isodisomy. A concurrent maternal contamination exclusion test, analyzing polymorphic microsatellite markers on multiple chromosomes, showed an imbalance in favor of paternal alleles at all examined loci on cultured amniocytes and umbilical cord samples. This led to suspicion of mosaic GWpUPD, later confirmed by SNP-array, identifying a mosaic genome-wide paternal isodisomy affecting 60% of fetal cells. The assessment of mosaic GWpUPD requires multiple approaches beyond the current established diagnostic processes, also entertaining possible low-rate mosaicism. Clinical acumen and an integrated testing approach are the key to a successful diagnosis.

Germline and Mosaic Variants in PRKACA and PRKACB Cause a Multiple Congenital Malformation Syndrome.

PRKACA and PRKACB code for two catalytic subunits (Cα and Cß) 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 Cß subunits of PKA during human development.

Common atrium/atrioventricular canal defect and postaxial polydactyly: A mild clinical subtype of Ellis-van Creveld syndrome caused by hypomorphic mutations in the EVC gene.

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.

DNA Methylation in the Diagnosis of Monogenic Diseases.

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.

Spectrum of epilepsy and electroencephalogram patterns in idic (15) syndrome.

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 <50%) in 10.7%; and uncontrolled in 32%. One patient was not taking any anti-epileptic drugs by his parents' choice. Fourteen percent were on monotherapy; whereas the other 82% were on polytherapy. Seizures stopped at a median age of 5 years 5 months. The interictal electroencephalogram showed slow/sharp waves, and/or biphasic spikes-polyspikes, spike/wave complexes, and an excess of fast activity mainly over the fronto-temporal areas. Epilepsy is a major clinical challenge in patients with idic(15), associated with a poor prognosis in 55%. Frontal lobe seizures are a novel finding. © 2016 Wiley Periodicals, Inc.

p.Arg1809Cys substitution in neurofibromin is associated with a distinctive NF1 phenotype without neurofibromas.

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 cafè-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.

Spinal neurofibromatosis in a family with classical neurofibromatosis type 1 and a novel NF1 gene mutation.

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".

Four-year follow-up study in a NF1 boy with a focal pontine hamartoma.

Neurofibromatosis is a collective name for a group of genetic conditions in which benign tumours affect the nervous system. Type 1 is caused by a genetic mutation in the NF1 gene (OMIM 613113) and symptoms can vary dramatically between individuals, even within the same family. Some people have very mild skin changes, whereas others suffer severe medical complications. The condition usually appears in childhood and is diagnosed if two of the following are present: six or more café-au-lait patches larger than 1.5 cm in diameter, axillary or groin freckling, 2 or more Lisch nodules (small pigmented areas in the iris of the eye), 2 or more neurofibromas, optic pathway gliomas, bone dysplasia, and a first-degree family relative with Neurofibromatosis type 1. The pattern of inheritance is autosomal dominant, however, half of all NF1 cases are 'sporadic' and there is no family history. Neurofibromatosis type 1 is an extremely variable condition whose morbidity and mortality is largely dictated by the occurrence of the many complications that may involve any of the body systems. We describe a family affected by NF1 in whom genetic molecular analysis identified the same mutation in the son and father. Routine MRI showed pontine focal lesions in the eight-year-old son, though not in the father. We performed a four years follow-up study and at follow-up pontine hamartoma size remained unchanged in the son, and the father showed still no brain lesions, confirming thus an intra-familial phenotype variability.

Novel and recurrent EVC and EVC2 mutations in Ellis-van Creveld syndrome and Weyers acrofacial dyostosis.

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.

ATM gene alterations in chronic lymphocytic leukemia patients induce a distinct gene expression profile and predict disease progression.

BACKGROUND: The genetic characterization of chronic lymphocytic leukemia cells correlates with the behavior, progression and response to treatment of the disease. DESIGN AND METHODS: Our aim was to investigate the role of ATM gene alterations, their biological consequences and their value in predicting disease progression. The ATM gene was analyzed by denaturing high performance liquid chromatography and multiplex ligation probe amplification in a series of patients at diagnosis. The results were correlated with immunoglobulin gene mutations, cytogenetic abnormalities, ZAP-70 and CD38 expression, TP53 mutations, gene expression profile and treatment-free interval. RESULTS: Mutational screening of the ATM gene identified point mutations in 8/57 cases (14%). Multiplex ligation probe amplification analysis identified six patients with 11q deletion: all of them had at least 20% of deleted cells, analyzed by fluorescent in situ hybridization. Overall, ATM point mutations and deletions were detected in 14/57 (24.6%) cases at presentation, representing the most common unfavorable genetic anomalies in chronic lymphocytic leukemia, also in stage A patients. Patients with deleted or mutated ATM had a significantly shorter treatment-free interval compared to patients without ATM alterations. ATM-mutated cases had a peculiar gene expression profile characterized by the deregulation of genes involved in apoptosis and DNA repair. Finally, definition of the structure of the ATM-mutated protein led to a hypothesis that functional abnormalities are responsible for the unfavorable clinical course of patients carrying these point mutations. CONCLUSIONS: ATM alterations are present at diagnosis in about 25% of individuals with chronic lymphocytic leukemia; these alterations are associated with a peculiar gene expression pattern and a shorter treatment-free interval.

Familial spinal neurofibromatosis due to a multiexonic NF1 gene deletion.

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.

Evaluation of TP53 mutations with the AmpliChip p53 research test in chronic lymphocytic leukemia: correlation with clinical outcome and gene expression profiling.

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.

Bilateral (opercular and paracentral lobular) polymicrogyria and neurofibromatosis type 1.

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.

Identification of the second CFTR mutation in patients with congenital bilateral absence of vas deferens undergoing ART protocols.

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%.

Germline mosaicism in neurofibromatosis type 1 due to a paternally derived multi-exon deletion.

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.

Founder effects for ATM gene mutations in Italian Ataxia Telangiectasia families.

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.

Type 2 deiodinase polymorphism (threonine 92 alanine) predicts L-thyroxine dose to achieve target thyrotropin levels in thyroidectomized patients.

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, >or=0.1<0.5 mU/liter) serum TSH levels. MAIN OUTCOME MEASURES: DNA genotyping for D2 Thr92Ala variant and evaluation of T4 dose (microg/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 microg/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.

Gain-of-function RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy.

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.