Atrioventricular canal defect and genetic syndromes: The unifying role of sonic hedgehog.

The atrioventricular canal defect (AVCD) is a congenital heart defect (CHD) frequently associated with extracardiac anomalies (75%). Previous observations from a personal series of patients with AVCD and "polydactyly syndromes" showed that the distinct morphology and combination of AVCD features in some of these syndromes is reminiscent of the cardiac phenotype found in heterotaxy, a malformation complex previously associated with functional cilia abnormalities and aberrant Hedgehog (Hh) signaling. Hh signaling coordinates multiple aspects of left-right lateralization and cardiovascular growth. Being active at the venous pole the secondary heart field (SHF) is essential for normal development of dorsal mesenchymal protrusion and AVCD formation and septation. Experimental data show that perturbations of different components of the Hh pathway can lead to developmental errors presenting with partially overlapping manifestations and AVCD as a common denominator. We review the potential role of Hh signaling in the pathogenesis of AVCD in different genetic disorders. AVCD can be viewed as part of a "developmental field," according to the concept that malformations can be due to defects in signal transduction cascades or pathways, as morphogenetic units which may be altered by Mendelian mutations, aneuploidies, and environmental causes.

Intrafamiliar clinical variability of circumferential skin creases Kunze type caused by a novel heterozygous mutation of N-terminal TUBB gene.

Circumferential skin creases Kunze type (CSC-KT; OMIM 156610, 616734) is a rare disorder characterized by folding of excess skin, which leads to ringed creases, known as Michelin Tire Baby Syndrome (MTBS). CSC-KT patients also exhibit facial dysmorphism, growth retardation, intellectual disability (ID) and multiple congenital malformations. Recently, 2 heterozygous mutations in TUBB gene and 4 mutations (both homozygous and heterozygous) in MAPRE2 gene were identified in 3 and 4 CSC-KT patients, respectively. In the 3 TUBB gene-related CSC-KT patients, all mutations fall in the N-terminal gene domain and were de novo. Mutations in the C-terminal of TUBB gene have been associated to microcephaly and structural brain malformation, in the absence of CSC-KT features. We report a 9-year-old boy with a diagnosis of CSC-KT based on MTBS, facial dysmorphism, microcephaly, severe ID, cortical atrophy and corpus callosum hypoplasia. Sanger sequencing identified a novel heterozygous c.218T>C (p.Met73Thr) mutation in the N-terminal of TUBB gene, that was inherited from the mother affected by isolated MTBS. This is the first report of inherited TUBB gene-related CSC-KT resulting from a novel heterozygous mutation in the N-terminal domain. Present data support the role of TUBB mutations in CSC-KT and definitely includes CSC-KT syndrome within the tubulinopathies.

Expanding the clinical and molecular spectrum of PRMT7 mutations: 3 additional patients and review.

Protein arginine methyltransferase 7 (PRMT7) is a member of a family of enzymes that catalyze the transfer of methyl groups from S-adenosyl-l-methionine to nitrogen atoms on arginine residues. Arginine methylation is involved in multiple biological processes, such as signal transduction, mRNA splicing, transcriptional control, DNA repair, and protein translocation. Currently, 7 patients have been described harboring compound heterozygous or homozygous variants in the PRMT7 gene, causing a novel intellectual disability syndrome, known as SBIDDS syndrome (Short Stature, Brachydactyly, Intellectual Developmental Disability, and Seizures). We report on 3 additional patients from 2 consanguineous families with severe/moderate intellectual disability, short stature, brachydactyly and dysmorphisms. Exome sequencing revealed 2 novel homozygous mutations in PRMT7. Our findings expand the clinical and molecular spectrum of homozygous PRMT7 mutations, associated to the SBIDDS syndrome, showing a possible correlation between the type of mutation and the severity of the phenotype.

Biallelic mutations in DYNC2LI1 are a rare cause of Ellis-van Creveld syndrome.

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.

Syndromic non-compaction of the left ventricle: associated chromosomal anomalies.

Non-compaction of the left ventricle (NCLV) is a cardiomyopathy characterized by prominent left ventricular trabeculae and deep intertrabecular recesses. Associated extracardiac anomalies occur in 14-66% of patients of different series, while chromosomal anomalies were reported in sporadic cases. We investigated the prevalence of chromosomal imbalances in 25 syndromic patients with NCLV, using standard cytogenetic, subtelomeric fluorescent in situ hybridization, and array-comparative genomic hybridization (CGH) analyses. Standard chromosome analysis disclosed an abnormality in three (12%) patients, including a 45,X/46,XX mosaic, a 45,X/46,X,i(Y)(p11) mosaic, and a de novo Robertsonian 13;14 translocation in a child affected by hypomelanosis of Ito. Cryptic chromosome anomalies were found in six (24%) cases, including 1p36 deletion in two patients, 7p14.3p14.1 deletion, 18p subtelomeric deletion, 22q11.2 deletion associated with velo-cardio-facial syndrome, and distal 22q11.2 deletion, each in one case. These results recommend accurate clinical evaluation of patients with NCLV, and suggest that chromosome anomalies occur in about one third of syndromic NCLV individuals, without metabolic/neuromuscular disorder. Array-CGH analysis should be included in the diagnostic protocol of these patients, because different submicroscopic imbalances are causally associated with this disorder and can pinpoint candidate genes for this cardiomyopathy.

Cystinuria caused by mutations in rBAT, a gene involved in the transport of cystine.

Cystinuria is a classic heritable aminoaciduria that involves the defective transepithelial transport of cystine and dibasic amino acids in the kidney and intestine. Six missense mutations in the human rBAT gene, which is involved in high-affinity transport of cystine and dibasic amino acids in kidney and intestine, segregate with cystinuria. These mutations account for 30% of the cystinuria chromosomes studied. Homozygosity for the most common mutation (M467T) was detected in three cystinuric siblings. Mutation M467T nearly abolished the amino acid transport activity induced by rBAT in Xenopus oocytes. These results establish rBAT as a cystinuria gene.

Mutations in a new gene in Ellis-van Creveld syndrome and Weyers acrodental dysostosis.

Ellis-van Creveld syndrome (EvC, MIM 225500) is an autosomal recessive skeletal dysplasia characterized by short limbs, short ribs, postaxial polydactyly and dysplastic nails and teeth. Congenital cardiac defects, most commonly a defect of primary atrial septation producing a common atrium, occur in 60% of affected individuals. The disease was mapped to chromosome 4p16 in nine Amish subpedigrees and single pedigrees from Mexico, Ecuador and Brazil. Weyers acrodental dysostosis (MIM 193530), an autosomal dominant disorder with a similar but milder phenotype, has been mapped in a single pedigree to an area including the EvC critical region. We have identified a new gene (EVC), encoding a 992-amino-acid protein, that is mutated in individuals with EvC. We identified a splice-donor change in an Amish pedigree and six truncating mutations and a single amino acid deletion in seven pedigrees. The heterozygous carriers of these mutations did not manifest features of EvC. We found two heterozygous missense mutations associated with a phenotype, one in a man with Weyers acrodental dysostosis and another in a father and his daughter, who both have the heart defect characteristic of EvC and polydactyly, but not short stature. We suggest that EvC and Weyers acrodental dysostosis are allelic conditions.

Genetic prediction of common diseases. Still no help for the clinical diabetologist!

Genome-wide association studies (GWAS) have identified several loci associated with many common, multifactorial diseases which have been recently used to market genetic testing directly to the consumers. We here addressed the clinical utility of such GWAS-derived genetic information in predicting type 2 diabetes mellitus (T2DM) and coronary artery disease (CAD) in diabetic patients. In addition, the development of new statistical approaches, novel technologies of genome sequencing and ethical, legal and social aspects related to genetic testing have been also addressed. Available data clearly show that, similarly to what reported for most common diseases, genetic testing offered today by commercial companies cannot be used as predicting tools for T2DM and CAD. Further studies taking into account the complex interaction between genes as well as between genetic and non-genetic factors, including age, obesity and glycemic control which seem to modify genetic effects on the risk of T2DM and CAD, might mitigate such negative conclusions. Also, addressing the role of relatively rare variants by next generation sequencing may help identify novel and strong genetic markers with an important role in genetic prediction. Finally, statistical tools concentrated on reclassifying patients might be a useful application of genetic information for predicting many common diseases. By now, prediction of such diseases, including those of interest for the clinical diabetologist, have to be pursued by using traditional clinical markers which perform well and are not costly.

Congenital heart defects in the recurrent 2q13 deletion syndrome.

The recurrent 2q13 deletion syndrome is a rare genetic disorder associated with developmental delay, cardiac and urogenital malformations, and minor facial anomalies. Congenital heart defects (CHDs) are the most frequent malformations associated with del2q13. Experimental studies in zebrafish suggest that two genes mapping within the 2q13 critical region (FBLN7 and TMEM87B) could confer susceptibility to congenital heart defects in affected individuals. We reviewed the cardiac characteristics in four patients with 2q13 deletion admitted to our hospitals, and in published patients. Two of our patients had congenital heart defects, consisting in partial anomalous pulmonary venous connection, ostium secundum atrial septal defect ostium secundum, and small muscular ventricular septal defect in one of them, and aortic valve insufficiency with partial fusion of two commissures (incomplete bicuspid aortic valve) and mitral valve insufficiency due to trivial mitral valve prolapse in the other. The anatomic types of CHD in del2q13 syndrome are highly variable and distributed widely, including laterality defects, complex atrioventricular septal defect, septal anomalies, and cardiomyopathies. Cardiac evaluation should be part of the clinical workup at diagnosis of 2q13 deletion.

New mutations in ZFPM2/FOG2 gene in tetralogy of Fallot and double outlet right ventricle.

Conotruncal defects (CTDs) represent 15-20% of all congenital heart defects. Mutations in a number of genes have been associated with CTD in humans and animal models. We investigated the occurrence and the prevalence of GATA4, NKX2.5, ZFPM2/FOG2, GDF1, and ISLET1 gene mutations in a large cohort of individuals with CTD, including tetralogy of Fallot with or without pulmonary atresia (TOF, 178 patients), double outlet right ventricle (DORV, 13 patients), and truncus arteriosus (11 patients). Denaturing high-performance liquid chromatography (DHPLC) analysis followed by bidirectional sequencing disclosed no putative pathogenic mutation in GATA4, ISLET1, and GDF1 genes. Two novel (Ile227Val, Met544Ile) and one previously reported (Glu30Gly) possibly pathogenic missense variants were identified in the ZFPM2/FOG2 gene in 3 sporadic patients of 202 (1.5%) with CTD, including 1 of 178 (0.6%) with TOF and 2 of 13 (15.4%) with DORV. Mutation analysis also detected one known missense change (Arg25Cys) in NKX2.5 gene in two (1.1%) sporadic patients with TOF. These sequence alterations were found to be absent in 500 population-matched controls. In conclusion, the present results (i) indicate and confirm that mutations in the GATA4, GDF1, and ISLET1 genes are not major determinants in the pathogenesis of TOF, (ii) provide supportive evidence of an association between ZFPM2/FOG2 gene and TOF/DORV, and (iii) provide additional examples of the possible contribution of the Arg25Cys change in the NKX2.5 to a small number of TOF cases.

TRIB3 R84 variant affects glucose homeostasis by altering the interplay between insulin sensitivity and secretion.

AIMS/HYPOTHESIS: The results of studies on the genetics of complex traits need to be replicated and to reach robust statistical significance before they can be considered as established. We here tried to replicate the previously reported association between the TRIB3 Q84R polymorphism (rs2295490) and glucose homeostasis. METHODS: Three samples of Europeans with fasting glucose <7.0 mmol/l were studied. In sample 1 (n=791), the association between TRIB3 Q84R and impaired glucose regulation (IGR; defined as impaired fasting glucose and/or impaired glucose tolerance and/or type 2 diabetes by OGTT) and insulin sensitivity (ISI), and its interplay with early-phase insulin secretion (i.e. disposition index [DI]) were analysed. Sample 2 (n=374) and sample 3 (n=394) were used to replicate the association with IGR and insulin sensitivity (by glucose clamp), respectively. Genotyping was performed by TaqMan allele discrimination. RESULTS: R84 carriers were at higher risk of IGR: OR for the additive model 1.54, p=0.004, and 1.63, p=0.027, in samples 1 and 2, respectively. In sample 1, both ISI (p=0.005) and DI (p=0.043) were progressively lower from QQ to QR and RR individuals. A 'triangulation approach' indicated that the association with IGR was mostly mediated by DI rather than by ISI changes (i.e. being the expected ORs 1.51 and 1.25, respectively). In sample 3, glucose disposal was 38.8+/-17.7, 33.8+/-14.4, and 31.6+/-13.3 micromol min(-1)kg(-1), p=0.022, in QQ, QR and RR individuals, respectively. CONCLUSIONS/INTERPRETATION: Our data confirm that the TRIB3 R84 variant affects glucose homeostasis and suggest this effect is due to an alteration of the interplay between insulin sensitivity and secretion.

A novel homozygous splice site mutation in the HPGD gene causes mild primary hypertrophic osteoarthropathy.

OBJECTIVES: Homozygous mutations in HPGD gene, encoding 15-hydroxyprostaglandin dehydrogenase, have recently been associated with primary hypertrophic osteoarthropathy (PHO). So far, only 7 HPGD alterations are known. In order to expand this mutational spectrum and better delineate the HPGD-related phenotype, we report the clinical and molecular characterisation of a 13-year-old boy and compare his features to known mutated patients. METHODS: The HPGD gene exons 1-7 and exon-intron junctions were analysed by direct sequencing. Previously published HPGD-mutated patients were systematically reviewed based on the original clinical description. RESULTS: A novel homozygous c.217+1G>A mutation affecting the obligatory donor splice site of HPGD exon 2 was identified in our proband who showed a mild form of PHO. Review of HPGD-mutated patients outlined all patients manifested digital clubbing, periostosis and acro-osteolysis. Hyperhidrosis (92%), arthralgia (65%) and eczema (33%) were variably associated features. Pachydermia (54%) was mild and mostly limited to palms and sole; cutis vertigis gyrata, blepharoptosis and severe skin thickening were never observed. Besides digital clubbing, PHO infants often presented patent ductus arteriosus (PDA) (32%) and delayed cranial sutures closure (55%). CONCLUSIONS: The present findings broaden the allelic spectrum of HPGD gene to include a novel c.217+1G>A mutation. Mutated patients display a homogeneous phenotype mainly consisting in digital clubbing, periostosis, acro-osteolysis, hyperhidrosis and mild pachydermia. Earliest manifestations include delayed closure of the cranial sutures and PDA. In conclusion, the information reported herein would facilitate the diagnosis of PHO due to HPGD mutations.

IRS1 G972R polymorphism and type 2 diabetes: a paradigm for the difficult ascertainment of the contribution to disease susceptibility of 'low-frequency-low-risk' variants.

AIMS/HYPOTHESIS: The aim of the study was to determine the association between IRS1 G972R polymorphism and type 2 diabetes; published data concerning this association have been conflicting. To obtain further insight into this topic, we performed a meta-analysis of all available case-control studies. METHODS: We performed a meta-analysis of 32 studies (12,076 cases and 11,285 controls). RESULTS: The relatively infrequent R972 variant was not significantly associated with type 2 diabetes (OR 1.09, 95% CI 0.96-1.23, p = 0.184 under a dominant model). Some evidence of heterogeneity was observed across studies (p = 0.1). In the 14 studies (9,713 individuals) in which the mean age at type 2 diabetes diagnosis was available, this variable explained 52% of the heterogeneity (p = 0.03). When these studies were subdivided into tertiles of mean age at diagnosis, the OR for diabetes was 1.48 (95% CI 1.17-1.87), 1.22 (95% CI 0.97-1.53) and 0.88 (95% CI 0.68-1.13) in the youngest, intermediate and oldest tertile, respectively (p = 0.0022 for trend of ORs). CONCLUSIONS/INTERPRETATION: Our findings illustrate the difficulties of ascertaining the contribution of 'low-frequency-low-risk' variants to type 2 diabetes susceptibility. In the specific context of the R972 variant, approximately 200,000 study individuals would be needed to have 80% power to identify a 9% increase in diabetes risk at a genome-wide significance level. Under these circumstances, a strategy aimed at improving outcome definition and decreasing its heterogeneity may critically enhance our ability to detect genetic effects, thereby decreasing the required sample size. Our data suggest that focusing on early-onset diabetes, which is characterised by a stronger genetic background, may be part of such a strategy.

Polymorphism of the IRGM gene might predispose to fistulizing behavior in Crohn's disease.

OBJECTIVES: Recently, genome-wide association analyses have identified single nucleotide polymorphisms in the IRGM gene (rs1000113 and rs4958847) as strong candidate susceptibility factors for Crohn's disease (CD). The aim of our study was to test whether these variants are associated with inflammatory bowel disease (IBD) in adult- and childhood-onset Italian patients. METHODS: Allele and genotype frequencies of rs1000113 and rs4958847 were determined in 823 CD (265 younger than 19 years at diagnosis), 353 ulcerative colitis (UC) (130 younger than 19 years at diagnosis), and 578 controls. Genotype distributions were examined both within IBD clinical sub-phenotypes and CARD15 genotypes. RESULTS: rs1000113 and rs4958847 were both associated with adult-onset (P=2 x 10(-4); P=2.5 x 10(-3), respectively) and childhood-onset (P=4 x 10(-4); P=8 x 10(-3), respectively) CD cohorts. Similarly, the genotype frequencies remained significantly different for both variants (adult rs1000113, P=1 x 10(-4); rs4958847, P=1 x 10(-3); pediatric rs1000113, P=2.3 x 10(-4); rs4958847, P=9.6 x 10(-3)). At logistic regression, the rs4958847 polymorphism was associated with fistulizing behavior (P=0.037, OR=1.54, CI=1.02-2.31) and perianal fistulas (P=0.045, OR=1.55, CI=1.01-2.38). Conversely, no association with UC and sub-phenotypes was shown. CONCLUSIONS: We replicated the previously reported associations between CD and rs1000113 and rs4958847, confirming that IRGM is a susceptibility locus only for CD, either adult- or early-onset in the Italian population; furthermore, we have also shown its influence on specific clinical features (fistulizing disease).

Joubert syndrome with bilateral polymicrogyria: clinical and neuropathological findings in two brothers.

Joubert syndrome (JS) is characterized by hypotonia, ataxia, developmental delay, and a typical neuroimaging finding, the so-called "molar tooth sign" (MTS). The association of MTS and polymicrogyria (PMG) has been reported as a distinct JS-related disorder (JSRD). So far, five patients have been reported with this phenotype, only two of them being siblings. We report on one additional family, describing a living child with JS and PMG, and the corresponding neuropathological picture in the aborted brother. No mutations were detected in the AHI1 gene, the only so far associated with the JS + PMG phenotype. Moreover, linkage analysis allowed excluding all known gene loci, suggesting further genetic heterogeneity.

Normal cognitive functions in joubert syndrome.

Developmental delay and subsequent impaired cognitive functions are present in almost all patients with Joubert syndrome (JS). We report on a 20-year-old woman with mild clinical signs of JS (minimal truncal ataxia and oculomotor apraxia) but typical molar tooth sign on neuroimaging, normal full scale (IQ=93), verbal (IQ=93), and performance intelligence quotient (IQ=94). Only minor difficulties in visual-spatial organization and in some executive functions could be detected. This pattern of deficits is partly reminiscent of the cerebellar cognitive affective syndrome. Her diagnosis was only reached following the diagnosis of JS in two brothers with severe cognitive impairment. Molecular investigations demonstrated a homozygous mutation in the INPP5E gene. This exceptional observation confirms that normal cognitive functions are possible in JS and corroborates the well known intrafamilial variability.

Gender-stratified analysis of DLG5 R30Q in 4707 patients with Crohn disease and 4973 controls from 12 Caucasian cohorts.

BACKGROUND: DLG5 p.R30Q has been reported to be associated with Crohn disease (CD), but this association has not been replicated in most studies. A recent analysis of gender-stratified data from two case-control studies and two population cohorts found an association of DLG5 30Q with increased risk of CD in men but not in women and found differences between 30Q population frequencies for males and females. Male-female differences in population allele frequencies and male-specific risk could explain the difficulty in replicating the association with CD. METHODS: DLG5 R30Q genotype data were collected for patients with CD and controls from 11 studies that did not include gender-stratified allele counts in their published reports and tested for male-female frequency differences in controls and for case-control frequency differences in men and in women. RESULTS: The data showed no male-female allele frequency differences in controls. An exact conditional test gave marginal evidence that 30Q is associated with decreased risk of CD in women (p = 0.049, OR = 0.87, 95% CI 0.77 to 1.00). There was also a trend towards reduced 30Q frequencies in male patients with CD compared with male controls, but this was not significant at the 0.05 level (p = 0.058, OR = 0.87, 95% CI 0.74 to 1.01). When data from this study were combined with previously published, gender-stratified data, the 30Q allele was found to be associated with decreased risk of CD in women (p = 0.010, OR = 0.86, 95% CI 0.76 to 0.97), but not in men. CONCLUSION: DLG5 30Q is associated with a small reduction in risk of CD in women.

RPGRIP1L mutations are mainly associated with the cerebello-renal phenotype of Joubert syndrome-related disorders.

Joubert syndrome-related disorders (JSRDs) are autosomal recessive pleiotropic conditions sharing a peculiar cerebellar and brainstem malformation known as the 'molar tooth sign' (MTS). Recently, mutations in a novel ciliary gene, RPGRIP1L, have been shown to cause both JSRDs and Meckel-Gruber syndrome. We searched for RPGRIP1L mutations in 120 patients with proven MTS and phenotypes representative of all JSRD clinical subgroups. Two homozygous mutations, the previously reported p.T615P in exon 15 and the novel c.2268_2269delA in exon 16, were detected in 2 of 16 families with cerebello-renal presentation ( approximately 12%). Conversely, no pathogenic changes were found in patients with other JSRD phenotypes, suggesting that RPGRIP1L mutations are largely confined to the cerebello-renal subgroup, while they overall represent a rare cause of JSRD (<2%).

Deletions of NF1 gene and exons detected by multiplex ligation-dependent probe amplification.

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 encompassing >or=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 encompassing >or=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.