Germline testing and genetic counseling in biliary tract cancer: an operative proposal to improve the state of art.

INTRODUCTION: A genetic predisposition seems to be involved in biliary tract cancer, but the prevalence of germline mutations in BTC remains unclear, and the therapeutic role of the germline pathologic variants is still unknown. AREA COVERED: The aim of the present work is to systematically review the data available on the hereditary predisposition of biliary tract cancer by a specific research on PubMed, in order to highlight the most important critical points and to define the current possible role of germinal testing and genetic counseling in this setting of patients. EXPERT OPINION: Basing on data already available, we decided to start in our institution a specific genetic protocol focused on biliary tract cancer patients, which includes genetic counseling and, if indicated, germline test. The inclusion criteria are: 1) Patient with personal history of oncologic disease other than BTC, 2) Patient with familiar history of oncologic disease (considering relatives of first and second grade), 3) Patient with ≤ 50 years old, 4) Patient presenting a somatic mutation in genes involved in DNA damage repair pathways and mismatch repair. The aim of the presented protocol is to identify germline pathogenic variants with prophylactic and therapeutic impact, and to collect and integrate a significant amount of clinical, familial, somatic, and genetic data.

Behavioral Phenotype, Electroclinical Features, and Treatment Options in Twins with Lrp2 Candidate Variants (Donnay-Barrow/Foar Syndrome).

The LRP2 gene encodes megalin (LRP-2/GP330), a large single-spanning transmembrane glycoprotein that serves as a multiligand endocytotic receptor and mediates the reabsorption of albumin in the proximal renal tubule. LRP2 is implicated in an autosomal recessive disorder characterized by dimorphisms, ocular anomalies, sensorineural deafness, proteinuria, epilepsy, and intellectual disability: a clinical condition called Donnai-Barrow syndrome (DBS) or facio-oculo-acoustico-renal (FOAR) syndrome. Pathogenic variants in LRP2 have been reported in fewer than 60 patients, but a detailed description of seizures, electroencephalographic patterns, imaging findings, behavioral phenotype, and long-term follow-up is still needed. We provide a clinical report of two mono-chorionic twins with LRP2-related disease manifesting developmental delay, autistic features, seizures, proteinuria, and sleep disorders. By sequencing clinical exome, LRP2 candidate rare variants, c.6815G > A, p. (Arg2272His), inherited from the mother and c.12725A > G, p. (Asp4242Gly), inherited from the father, were identified. During follow-up, at the age of 7, the main clinical features of the patients included insomnia, autistic features, severe psychomotor delay, and absent speech. The patients were under treatment with risperidone, antiseizure medications (ASMs), and supplementation of alpha-lactalbumin for self-injury and sleep disturbance. Our study confirmed the wide spectrum of behavioral and neurological and psychiatric features of this rare condition, suggesting new treatment options.

Expanding the spectrum of neonatal-onset AIFM1-associated disorders.

OBJECTIVES: Pathogenic variants in AIFM1 have been associated with a wide spectrum of disorders, spanning from CMT4X to mitochondrial encephalopathy. Here we present a novel phenotype and review the existing literature on AIFM1-related disorders. METHODS: We performed EEG recordings, brain MRI and MR Spectroscopy, metabolic screening, echocardiogram, clinical exome sequencing (CES) and family study. Effects of the variant were established on cultured fibroblasts from skin punch biopsy. RESULTS: The patient presented with drug-resistant, electro-clinical, multifocal seizures 6 h after birth. Brain MRI revealed prominent brain swelling of both hemispheres and widespread signal alteration in large part of the cortex and of the thalami, with sparing of the basal nuclei. CES analysis revealed the likely pathogenic variant c.5T>C; p.(Phe2Ser) in the AIFM1 gene. The affected amino acid residue is located in the mitochondrial targeting sequence. Functional studies on cultured fibroblast showed a clear reduction in AIFM1 protein amount and defective activities of respiratory chain complexes I, III and IV. No evidence of protein mislocalization or accumulation of precursor protein was observed. Riboflavin, Coenzyme Q10 and thiamine supplementation was therefore given. At 6 months of age, the patient exhibited microcephaly but did not experience any further deterioration. He is still fed orally and there is no evidence of muscle weakness or atrophy. INTERPRETATION: This is the first AIFM1 case associated with neonatal seizures and diffuse white matter involvement with relative sparing of basal ganglia, in the absence of clinical signs suggestive of myopathy or motor neuron disease.

Case Report - Multinodular goiter in a patient with Congenital Hypothyroidism and Bannayan-Riley-Ruvalcaba syndrome: the possible synergic role of TPO and PTEN mutation.

We report the case of a paediatric female patient affected by Bannayan-Riley-Ruvalcaba syndrome (BRRS) and congenital hypothyroidism (CH) with homozygous mutation of the TPO gene. She underwent total thyroidectomy at the age of seven years because of the development of a multinodular goiter. BRRS patients present an increased risk of benign and malignant thyroid disease since childhood because of inactivating mutation of PTEN, an onco-suppressor gene. Instead, homozygous mutations in the TPO gene can be associated with severe forms of hypothyroidism with goiter; previous studies have described cases of follicular and papillary thyroid cancer in CH patients with TPO mutation despite a perfectly controlled thyroid function with Levothyroxine therapy. To our knowledge, this is the first case that describes the possible synergic role of coexisting mutation of both TPO and PTEN in the development of multinodular goiter underlining the importance of a tailored surveillance program in these patients, especially during childhood.

PRPH2-Associated Retinopathy: Novel Variants and Genotype-Phenotype Correlations.

PURPOSE: A broad spectrum of autosomal-dominant inherited retinal diseases (IRDs), ranging from mild macular pattern dystrophy to severe cone-rod degeneration, is associated with PRPH2 variants (peripherinopathies). We present detailed clinical and molecular characterization of patients affected by peripherinopathies, aiming to expand the mutational spectrum, and propose novel genotype-phenotype correlations. DESIGN: Observational, retrospective case series. PARTICIPANTS: Patients with an IRD related to a molecularly proven PRPH2 variant. METHODS: Data from ophthalmic examinations and retinal imaging were collected for each follow-up visit. The standard imaging protocol included OCT, blue-light autofluorescence, near-infrared autofluorescence, and ultra-widefield fundus imaging. Genetic analysis was performed with a genomic approach by next-generation sequencing. MAIN OUTCOME MEASURES: Results of ophthalmic examination, retinal imaging, and molecular genetic analysis. RESULTS: Overall, a total of 19 patients with an IRD and a (likely) pathogenic PRPH2 variant were identified. Their age at presentation had a median of 48 years, whereas the symptomatic disease onset was in their 30s or 40s in 74% of cases. The median follow-up time was 4 years. Clinically, 6 patients were diagnosed with cone-rod dystrophy and 13 with pattern dystrophy. Among the 13 PRPH2 pathogenic variants identified in our cohort, 7 were missense, 3 nonsense, 2 frame shifting, and 1 splice site. Missense variants in the D2 loop were associated with cone-rod dystrophies and poor visual prognosis, whereas predicted loss-of-function alleles with pattern dystrophies and retention of a good visual function into adulthood. Overall, the following 7 variants were novel and never associated to a clinical phenotype: c.68delT, c.290G>A, c.413T>G, c.642C>G, c.702_706dupCAGTT, c.771_772delinsGA, and c.850C>G. CONCLUSIONS: Here, we report the findings of a retrospective case series that provided a detailed clinical and molecular characterization of 19 patients harboring 13 different PRPH2 pathogenic variants, 7 of which were previously unreported, expanding the mutational spectrum of the PRPH2 gene. Loss-of-function variants might be preferentially associated with mild-pattern dystrophies, whereas missense dominant-negative variants might be preferentially associated with severely blinding cone-rod degenerations. Further studies are needed to better define the pathogenetic mechanisms and the functional effects of most variants to allow the development of successful gene therapy. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

Multimodal imaging evaluation of occult macular dystrophy associated with a novel RP1L1 variant.

Purpose: Occult Macular Dystrophy (OMD) is an autosomal dominant inherited retinal dystrophy caused by mutations in the retinitis pigmentosa 1-like 1 (RP1L1) gene. The present study describes a novel RP1L1 variant, identified for the first time in two Italian sisters diagnosed with OMD, along with multimodal imaging features, including Optical Coherence Tomography (OCT) Angiography. Methods: We performed multimodal imaging including spectral-domain OCT, blue light autofluorescence (BAF), infrared autofluorescence (IRAF), swept-source OCT Angiography (OCTA), full-field and multifocal electroretinography. Genetic analysis was performed using Next-Generation Sequencing. Pathogenic potential of nonsynonymous novel variants was scored with two in silico algorithms. Results: Proband 1 (P1) and proband 2 (P2) were two Italian sisters of 61 and 56 years old. Both reported a history of progressive visual loss without fundoscopic alterations. P1 reported a 4-year history of rapid visual function worsening, and her best-corrected visual acuity (BCVA) was counting fingers in both eyes. P2 reported a 20-year history of mild but progressive visual acuity loss, and her BCVA was 1/10 and 2/10 respectively in her right and left eye. Structural OCT displayed disorganization of outer retinal bands at the macula and foveal cavitation; loss of foveal photoreceptors was remarkably evident on en-face OCT slabs. OCTA quantitative analysis found that vessel density was reduced both at SCP and DCP while choriocapillaris blood flow was relatively spared. Genetic analysis found the same rare dominant c.2873G > C, p.Arg958Pro variant in the RP1L1 gene. The substitution was regarded as moderately radical according to Grantham score while PolyPhen2 classified the amino acidic substitution as probably damaging. Conclusions and importance: Our study expands the mutational spectrum of RP1L1 gene: the rare c.2873G > C, p.Arg958Pro missense variant may be considered a new pathogenic variant for OMD, the first to be identified exclusively in an Italian family. Moreover, our quantitative OCTA data suggest that OMD is characterized by a rarefaction of superficial and deep capillary plexus.

Transcutaneous electrical stimulation therapy and genetic analysis in Dercum's disease: A pilot study.

ABSTRACT: Dercum's disease (DD), or adiposis dolorosa, is a rare condition of unknown etiology characterized by growth of painful subcutaneous adipose tissue. No specific treatment exists. Pain is often invalidating and resistant to analgesic drugs. We tested the efficacy of Frequency Rhythmic Electrical Modulation System (FREMS) therapy on pain relief. Subcutaneous biopsies were performed for genetic analysis.Nine DD patients were enrolled. Five cycles of FREMS at 3-month intervals during 1 year were administered. Visual analogue scale (VAS), Bartel Index Questionnaire and Short Form 36 questionnaire were used to measure pain and general health status at baseline, 6 and 12 months. Dual-energy X-ray absorptiometry (DEXA) quantified fat mass. Next-Generation Sequencing (NGS) was performed on adipose tissue biopsies and peripheral blood sample to search for somatic variants and specific protein pathway mutation.Seven patients were included in the final analysis. FREMS induced a reduction in VAS score (from 92 to 52.5, P = .0597) and a significant improvement in SF-36 domains (Physical functioning, Role limitation due to physical health, Body pain, Vitality, Social functioning, P < .05). No modification in anthropometrics and DEXA values was observed. The analysis of the mitochondrial Displacement loop (D-loop) region confirmed the clonality of all lipomatous lesions. The presence of the mitochondrially encoded tRNA-Lysine (MT-TK) m.8344A>G variant, occasionally identified in patients with multiple symmetric lipomatosis, was excluded in all subjects. On the other hand, we observed variants in genes belonging to signaling pathways involved in cell cycle and proliferation (Phosphoinositide 3-kinase/AKT/mTOR, MAPK/ERK, and Hippo).FREMS can be a useful tool to alleviate pain and improve overall quality of life in patients with DD. Genetic analysis highlighted the molecular heterogeneity of lipomas.

Newborn Screening for Congenital Hypothyroidism: the Benefit of Using Differential TSH Cutoffs in a 2-Screen Program.

CONTEXT: Analysis of a 2-screen program for congenital hypothyroidism (CH) was performed using differential dried-blood spot thyrotropin (bTSH) cutoffs of 10 mU/L at first screening (all infants) and 5 mU/L at second screening (selected infants). OBJECTIVES: This work aimed to characterize CH infants identified by the second screening and compare infants with bTSH of 5.0 to 9.9 and 10 mU/L or greater on second screening. DESIGN AND PATIENTS: Maternal and neonatal clinical features were retrospectively analyzed for 119 CH babies detected on the second screen in the Lombardy region of Italy, 2007 to 2014. RESULTS: Fifty-two (43.7%) of the 119 CH neonates showed bTSH values ranging from 5.0 to 9.9 mU/L at the second screening (low bTSH group) and 67 (56.3%) bTSH of 10.0 mU/L or greater (high bTSH group). The frequency of thyroid dysgenesis and eutopic gland was similar in both groups, as was the frequency of permanent and transient CH. Moreover, a high frequency of extrathyroidal malformations was found in both groups. The percentage of preterm infants (57.7% vs 23.9%, P < .001) and infants admitted to the neonatal intensive care unit (50.0% vs 17.9%, P < .001) was significantly higher in the low vs the high bTSH group. In addition, maternal treatment with glucocorticoids in pregnancy was significantly more frequent in the low bTSH group than in the high bTSH group (11.5% vs 1.5%, P = .042), as well as maternal hypothyroidism and/or goiter (26.9% vs 10.4%, P = .036). CONCLUSIONS: This study has demonstrated that a lower TSH cutoff at the second screening can detect additional cases of CH and that a second bTSH cutoff of 5.0 mU/L is appropriate for identifying preterm newborns and babies with associated risk factors.

Oral and Fecal Microbiota in Lynch Syndrome.

BACKGROUND: The role of microbiota in Lynch syndrome (LS) is still under debate. We compared oral and fecal microbiota of LS saliva and stool samples with normal healthy controls (NHC). METHODS: Total DNA was purified from feces and saliva to amplify the V3-V4 region of the 16s rRNA gene. Sequences with a high-quality score and length >250 bp were used for taxonomic analysis with QIIME software. RESULTS: Compared to NHC, LS fecal samples demonstrated a statistically significant increase of Bacteroidetes and Proteobacteria and a significant decrease of Firmicutes at the phylum level and of Ruminococcaceae at the family level. Moreover, LS oral samples exhibited a statistically significant increase of Veillonellaceae and Leptotrichiaceae and a statistically significant decrease of Pasteurellaceae. A beta-diversity index allowed differentiation of the two groups. CONCLUSIONS: A peculiar microbial signature is associated with LS, similar to that of sporadic colorectal cancer and Crohn's disease. These data suggest a possible role of proinflammatory bacteria in tumor development in a condition of genetic predisposition, such as LS.

The phenotypic variations of multi-locus imprinting disturbances associated with maternal-effect variants of NLRP5 range from overt imprinting disorder to apparently healthy phenotype.

BACKGROUND: A subset of individuals affected by imprinting disorders displays multi-locus imprinting disturbances (MLID). MLID has been associated with maternal-effect variants that alter the maintenance of methylation at germline-derived differentially methylated regions (gDMRs) in early embryogenesis. Pedigrees of individuals with MLID also include siblings with healthy phenotype. However, it is unknown if these healthy individuals have MLID themselves or if their methylation patterns differ from those associated with imprinting disorders, and in general, if MLID affects the clinical phenotype. METHODS: We have investigated gDMR methylation by locus-specific and whole-genome analyses in a family with multiple pregnancy losses, a child with Beckwith-Wiedemann syndrome (BWS) and a further child with no clinical diagnosis of imprinting disorder or other pathologies. RESULTS: We detected MLID with different methylation profiles in the BWS-affected and healthy siblings. Whole-exome sequencing demonstrated the presence of novel loss-of-function variants of NLRP5 in compound heterozygosity in the mother. The methylation profiles of the two siblings were compared with those of other cases with MLID and control groups by principal component analysis and unsupervised hierarchical clustering, but while their patterns were clearly separated from those of controls, we were unable to cluster those associated with specific clinical phenotypes among the MLID cases. CONCLUSION: The identification of two novel maternal-effect variants of NLRP5 associated with poly-abortivity and MLID adds further evidence to the role of this gene in the maintenance of genomic imprinting in early embryos. Furthermore, our results demonstrate that within these pedigrees, MLID can also be present in the progeny with healthy phenotype, indicating that some sort of compensation occurs between altered imprinted loci in these individuals. The analysis of larger cohorts of patients with MLID is needed to formulate more accurate epigenotype-phenotype correlations.

A novel truncating variant of GLI2 associated with Culler-Jones syndrome impairs Hedgehog signalling.

BACKGROUND: GLI2 encodes for a transcription factor that controls the expression of several genes in the Hedgehog pathway. Mutations in GLI2 have been described as causative of a spectrum of clinical phenotypes, notably holoprosencephaly, hypopituitarism and postaxial polydactyl. METHODS: In order to identify causative genetic variant, we performed exome sequencing of a trio from an Italian family with multiple affected individuals presenting clinical phenotypes in the Culler-Jones syndrome spectrum. We performed a series of cell-based assays to test the functional properties of mutant GLI2. RESULTS: Here we report a novel deletion c.3493delC (p.P1167LfsX52) in the C-terminal activation domain of GLI2. Functional assays confirmed the pathogenicity of the identified variant and revealed a dominant-negative effect of mutant GLI2 on Hedgehog signalling. CONCLUSIONS: Our results highlight the variable clinical manifestation of GLI2 mutations and emphasize the value of functional characterisation of novel gene variants to assist genetic counselling and diagnosis.

[Cancer genetic counseling and quality of life: the effect of coping strategies and psychopathological symptoms during pre-test genetic counseling]. / Consulenza genetica oncologica e qualità di vita: il ruolo delle strategie di coping e della sintomatologia psicopatologica nella fase pre-testale.

UNLABELLED: The cancer genetic counseling (CGC) identifies genetic mutations for hereditary neoplastic diseases, but little is known on its psychological effects on subjects. METHODS: The present study involved women who underwent genetic counseling for breast or ovarian cancer: 19 unaffected, 43 current patients, and 28 past patients. The aim of the study was to examine the relation between coping strategies and the quality of life during genetic counseling before testing, considering the effects of psychopathological symptoms and the health status. RESULTS: Results showed that the use of avoidance strategies led to a decrease in quality of life, and that this relationship was entirely mediated by the intensity of psychopathological symptoms, while the health status did not show any effect on it. CONCLUSIONS: The study, which is the first in Italy, suggests the importance of assessing coping strategies in subjects who undergo the CGO to identify individuals who are at risk of decrease of psychological well-being. Indeed, psychological counselling improving coping strategies could preserve the psychological well-being of individuals.

Reduction in catheter-related infections after switching from povidone-iodine to chlorhexidine for the exit-site care of tunneled central venous catheters in children on hemodialysis.

Only a few studies have investigated the optimal exit site management of tunneled central venous catheters (CVCs) in pediatric patients on chronic hemodialysis (HD). The aim of this study was to assess the efficacy of chlorhexidine solutions and a 5% povidone-iodine solution on the incidence of CVC-related infections in children on HD. The incidence of exit-site infection (ESI), tunnel infection (TI), and bloodstream infection (BSI) was assessed in two groups of tunneled CVCs. The iodopovidone group consisted of 14 CVCs used between 1 January 2011 and 30 June 2012 in 10 children, whose median age at the time of CVC placement was 11.8 years (range 1.2-19.2): 5% povidone-iodine was used for CVC exit-site care. From 1 August 2012 to 31 January 2014, 0.5% chlorhexidine gluconate/70% isopropyl alcohol was used for the exit site, and 2% chlorhexidine gluconate/70% isopropyl alcohol spray for the hub in 13 CVCs was used in 10 patients (chlorhexidine group), whose median age at the time of CVC placement was 10 years (range 1.2-19.2). Ten episodes of ESI were diagnosed in the iodopovidone group (incidence 3.4/1000 CVC days), and only one in the chlorhexidine group (incidence 0.36/1000 CVC days, P = 0.008). One TI was observed in the iodopovidone group (0.34/1000 CVC days), and none in the chlorhexidine group. The incidence of BSIs decreased from 1.7/1000 CVC days (5 cases) to 0.36/1000 CVC days (1 case, P = 0.06) after switching to chlorhexidine. Two CVCs were lost due to CVC-related infections in the iodopovidone group, whereas no CVC was lost due to infections in the chlorhexidine group. In comparison with 5% povidone-iodine, the use of chlorhexidine gluconate was associated with a reduction in the incidence of ESI, TI, and BSI in children on HD.

Molecular analysis, pathogenic mechanisms, and readthrough therapy on a large cohort of Kabuki syndrome patients.

Kabuki syndrome (KS) is a multiple congenital anomalies syndrome characterized by characteristic facial features and varying degrees of mental retardation, caused by mutations in KMT2D/MLL2 and KDM6A/UTX genes. In this study, we performed a mutational screening on 303 Kabuki patients by direct sequencing, MLPA, and quantitative PCR identifying 133 KMT2D, 62 never described before, and four KDM6A mutations, three of them are novel. We found that a number of KMT2D truncating mutations result in mRNA degradation through the nonsense-mediated mRNA decay, contributing to protein haploinsufficiency. Furthermore, we demonstrated that the reduction of KMT2D protein level in patients' lymphoblastoid and skin fibroblast cell lines carrying KMT2D-truncating mutations affects the expression levels of known KMT2D target genes. Finally, we hypothesized that the KS patients may benefit from a readthrough therapy to restore physiological levels of KMT2D and KDM6A proteins. To assess this, we performed a proof-of-principle study on 14 KMT2D and two KDM6A nonsense mutations using specific compounds that mediate translational readthrough and thereby stimulate the re-expression of full-length functional proteins. Our experimental data showed that both KMT2D and KDM6A nonsense mutations displayed high levels of readthrough in response to gentamicin treatment, paving the way to further studies aimed at eventually treating some Kabuki patients with readthrough inducers.

De novo unbalanced translocations in Prader-Willi and Angelman syndrome might be the reciprocal product of inv dup(15)s.

The 15q11-q13 region is characterized by high instability, caused by the presence of several paralogous segmental duplications. Although most mechanisms dealing with cryptic deletions and amplifications have been at least partly characterized, little is known about the rare translocations involving this region. We characterized at the molecular level five unbalanced translocations, including a jumping one, having most of 15q transposed to the end of another chromosome, whereas the der(15)(pter->q11-q13) was missing. Imbalances were associated either with Prader-Willi or Angelman syndrome. Array-CGH demonstrated the absence of any copy number changes in the recipient chromosome in three cases, while one carried a cryptic terminal deletion and another a large terminal deletion, already diagnosed by classical cytogenetics. We cloned the breakpoint junctions in two cases, whereas cloning was impaired by complex regional genomic architecture and mosaicism in the others. Our results strongly indicate that some of our translocations originated through a prezygotic/postzygotic two-hit mechanism starting with the formation of an acentric 15qter->q1::q1->qter representing the reciprocal product of the inv dup(15) supernumerary marker chromosome. An embryo with such an acentric chromosome plus a normal chromosome 15 inherited from the other parent could survive only if partial trisomy 15 rescue would occur through elimination of part of the acentric chromosome, stabilization of the remaining portion with telomere capture, and formation of a derivative chromosome. All these events likely do not happen concurrently in a single cell but are rather the result of successive stabilization attempts occurring in different cells of which only the fittest will finally survive. Accordingly, jumping translocations might represent successful rescue attempts in different cells rather than transfer of the same 15q portion to different chromosomes. We also hypothesize that neocentromerization of the original acentric chromosome during early embryogenesis may be required to avoid its loss before cell survival is finally assured.

Molecular mechanisms generating and stabilizing terminal 22q13 deletions in 44 subjects with Phelan/McDermid syndrome.

In this study, we used deletions at 22q13, which represent a substantial source of human pathology (Phelan/McDermid syndrome), as a model for investigating the molecular mechanisms of terminal deletions that are currently poorly understood. We characterized at the molecular level the genomic rearrangement in 44 unrelated patients with 22q13 monosomy resulting from simple terminal deletions (72%), ring chromosomes (14%), and unbalanced translocations (7%). We also discovered interstitial deletions between 17-74 kb in 9% of the patients. Haploinsufficiency of the SHANK3 gene, confirmed in all rearrangements, is very likely the cause of the major neurological features associated with PMS. SHANK3 mutations can also result in language and/or social interaction disabilities. We determined the breakpoint junctions in 29 cases, providing a realistic snapshot of the variety of mechanisms driving non-recurrent deletion and repair at chromosome ends. De novo telomere synthesis and telomere capture are used to repair terminal deletions; non-homologous end-joining or microhomology-mediated break-induced replication is probably involved in ring 22 formation and translocations; non-homologous end-joining and fork stalling and template switching prevail in cases with interstitial 22q13.3. For the first time, we also demonstrated that distinct stabilizing events of the same terminal deletion can occur in different early embryonic cells, proving that terminal deletions can be repaired by multistep healing events and supporting the recent hypothesis that rare pathogenic germline rearrangements may have mitotic origin. Finally, the progressive clinical deterioration observed throughout the longitudinal medical history of three subjects over forty years supports the hypothesis of a role for SHANK3 haploinsufficiency in neurological deterioration, in addition to its involvement in the neurobehavioral phenotype of PMS.

XX males SRY negative: a confirmed cause of infertility.

BACKGROUND: SOX9 is a widely expressed transcription factor playing several relevant functions during development and essential for testes differentiation. It is considered to be the direct target gene of the protein encoded by SRY and its overexpression in an XX murine gonad can lead to male development in the absence of Sry. Recently, a family was reported with a 178 kb duplication in the gene desert region ending about 500 kb upstream of SOX9 in which 46,XY duplicated persons were completely normal and fertile whereas the 46,XX ones were males who came to clinical attention because of infertility. METHODS AND RESULTS: We report a family with two azoospermic brothers, both 46,XX, SRY negative, having a 96 kb triplication 500 kb upstream of SOX9. Both subjects have been analyzed trough oligonucleotide array-CGH and the triplication was confirmed and characterised through qPCR, defining the minimal region of amplification upstream of SOX9 associated with 46,XX infertile males, SRY negative. CONCLUSIONS: Our results confirm that even in absence of SRY, complete male differentiation may occur, possibly driven by overexpression of SOX9 in the gonadal ridge, as a consequence of the amplification of a gene desert region. We hypothesize that this region contains gonadal specific long-range regulation elements whose alteration may impair the normal sex development. Our data show that normal XX males, with alteration in copy number or, possibly, in the critical sequence upstream to SOX9 are a new category of infertility inherited in a dominant way with expression limited to the XX background.

A 12Mb deletion at 7q33-q35 associated with autism spectrum disorders and primary amenorrhea.

An interstitial deletion of about 12Mb at 7q33-q36 was found in an adult female affected by autism and primary amenorrhea. Two genes, CNTNAP2 and NOBOX, both contained within the deletion region, have been recently associated with autism susceptibility and premature ovarian failure, respectively. Our findings reinforce the hypothesis that haploinsufficiency of both these genes is sufficient for autism development and occurrence of primary amenorrhea, confirming a previous case in which CNTNAP2 had been disrupted by a chromosome inversion and possibly enlarging the phenotype of ovarian function disturbances already demonstrated for NOBOX mutations.

13q Deletion and central nervous system anomalies: further insights from karyotype-phenotype analyses of 14 patients.

BACKGROUND: Chromosome 13q deletion is associated with varying phenotypes, which seem to depend on the location of the deleted segment. Although various attempts have been made to link the 13q deletion intervals to distinct phenotypes, there is still no acknowledged consensus correlation between the monosomy of distinct 13q regions and specific clinical features. METHODS: 14 Italian patients carrying partial de novo 13q deletions were studied. Molecular-cytogenetic characterisation was carried out by means of array-comparative genomic hybridisation (array-CGH) or fluorescent in situ hybridisation (FISH). RESULTS: Our 14 patients showed mental retardation ranging from profound-severe to moderate-mild: eight had central nervous system (CNS) anomalies, including neural tube defects (NTDs), six had eye abnormalities, nine had facial dysmorphisms and 10 had hand or feet anomalies. The size of the deleted regions varied from 4.2 to 75.7 Mb. CONCLUSION: This study is the first systematic molecular characterisation of de novo 13q deletions, and offers a karyotype-phenotype correlation based on detailed clinical studies and molecular determinations of the deleted regions. Analyses confirm that patients lacking the 13q32 band are the most seriously affected, and critical intervals have been preliminarily assigned for CNS malformations. Dose-sensitive genes proximal to q33.2 may be involved in NTDs. The minimal deletion interval associated with the Dandy-Walker malformation (DWM) was narrowed to the 13q32.2-33.2 region, in which the ZIC2 and ZIC5 genes proposed as underlying various CNS malformations are mapped.

Direct duplication 12p11.21-p13.31 mediated by segmental duplications: a new recurrent rearrangement?

We describe the characterization of an interstitial duplication of 12p, dup(12)(p11.21p13.31), by array-CGH and FISH in a patient with mental retardation and dysmorphic features. The sequence analysis of the breakpoints revealed the presence of homologous low copy repeats (LCRs) flanking the duplication region, thus suggesting that they have mediated the rearrangement. Pip-maker analysis showed that a third cluster of homologous LCRs lie distally to the two mediating the 12p duplication. We hypothesize that this duplication might be a new recurrent rearrangement and that, thanks to the different orientations of the homologous regions lying within each cluster, the three clusters are responsible for at least some of the several 12p aneuploidies reported in the literature such as direct and inverted duplications, deletions and supernumerary analphoid chromosomes. Moreover, we excluded that polymorphic inversions between these three clusters are present in the normal population.