Copy number variants analysis in a cohort of isolated and syndromic developmental delay/intellectual disability reveals novel genomic disorders, position effects and candidate disease genes.

BACKGROUND: Array-comparative genomic hybridization (array-CGH) is a widely used technique to detect copy number variants (CNVs) associated with developmental delay/intellectual disability (DD/ID). AIMS: Identification of genomic disorders in DD/ID. MATERIALS AND METHODS: We performed a comprehensive array-CGH investigation of 1,015 consecutive cases with DD/ID and combined literature mining, genetic evidence, evolutionary constraint scores, and functional information in order to assess the pathogenicity of the CNVs. RESULTS: We identified non-benign CNVs in 29% of patients. Amongst the pathogenic variants (11%), detected with a yield consistent with the literature, we found rare genomic disorders and CNVs spanning known disease genes. We further identified and discussed 51 cases with likely pathogenic CNVs spanning novel candidate genes, including genes encoding synaptic components and/or proteins involved in corticogenesis. Additionally, we identified two deletions spanning potential Topological Associated Domain (TAD) boundaries probably affecting the regulatory landscape. DISCUSSION AND CONCLUSION: We show how phenotypic and genetic analyses of array-CGH data allow unraveling complex cases, identifying rare disease genes, and revealing unexpected position effects.

Monocytes of patients with amyotrophic lateral sclerosis linked to gene mutations display altered TDP-43 subcellular distribution.

AIMS: Cytoplasmic accumulation of the nuclear protein transactive response DNA-binding protein 43 (TDP-43) is an early determinant of motor neuron degeneration in most amyotrophic lateral sclerosis (ALS) cases. We previously disclosed this accumulation in circulating lymphomonocytes (CLM) of ALS patients with mutant TARDBP, the TDP-43-coding gene, as well as of a healthy individual carrying the parental TARDBP mutation. Here, we investigate TDP-43 subcellular localization in CLM and in the constituent cells, lymphocytes and monocytes, of patients with various ALS-linked mutant genes. METHODS: TDP-43 subcellular localization was analysed with western immunoblotting and immunocytofluorescence in CLM of healthy controls (n = 10), patients with mutant TARDBP (n = 4, 1 homozygous), valosin-containing protein (VCP; n = 2), fused in sarcoma/translocated in liposarcoma (FUS; n = 2), Cu/Zn superoxide dismutase 1 (SOD1; n = 6), chromosome 9 open reading frame 72 (C9ORF72; n = 4), without mutations (n = 5) and neurologically unaffected subjects with mutant TARDBP (n = 2). RESULTS: TDP-43 cytoplasmic accumulation was found (P < 0.05 vs. controls) in CLM of patients with mutant TARDBP or VCP, but not FUS, in line with TDP-43 subcellular localization described for motor neurons of corresponding groups. Accumulation also characterized CLM of the healthy individuals with mutant TARDBP and of some patients with mutant SOD1 or C9ORF72. In 5 patients, belonging to categories described to carry TDP-43 mislocalization in motor neurons (3 C9ORF72, 1 TARDBP and 1 without mutations), TDP-43 cytoplasmic accumulation was not detected in CLM or in lymphocytes but was in monocytes. CONCLUSIONS: In ALS forms characterized by TDP-43 mislocalization in motor neurons, monocytes display this alteration, even when not manifest in CLM. Monocytes may be used to support diagnosis, as well as to identify subjects at risk, of ALS and to develop/monitor targeted treatments.

A case report of AA amyloidosis associated with familial periodic fever syndrome diagnosed after kidney transplantation: never say never.

Recurrent or "de novo" AA amyloidosis in the renal allograft is rarely described. We describe a case of severe nephrotic syndrome in a recipient of a kidney graft with a previous diagnosis of polycystic nephropathy caused by AA amyloidosis diagnosed only after the renal transplantation. The disease was possibly a tumor necrosis factor receptor-associated periodic syndrome (TRAPS). TRAPS is a rare hereditary inflammatory disease never reported to the best of our knowledge, as a de novo diagnosis in the transplantation setting. Biopsy of the renal graft, indicated for the onset of heavy proteinuria, and genetic investigation provided the clues for diagnosis.

NEMO syndrome (incontinentia pigmenti) and systemic lupus erythematosus: a new disease association.

Congenital diseases are increasingly being recognised in adults because of clinical mimicry, variable clinical picture or rarity of the disease; pregnancy is a valuable diagnostic occasion. The present case is the first report of an association report between NEMO syndrome (an acronym of the mutated, non-functioning gene, NF-kB essential modulator), a rare X-linked disease, characterised by developmental anomalies, immunodepression and skin lesions, and systemic lupus erythematosus (SLE). A 35-year-old patient affected by SLE sought clinical advice in the 8th week of gestation. The diagnosis of SLE dated back to the age of 24, when multisystemic manifestations (pleuropericarditis, weight loss, alopecia, skin involvement, joint pain, kidney involvement) were observed. She had been treated with steroids since 1999; immunosuppressive drugs had been added for short periods. Developmental anomalies were present, including oligodontia, retinal problems, anomalies of the corpus callosum and pes planovalgus. Family history included multiple miscarriages, dental malformations and oligodontia and skin blistering in the first months of life. On these bases, incontinentia pigmenti (IP; or NEMO syndrome) was diagnosed and confirmed by genetic testing. The NEMO gene is implicated in immune deficiencies as well as in autoimmune diseases. This report may suggest a role for NF-kB essential modulator in the pathogenesis of SLE, in the context of the complex immunologic deficiencies increasingly associated with autoimmune diseases.

Prevalence of SOD1 mutations in the Italian ALS population.

BACKGROUND: Five to 10% of amyotrophic lateral sclerosis (ALS) cases are reported to be familial (FALS), and mutations of SOD1 account for 20% of these cases. However, estimates of SOD1 mutation prevalence have been exclusively based on case series and clinic referral cohorts. OBJECTIVE: To assess the frequency and nature of SOD1 mutations in a large population-based cohort of Italian patients diagnosed with ALS over a 6-year period. METHODS: All ALS cases incident in Piemonte and Valle d'Aosta, Italy, are collected through a prospective epidemiologic register. Almost all patients with ALS resident in the largest province of Piemonte (Turin) have been evaluated for SOD1 mutations in the 6-year period 2000 through 2005. RESULTS: During the study period, 386 residents of Turin province were diagnosed with ALS (mean crude incidence rate of 2.9/100,000/year). Twenty-two patients (5.7%) had a positive family history of ALS. SOD1 analysis was performed in 325 patients (84.2% of the whole cohort), including all FALS cases. Five patients carried a SOD1 coding mutation, three with a family history of ALS (13.6% of FALS) and two in sporadic cases (0.7% of sporadic ALS). CONCLUSIONS: In this population-based series, the frequency of familial amyotrophic lateral sclerosis (FALS) was lower than that reported in series from ALS referral centers. While the frequency of SOD1 mutations in FALS was similar to the data reported in the literature, only 0.7% of sporadic ALS cases had a SOD1 mutation. Our data indicate that studies from referral centers may overestimate the frequency of FALS and of SOD1 mutations in sporadic ALS.

Progranulin mutations and amyotrophic lateral sclerosis or amyotrophic lateral sclerosis-frontotemporal dementia phenotypes.

OBJECTIVE: Mutations in the progranulin (PGRN) gene were recently described as the cause of ubiquitin positive frontotemporal dementia (FTD). Clinical and pathological overlap between amyotrophic lateral sclerosis (ALS) and FTD prompted us to screen PGRN in patients with ALS and ALS-FTD. METHODS: The PGRN gene was sequenced in 272 cases of sporadic ALS, 40 cases of familial ALS and in 49 patients with ALS-FTD. RESULTS: Missense changes were identified in an ALS-FTD patient (p.S120Y) and in a single case of limb onset sporadic ALS (p.T182M), although the pathogenicity of these variants remains unclear. CONCLUSION: PGRN mutations are not a common cause of ALS phenotypes.

The IVS1 +319 t>a of SOD1 gene is not an ALS causing mutation.

Amyotrophic lateral sclerosis (ALS) is caused by mutations in the gene for Cu/Zn superoxide dismutase (SOD1) in 10% of familial and sporadic cases. During the SOD1 analysis of 9 FALS and 121 SALS, in only one sporadic case we found the exonic mutation N19S; in 15 SALS patients we found a 319t>a variation in IVS1 sequence, at 108 bp upstream from exon 2. This variation has an unusually high frequency of 11% and is always in linkage disequilibrium with a described polymorphism in IVS3, +34a>c. The 319t>a variation is classified in two different public databases, HGMD and The ALS Online Database, as a splicing mutation and not as a polymorphism. The unusually high frequency of this mutation in our patients prompted us to determinate its frequency in 130 age- and gender- matched healthy controls and in 54 patients with Alzheimer's disease. We found again linkage disequilibrium with the polymorphism in intron 3, and the frequency of 11% and 7.8%, respectively. These results strongly support the idea that the IVS1 +319 t>a alone is not an ALS causing mutation, and that special care must be taken in the interpretation of data from mutations databases for correct genetic counselling.

Feasibility study for a microchip-based approach for noninvasive prenatal diagnosis of genetic diseases.

Fetal DNA in maternal plasma may represent a source of genetic material for prenatal noninvasive diagnosis of genetic diseases. We evaluated a cohort of physiological pregnancies to determine if fetal DNA can be retrieved at any gestational week in sufficient quantity to be analyzed with advanced mutation detection technologies. We performed fetal DNA quantification by real-time polymerase chain reaction (PCR) on the SRY gene in 356 women sampled from 6 to 40 gestational weeks. Fetal DNA was retrieved at any week. All female fetuses were correctly identified. In 5 of 188 (2.6%) male-bearing pregnancies, no amplification was obtained. For noninvasive testing, complete clearance of fetal DNA after delivery is mandatory. Long-term persistence was not detected in women with previous sons or abortions. These findings confirm that maternal plasma may represent the optimal source of fetal genetic material. For noninvasive diagnosis of genetic diseases, we evaluated microchip technology. The detection limit for a minority allele determined by diluting a mutated DNA into a wild-type plasma sample was 5 genome equivalents, indicating that the test might be applied to the identification of paternally inherited fetal alleles in maternal plasma. The addition of peptide nucleic acids (PNAs) to either the PCR reaction or the chip hybridization mixture allowed approximately 50% inhibition of wild-type allele signals.

Severe and long-lasting disruption of T-cell receptor diversity in human myeloma after high-dose chemotherapy and autologous peripheral blood progenitor cell infusion.

Vaccine-based strategies are currently under investigation as a means of inducing tumour-specific immune responses and improving the clinical outcome of multiple myeloma (MM) patients in remission after high-dose chemotherapy and peripheral blood progenitor cell (PBPC) infusion. The immune competence of these patients was investigated by determining the overall diversity of the T-cell receptor (TCR) repertoire in the peripheral blood (PB) and bone marrow (BM). The average time after transplantation was 13 months. The clonality and reciprocal usage of BV gene segments (TCRBV repertoire) was estimated at the cDNA level and membrane protein expression. The TCRBV repertoire of MM was severely disrupted compared with age-matched normal donors. On average, one-third of the total repertoire in both the PB and the BM consisted of T cells expressing oligoclonal TCRbeta transcripts. Flow cytometry showed an increased frequency of abnormally expanded BV subfamilies at both sites. BV expansions were predominantly CD8+ and had the phenotype of antigen-experienced memory T cells as well as T cells with the naive phenotype. Oligoclonality was not restricted to phenotypically expanded BV subfamilies, but also involved normally represented BV subfamilies. The TCR repertoire of MM in remission was then compared with monoclonal gammopathy of undetermined significance (MGUS) and MM patients at diagnosis. The degree of TCR diversity was similar in age-matched normal donors and MGUS, but progressively decreased from MGUS to MM at diagnosis and then to MM in remission. These data indicate that: (1) there is a long-lasting and severe disruption of TCR diversity after high-dose chemotherapy and PBPC infusion, and (2) the extent of TCR disruption may affect the clinical outcome of vaccine-based strategies delivered at the stage of minimal residual disease.

Molecular basis of childhood deafness resulting from mutations in the GJB2 (connexin 26) gene.

Mutations in the GJB2 gene have been identified in many patients with childhood deafness, 35delG being the most common mutation in Caucasoid populations. We have analyzed a total of 576 families/unrelated patients with recessive or sporadic deafness from Italy and Spain, 193 of them being referred as autosomal recessive, and the other 383 as apparently sporadic cases (singletons). Of the 1,152 unrelated GJB2 chromosomes analyzed from these patients, 37% had GJB2 mutations. Twenty-three different mutations were detected (1 in-frame deletion, 4 nonsense, 5 frameshift, and 13 missense mutations). Mutation 35delG was the most common, accounting for 82% of all GJB2 deafness alleles. The relative frequency of 35delG in Italy and Spain was different, representing 88% of the alleles in Italian patients and only 55% in the Spanish cases. Eight non-35delG mutations were detected more than once (V37I, E47X, 167delT, L90P, 312de114, 334delAA, R143W, and R184P), with relative frequencies ranging between 0.5 and 1.6% of the GJB2 deafness alleles. The information based on conservation of amino acid residues, coexistence with a second GJB2 mutation or absence of the mutation in non-deaf control subjects, suggests that most of these missense changes should be responsible for the deafness phenotype.

A pilot C282Y hemochromatosis screening in Italian newborns by TaqMan technology.

Hereditary hemochromatosis (HH) is a disorder of iron metabolism that leads to iron overload in middle age and can be caused by homozygosity for the C282Y mutation in the HFE gene. Preliminary studies have estimated the frequency of this mutation at 0.5-1% in Italy, but this has not been verified on a large sample. We analyzed 1,331 Italian newborns for the C282Y mutation in the HFE gene using dried blood spots (DBS) from the Neonatal Screening Center in Turin, Italy. The mutation was assessed using a semi-automatable 5'-nuclease assay (TaqMan technology). We detected 55 heterozygotes and no homozygotes in our sampling, resulting in an overall frequency of 2.1% +/- 0.6 for the C282Y allele. Differences in allele frequency were observed, and ranged from 2.7% +/- 1.3 in samples from Northern Italy, to 1.7% +/- 0.9 in samples from Central-Southern Italy. The low frequency of the at-risk genotype for iron overload suggests that genetic screening for HFE in Italy would not be cost effective. The present study, in addition to defining C282Y frequency, documents detection of the major HFE mutation on routine DBS samples from neonatal screening programs using a semi-automatable, rapid, reliable, and relatively inexpensive approach.

Analysis of 31 CFTR mutations by polymerase chain reaction/oligonucleotide ligation assay in a pilot screening of 4476 newborns for cystic fibrosis.

OBJECTIVES: Molecular biological testing for genetic diseases has grown rapidly, but speed, accuracy, specificity, sensitivity, throughput, and cost become more important as large scale screening is considered. This is a pilot study of an assay for the simultaneous detection of up to 31 cystic fibrosis mutations in a multicentre population based screening of 4476 Italian newborns. METHODS: The assay is a polymerase chain reaction, followed by an oligonucleotide ligation assay (PCR/OLA) and finally a sequence coded separation. It allows the detection of up to 31 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Guthrie spots were used as a source of template DNA. RESULTS: 144 carriers were detected during the analysis of 4476 samples, which translates into a carrier frequency of 1/31.1. Forty two carriers were detected from 1341 samples in Pavia (1/31.9), 53 from 1574 in Turin (1/29.7), and 49 from 1561 in San Giovanni Rotondo (1/31.8). Fifteen different mutations were detected, the most common being delta F508 (0.625). Other common mutations included G542X (16 of 144), which was particularly common in southern Italy (14 of 49), N1303K (8 of 144), and R117H (8 of 144), detected only in the northern centres. CONCLUSIONS: PCR/OLA is a robust, accurate, user friendly method for cystic fibrosis screening of newborns using blood spots in a semiautomated way at a low cost per mutation (0.8 Euro).

Long first metacarpal in monozygotic twins with probable Baller-Gerold syndrome.

We report on a pair of monozygotic twins with probable Baller-Gerold syndrome (BGS). Twin A had severe coronal craniosynostosis. Twin B had right radioulnar and ipsilateral first metacarpal hypoplasia. Both had bilateral fifth finger clinodactyly. Assuming that the twins were truly monozygotic, a single genetic disorder (i.e., BGS) could explain the variable expression. Together the twins have the typical anomalies of BGS. The diagnosis was supported by the metacarpophalangeal profile (MPP) which confirmed hypoplasia of the first right metacarpal in Twin A and bilateral fifth finger brachymesophalangy in both twins. Furthermore, the MPP showed an unexpected abnormal lengthening of the first metacarpal (unilateral in Twin A and bilateral in Twin B), a previously undetected radial ray defect in BGS. These findings suggest the possibility that the MPP may assist recognition of mild cases of BGS such as those with apparently isolated craniosynostosis or isolated upper limbs defects.

Multiple independent molecular etiology for limb-girdle muscular dystrophy type 2A patients from various geographical origins.

Limb-girdle muscular dystrophies (LGMDs) are a group of neuromuscular diseases presenting great clinical heterogeneity. Mutations in CANP3, the gene encoding muscle-specific calpain, were used to identify this gene as the genetic site responsible for autosomal recessive LGMD type 2A (LGMD2A; MIM 253600). Analyses of the segregation of markers flanking the LGMD2A locus and a search for CANP3 mutations were performed for 21 LGMD2 pedigrees from various origins. In addition to the 16 mutations described previously, we report 19 novel mutations. These data indicate that muscular dystrophy caused by mutations in CANP3 are found in patients from all countries examined so far and further support the wide heterogeneity of molecular defects in this rare disease.

Systematic use of dystrophin testing in muscle biopsies: results in 201 cases.

We studied dystrophin with both immunohistochemistry and immunoblotting in 201 muscle biopsies stored in liquid nitrogen during the period 1985-92. The systematic use of dystrophin testing combined with DNA analysis and with 3-10 years follow-up of the patients yielded a significant modification of the diagnoses made previously and identified dystrophinopathies with unusual expression and course. Seventeen out of 152 (11.18%) diagnoses in males and 8 out of 49 (16.32%) in females were modified by dystrophin testing. Most diagnostic errors (9 out of 27 diagnoses) were in the group Becker muscular dystrophy-limb girdle muscular dystrophy, confirming the clinical overlap of the two diseases. Unusual expressions of dystrophinopathy included muscular dystrophy with early elbow contractures (two patients), recurrent myoglobinuria (one patient), dilating cardiomyopathy (two patients), myoglobinuria and associated dilating cardiomyopathy (one patient), very late-onset benign myopathy (two patients and one manifesting carrier) and congenital myopathy (one manifesting carrier). In the group 'idiopathic hyper-CKaemia', we did not find any dystrophinopathy in 34 males, whereas five out of nine females were found to be carriers. Immunohistochemical analysis of dystrophin using the monoclonal antibody against the C-terminus detected 99% of protein defects and was found to be the most cost-effective way of revealing dystrophinopathies. The combined use of immunohistochemical analysis with the antibody against the C-terminus and immunoblotting with the antibody against the core of the protein appears to be a highly reliable diagnostic approach (100% detection rate).

Retinitis pigmentosa, hypopituitarism, nephronophthisis, and mild skeletal dysplasia (RHYNS): a new syndrome?

We report on a 17 6/12-year-old boy with nephronophthisis, retinitis pigmentosa, left upper eyelid ptosis, enopthalmos, transmissive deafness, GH and TSH deficiency, and mild skeletal dysplasia. A similar case was reported by Bianchi et al. [1988: Helv Paediatr Acta 43:449-455] in another Italian patient. Here we confirm the previous observations and argue that both patients might be affected by a new syndrome.

Autosomal dominant polycystic kidney disease (ADPKD) in an Italian family carrying a novel nonsense mutation and two missense changes in exons 44 and 45 of the PKD1 Gene.

Sixty-seven Italian patients with autosomal dominant polycystic kidney disease (ADPKD) were screened for mutations in the 3' unique region of the PKD1 gene, using heteroduplex DNA analysis. Novel aberrant bands were detected in 3 patients from the same family. DNA sequencing showed a C to T transition in exon 44 (C12269T), resulting in a premature stop codon (R4020X), predicted to impair the synthesis of the putative intracytoplasmic C-terminus tail of the PKD1 protein, polycystin. The mutation also generates a novel DdeI restriction site, and the abnormal restriction pattern was observed both on genomic DNA and on cDNA from the affected relatives, indicating that this is indeed the pathogenetic molecular lesion. Reverse transcriptase-polymerase chain reaction (RT-PCR) performed on lymphocyte mRNA showed that the mutant transcript is normally present and stable. No aberrantly spliced mRNAs were detected. Interestingly, the mutant PKD1 chromosome in this family also bears two missense mutations downstream (A12341G and C12384T), not found in the other ADPKD families studied.

Prenatal diagnosis of limb-girdle muscular dystrophy type 2A.

A branch of a highly inbred family was referred for prenatal counseling with an initial misdiagnosis of Becker Muscular Dystrophy (BMD) due to the limited clinical and laboratory data obtained in pre-dystrophin era and hidden family information. In a second branch of the family with a diagnosis of limb-girdle muscular dystrophy type 2A (LGMD2A) molecular studies revealed a homozygous 550 delta A mutation in the calcium-activated neutral protease 3 (calpain 3, CANP3) gene in the affected members. Finally, in the third branch of the family, it turned out that both parents were heterozygous for the 550 delta A mutation and the 13-week-old fetus was homozygous. The same mutation subsequently also was found in the first branch of the family. The parents were informed that the risk of their child of developing the disease would be very high given that he was carrying the same homozygous mutation of the other affected members. They were informed also that in another population (in Reunion Island) the same disease does not necessarily follow such a simple pattern of inheritance. After counseling the parents decided to terminate the pregnancy.

Analysis of the rhodopsin and peripherin/RDS gene in two families with pattern dystrophy of the retinal pigment epithelium.

Mutations of the peripherin/retinal degeneration slow (RDS) gene have been reported in autosomal dominant retinitis pigmentosa and variable forms of pattern dystrophy of the retinal pigment epithelium. We screened the rhodopsin and the peripherin/RDS gene in the members of two families who presented the clinical features of pattern dystrophy of the retinal pigment epithelium transmitted as an autosomal dominant trait. No migration patterns were detected in single strand conformation polymorphism or hydrolink gels. Both the rhodopsin and the peripherin/RDS gene were normal in one family. In the second, the proband had a normal rhodopsin gene and, although he passed a different haplotype to each of his affected daughters, there was no linkage with the peripherin/RDS gene. The origin of the retinal disturbance in our two pedigrees must therefore be sought, if indeed DNA is involved, elsewhere in the genome. Our findings provide additional evidence that pattern dystrophies of the retinal pigment epithelium may be pathogenically related in spite of different etiological origins. The genetic polymorphism can probably account for the wide range of phenotypes.