Recurrent copy number variations as risk factors for autism spectrum disorders: analysis of the clinical implications.

Chromosomal microarray analysis (CMA) is currently considered a first-tier diagnostic assay for the investigation of autism spectrum disorders (ASD), developmental delay and intellectual disability of unknown etiology. High-resolution arrays were utilized for the identification of copy number variations (CNVs) in 195 ASD patients of Greek origin (126 males, 69 females). CMA resulted in the detection of 65 CNVs, excluding the known polymorphic copy number polymorphisms also found in the Database of Genomic Variants, for 51/195 patients (26.1%). Parental DNA testing in 20/51 patients revealed that 17 CNVs were de novo, 6 paternal and 3 of maternal origin. The majority of the 65 CNVs were deletions (66.1%), of which 5 on the X-chromosome while the duplications, of which 7 on the X-chromosome, were rarer (22/65, 33.8%). Fifty-one CNVs from a total of 65, reported for our cohort of ASD patients, were of diagnostic significance and well described in the literature while 14 CNVs (8 losses, 6 gains) were characterized as variants of unknown significance and need further investigation. Among the 51 patients, 39 carried one CNV, 10 carried two CNVs and 2 carried three CNVs. The use of CMA, its clinical validity and utility was assessed.

SMA prenatal diagnosis: a modified protocol to help differentiation between deletions and gene conversion.

In SMA, unusual findings such as deletions restricted only to SMN1 exon 8, inspite of honozygous SMN1 exons 7-8 deletions in the family, may obscure final diagnosis. Application of a modified PCR procedure allowed discrimination between a deletion or a gene conversion event in a case of prenatal diagnosis.

Noonan syndrome and systemic lupus erythematosus in a patient with a novel KRAS mutation.

Noonan syndrome is characterised by distinct facial stigmata, short stature and congenital cardiopathy. It has a high genetic heterogeneity and mutations in six different genes can be involved. We report a patient with Noonan syndrome and a novel KRAS mutation who presents systemic lupus erythematosus.

Two cases of Sotos syndrome with novel mutations of the NSD1 gene.

Mutations and deletions of the NSD1 gene, located on chromosome 5q35, are responsible for over 90% of cases of Sotos syndrome. Fluorescent in situ hybridization analysis (FISH), MLPA or multiplex quantitative PCR allow detection of total/partial NSD1 deletions and direct sequencing allows detection of NSD1 mutations. We describe two boys with Sotos syndrome in whom PCR amplification and direct sequencing of the NSD1 gene identified two novel mutations not previously described: c.4736dupG in exon 12 and c.3938_3939insT in exon 7. In addition to the cardinal and major features of the syndrome (abnormal facial appearance, overgrowth, cardiac anomalies, renal anomalies, hypotonia, neonatal jaundice, seizures and brain MRI abnormalities) in both patients, one boy also had cryptorchidism and vertebral anomalies, features considered not common. Despite the wide range of possible combinations of phenotypic features, molecular analysis can correctly identify Sotos syndrome.

10 years' experience in fragile X testing among mentally retarded individuals in Greece: a molecular and epidemiological approach.

Fragile X syndrome, the second most common genetic cause of mental retardation, is due to the expansion of a trinucleotide repeat (CGG)n within the first exon of the FMR-1 gene. Molecular genetic analysis provides accurate diagnosis and facilitates genetic counselling and prenatal testing. Screening for the fragile X mutation in a sample of 3,888 individuals in Greece is reported: 1,755 children with non-specific mental retardation, 1,733 parents and other family members and 400 normal individuals. Molecular analysis allowed for the identification and characterization of 52 fragile X families confirming the clinical diagnosis in 57 males and 4 females. Sixty-six female carriers (6 mentally retarded) and 4 normal transmitting males were also identified. Four severely retarded males and their mothers carried unmethylated premutations, while a moderately retarded girl had a deletion of approximately equal to 150 bp. Overall sizing of the CGG repeat produced an allele distribution of 6-58 CGG repeats (mean 28-30), similar to that in other Caucasian populations.

Coinheritance of mutated SMN1 and MECP2 genes in a child with phenotypic features of spinal muscular atrophy (SMA) type II and Rett syndrome.

Spinal muscular atrophy (SMA) is a neuromuscular autosomal recessive disease characterized by progressive muscle weakness and atrophy combined with motor neuron degeneration caused by mutations in the SMN 1 gene locus (5q11.2-13.2). Rett syndrome (RS) is an X-linked dominant neurodevelopmental disorder caused by mutations in MECP2 (Xq28) and characterized by normal development until 6-12 months of age, followed by regression with loss of acquired skills, gradual onset of microcephaly, stereotypic hand movements and psychomotor delay. We report a 6-year-old girl who, at 2 years of age, presented with hypotonia, psychomotor delay, amyotrophy and areflexia of the lower extremities. Molecular DNA analysis (PCR-RFLP's) for SMA type II revealed that both exons 7 and 8 of SMN 1 gene were deleted. Over the past 4 years, onset of stereotypic hand-washing movements, epileptic seizures, microcephaly, hyperventilation/breath-holding attacks and severe psychomotor delay raised the suspicion of the coexistence of RS. DNA analysis (DGGE and sequencing) identified the hotspot missense mutation R306C (c.916C>T) in exon 4 of the MECP2 gene. The coinheritance of SMA and RS, two rare monogenic syndromes in the same patient, has not been previously reported. Thorough clinical evaluation in combination with DNA analysis, allowed accurate diagnosis, providing valuable information for the genetic counseling of the family.

Spontaneous ovulation in a true hermaphrodite with normal male phenotype and a rare 46,XX/47,XXY Klinefelter's mosaic karyotype.

BACKGROUND: Most true hermaphrodite patients--characterized by the presence of both ovarian and testicular tissue--demonstrate ambiguous genitalia and are diagnosed at birth, most commonly bearing a 46,XX karyotype. PATIENT AND METHODS: We report on a 13-year-old boy presenting with left scrotal hemorrhage. He had a left inguinal hernia, a palpable testis in the right, normal male external genitalia and significant gynecomastia. During operation, the left gonad and adjacent tissue were removed for histological examination, which revealed the presence of a normal ovary, rich in follicles and a ruptured corpus luteum, suggestive of spontaneous ovulation, with a normal ipsilateral adnexa and semi-uterus. Biopsy of the right gonad revealed a dysgenetic testicle. Endocrinological assessment postoperatively depicted high FSH, pubertal testosterone and low estradiol levels. Cytogenetic analysis in peripheral blood lymphocytes and FISH of the right gonad revealed a 46,XX (70-60%)/47,XXY (30-40%) karyotype, respectively, while molecular analysis verified the presence of SRY and azoospermia factor genes. CONCLUSION: The importance of full histological, cytogenetic and molecular investigation and of interdisciplinary approach in every single patient with sex differentiation disorders is highlighted by this rare case of spontaneous ovulation in a true hermaphrodite with normal male external genitalia and Klinefelter mosaicism.

Two high throughput technologies to detect segmental aneuploidies identify new Williams-Beuren syndrome patients with atypical deletions.

OBJECTIVE: To develop and compare two new technologies for diagnosing a contiguous gene syndrome, the Williams-Beuren syndrome (WBS). METHODS: The first proposed method, named paralogous sequence quantification (PSQ), is based on the use of paralogous sequences located on different chromosomes and quantification of specific mismatches present at these loci using pyrosequencing technology. The second exploits quantitative real time polymerase chain reaction (QPCR) to assess the relative quantity of an analysed locus. RESULTS: A correct and unambiguous diagnosis was obtained for 100% of the analysed samples with either technique (n = 165 and n = 155, respectively). These methods allowed the identification of two patients with atypical deletions in a cohort of 182 WBS patients. Both patients presented with mild facial anomalies, mild mental retardation with impaired visuospatial cognition, supravalvar aortic stenosis, and normal growth indices. These observations are consistent with the involvement of GTF2IRD1 or GTF2I in some of the WBS facial features. CONCLUSIONS: Both PSQ and QPCR are robust, easy to interpret, and simple to set up. They represent a competitive alternative for the diagnosis of segmental aneuploidies in clinical laboratories. They have advantages over fluorescence in situ hybridisation or microsatellites/SNP genotyping for detecting short segmental aneuploidies as the former is costly and labour intensive while the latter depends on the informativeness of the polymorphisms.

The phenotypic spectrum in patients with arginine to cysteine mutations in the COL2A1 gene.

BACKGROUND: The majority of COL2A1 missense mutations are substitutions of obligatory glycine residues in the triple helical domain. Only a few non-glycine missense mutations have been reported and among these, the arginine to cysteine substitutions predominate. OBJECTIVE: To investigate in more detail the phenotype resulting from arginine to cysteine mutations in the COL2A1 gene. METHODS: The clinical and radiographic phenotype of all patients in whom an arginine to cysteine mutation in the COL2A1 gene was identified in our laboratory, was studied and correlated with the abnormal genotype. The COL2A1 genotyping involved DHPLC analysis with subsequent sequencing of the abnormal fragments. RESULTS: Six different mutations (R75C, R365C, R519C, R704C, R789C, R1076C) were found in 11 unrelated probands. Each mutation resulted in a rather constant and site-specific phenotype, but a perinatally lethal disorder was never observed. Spondyloarthropathy with normal stature and no ocular involvement were features of patients with the R75C, R519C, or R1076C mutation. Short third and/or fourth toes was a distinguishing feature of the R75C mutation and brachydactyly with enlarged finger joints a key feature of the R1076C substitution. Stickler dysplasia with brachydactyly was observed in patients with the R704C mutation. The R365C and R789C mutations resulted in classic Stickler dysplasia and spondyloepiphyseal dysplasia congenita (SEDC), respectively. CONCLUSIONS: Arginine to cysteine mutations are rather infrequent COL2A1 mutations which cause a spectrum of phenotypes including classic SEDC and Stickler dysplasia, but also some unusual entities that have not yet been recognised and described as type II collagenopathies.

Clinical, genetic, and cellular analysis of 49 osteopetrotic patients: implications for diagnosis and treatment.

BACKGROUND: Osteopetrosis, a genetic disease characterised by osteoclast failure, is classified into three forms: infantile malignant autosomal recessive osteopetrosis (ARO), intermediate autosomal recessive osteopetrosis (IRO), and autosomal dominant osteopetrosis (ADO). METHODS: We studied 49 patients, 21 with ARO, one with IRO, and 27 with type II ADO (ADO II). RESULTS: Most ARO patients bore known or novel (one case) ATP6i (TCIRG1) gene mutations. Six ADO II patients had no mutations in ClCN7, the only so far recognised gene implicated, suggesting involvement of yet unknown genes. Identical ClCN7 mutations produced differing phenotypes with variable degrees of severity. In ADO II, serum tartrate resistant acid phosphatase was always elevated. Bone alkaline phosphatase (BALP) was generally low, but osteocalcin was high, suggesting perturbed osteoblast differentiation or function. In contrast, BALP was high in ARO patients. Elevated osteoclast surface/bone surface was noted in biopsies from most ARO patients. Cases with high osteoclasts also showed increased osteoblast surface/bone surface. ARO osteoclasts were morphologically normal, with unaltered formation rates, intracellular pH handling, and response to acidification. Their resorption activity was greatly reduced, but not abolished. In control osteoclasts, all resorption activity was abolished by combined inhibition of proton pumping and sodium/proton antiport. CONCLUSIONS: These findings provide a rationale for novel therapies targeting pH handling mechanisms in osteoclasts and their microenvironment.

Identification of autosomal supernumerary chromosome markers (SMCs) by fluorescent in situ hybridization (FISH).

Supernumerary marker chromosomes (SMCs) are rare chromosomal abnormalities resulting in partial trisomy of specific genomic regions with characteristic phenotypic effects. Twenty six cases with autosomal SMCs are reported. Four were identified prenatally and 22 postnatally in children, aged from 8 days to 15 years, who were referred for genetic evaluation because of various congenital anomalies and developmental delay. In 22 of the 26 cases, the SMCs were de novo, in two they were familial and in another two a 11;22 reciprocal translocation was revealed in the mothers. In only one patient was the SMC present in a mosaic form. Sequential fluorescent in situ hybridization studies (FISH) using Whole Chromosome Paint (WCP) probes were performed in order to determine the chromosomal origin of the SMCs. Sixteen of them originated from chromosome 15, five were shown to be an isochromosome 18p and one was derived from chromosome 22, but did not contain the DiGeorge/ VCFS critical region. In two instances, the SMCs were derivatives of chromosome 13 and in two the SMCs resulted from a 11;22 maternal translocation and contained material from both chromosomes 11 and 22. Molecular investigation of two of the patients with an SMC[15] revealed three copies of the SNRPN gene, but the diagnosis of PW/AS due to possible imprinting was excluded in both patients by a methylation-specific PCR. FISH and molecular studies have greatly facilitated the characterization of marker chromosomes. As more SMCs are classified, better genetic counseling and risk evaluation can be achieved.

Clinical manifestations in 17 Greek patients with Goldenhar syndrome.

Goldenhar (GS) syndrome is a well-recognised developmental disorder involving first and second branchial arches and characterized by considerable phenotypic variability. The present study presents clinical data on the morphologic features, hearing, ophthalmologic, orthopaedic, neurological, cardiovascular, genitourinary and gastrointestinal evaluation of 17 Greek patients (one pair of monozygotic twins) aged 20 days to 23 years with the clinical diagnosis of GS and with a normal karyotype. The most consistent findings were auricular defects (94%), followed by facial (76%) and ocular anomalies (65%), 70% unilateral, mainly right-sided. In the majority of our patients (90%) mandibular hypoplasia was ipsilateral to the dysplastic ear or the most severely affected ear in bilateral cases. Hearing loss, mainly conductive, was noted in 76% of our patients. Skeletal defects were evident in 23%, while cardiovascular, genitourinary and gastrointestinal in 18%, 23% and 12% respectively. The most frequent neurological manifestation was facial nerve paralysis (12%), while the incidence of mental retardation was higher (23%) than reported in the literature, presumably attributed to the severe hearing and vision loss. In a pair of monozygotic twins of our study discordance of clinical findings was noted. Precise evaluation of GS patients and multidisciplinary care management is necessary to avoid possible complications of many systems and to offer appropriate genetic counselling to the family.

Partial monosomy14q involving FOXG1 and NOVA1 in an infant with microcephaly, seizures and severe developmental delay.

BACKGROUND: FOXG1 gene mutations have been associated with the congenital variant of Rett syndrome (RTT) since the initial description of two patients in 2008. The on-going accumulation of clinical data suggests that the FOXG1-variant of RTT forms a distinguishable phenotype, consisting mainly of postnatal microcephaly, seizures, hypotonia, developmental delay and corpus callosum agenesis. CASE PRESENTATION: We report a 6-month-old female infant, born at 38 weeks of gestation after in vitro fertilization, who presented with feeding difficulties, irritability and developmental delay from the first months of life. Microcephaly with bitemporal narrowing, dyspraxia, poor eye contact and strabismus were also noted. At 10 months, the proband exhibited focal seizures and required valproic acid treatment. Array-Comparative Genomic Hybridization revealed a 4.09 Mb deletion in 14q12 region, encompassing the FOXG1 and NOVA1 genes. The proband presented similar feature with patients with 14q12 deletions except for dysgenesis of corpus callosum. Disruption of the NOVA1 gene which promotes the motor neurons apoptosis has not yet been linked to any human phenotypes and it is uncertain if it affects our patient's phenotype. CONCLUSIONS: Since our patient is the first reported case with deletion of both genes (FOXG1-NOVA1), thorough clinical follow up would further delineate the Congenital Rett-Variant phenotypes.

Detection of 22q11.2 deletion among 139 patients with Di George/Velocardiofacial syndrome features.

Cytogenetic and FISH analysis was performed in 139 patients to detect the pathognomonic of Di George/ Velocardiofacial syndrome (DGS/VFCS) deletion 22q11.2. An abnormal karyotype was revealed in 2/139 cases (47, XXY and 46, XX, 2p+). A deletion was found in 17/139 (12.2%) patients (14 males/ 3 females), inherited in 3 (2 maternal and 1 paternal). Patients with 22q11.2 deletion exhibited facial dysmorphic features (82%), congenital heart defects (70%), immunological problems (47%), multiple congenital anomalies (64%), hypocalcemia (47%), mental retardation/learning difficulties (35%), cleft palate/velopharyngeal insufficiency (23.5%), seizures/hypotonia (23%) and growth retardation (12%). Among 56/139 patients with detailed available clinical data, the 22q11.2 deletion was confirmed in all cases with hypocalcemia and in over half of the cases with multiple congenital anomalies, immunological problems and hypotonia/seizures (70%, 60% and 57%, respectively). Genetic reevaluation of 39 patients without the 22q11.2 deletion contributed to the classification of 14 (37%) under different syndromes, emphasizing the need for stricter referral criteria.

A female with Coffin-Lowry syndrome and "cataplexy".

Coffin-Lowry syndrome (CLS) is an X-linked semidominant condition, caused by mutations in the gene encoding the ribosomal protein S6 kinase-2 (RSK-2), a growth factor regulating protein kinase, which is mapped to Xp 22.2. The syndrome is mainly seen in males. It is manifested by moderate to severe mental retardation and characteristic facial, hand and skeletal malformations. We present a female patient with fully manifested CLS, confirmed by molecular analysis, who experienced daily drop episodes, diagnosed as "cataplexy". The episodes were precipitated by emotional or auditory stimuli and were significantly reduced, by selective serotonine re-uptake inhibitors.

Mutation spectrum and phenotypic manifestation in FSHD Greek patients.

Facioscapulohumeral muscular dystrophy (FSHD) is a genetic myopathy with a remarkable intra- and inter-familial clinical heterogeneity. This study reports the clinical and genetic analysis of 133 individuals from 71 unrelated Greek families based on a revised clinical severity score (rCSS) index which was developed for clinical assessment regarding the disease progression. A high ratio (31/62, 50%) of probands' family members was found to be asymptomatic or minimally affected gene carriers of a contracted 4q allele. Moreover, a notable clinical variability of FSHD is reported concerning the detection of an identical de novo 13 b EcoRI fragment in monozygotic twins, as well as indications of founder effect. This is the first survey that presents data of FSHD families from an East Mediterranean country supporting the speculation that the prevalence of disease might be significantly underestimated and that synergistic factors could play an essential role on the progression of the disease.

Dental abnormalities in Schimke immuno-osseous dysplasia.

Described for the first time in 1971, Schimke immuno-osseous dysplasia (SIOD) is an autosomal-recessive multisystem disorder that is caused by bi-allelic mutations of SMARCAL1, which encodes a DNA annealing helicase. To define better the dental anomalies of SIOD, we reviewed the records from SIOD patients with identified bi-allelic SMARCAL1 mutations, and we found that 66.0% had microdontia, hypodontia, or malformed deciduous and permanent molars. Immunohistochemical analyses showed expression of SMARCAL1 in all developing teeth, raising the possibility that the malformations are cell-autonomous consequences of SMARCAL1 deficiency. We also found that stimulation of cultured skin fibroblasts from SIOD patients with the tooth morphogens WNT3A, BMP4, and TGFß1 identified altered transcriptional responses, raising the hypothesis that the dental malformations arise in part from altered responses to developmental morphogens. To the best of our knowledge, this is the first systematic study of the dental anomalies associated with SIOD.

Severe Developmental Delay in a Patient with 7p21.1-p14.3 Microdeletion Spanning the TWIST Gene and the HOXA Gene Cluster.

We describe a patient with a rare interstitial deletion of chromosome 7p21.1-p14.3 detected by array-CGH. The deletion encompassed 74 genes and caused haploinsufficiency (or loss of allele) of 6 genes known to be implicated in different autosomal dominant genetic disorders: TWIST, DFNA5, CYCS, HOXA11, HOXA13, and GARS. The patient had several morphological abnormalities similar to Saethre-Chotzen syndrome (caused by TWIST mutations) including craniosynostosis of the coronal suture and anomalies similar to those seen in hand-foot-uterus syndrome (caused by HOXA13 mutations) including hypospadias. The combined phenotype of Saethre-Chotzen syndrome and hand-foot-uterus syndrome of our patient closely resembles a previously reported case with a cytogenetically visible small deletion spanning 7p21-p14.3. We therefore conclude that microdeletions of 7p spanning the TWIST gene and HOXA gene cluster lead to a clinically recognizable 'haploinsufficiency syndrome'.

Cohen syndrome: two new cases in siblings.

UNLABELLED: Cohen syndrome is a rare genetic disorder consisting of truncal obesity, hypotonia, mental retardation, characteristic facial appearance and ocular anomalies. Other diagnostic clinical features include narrow hands and feet, low growth parameters, neutropenia and chorioretinal dystrophy. We describe the similarities in the clinical and developmental profile of two siblings with Cohen syndrome, providing evidence for autosomal recessive inheritance in this condition. CONCLUSION: The diagnosis of Cohen syndrome should be suspected in mentally retarded children with the above characteristics. Neutropenia and ocular anomalies with high-grade myopia and chorioretinal dystrophy are also considered important findings and can aid in the clinical diagnosis especially at an early age.