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1.  Identification of a rare 17p13.3 duplication including the BHLHA9 and YWHAE genes in a family with developmental delay and behavioural problems 
BMC Medical Genetics  2012;13:93.
Background
Deletions and duplications of the PAFAH1B1 and YWHAE genes in 17p13.3 are associated with different clinical phenotypes. In particular, deletion of PAFAH1B1 causes isolated lissencephaly while deletions involving both PAFAH1B1 and YWHAE cause Miller-Dieker syndrome. Isolated duplications of PAFAH1B1 have been associated with mild developmental delay and hypotonia, while isolated duplications of YWHAE have been associated with autism. In particular, different dysmorphic features associated with PAFAH1B1 or YWHAE duplication have suggested the need to classify the patient clinical features in two groups according to which gene is involved in the chromosomal duplication.
Methods
We analyze the proband and his family by classical cytogenetic and array-CGH analyses. The putative rearrangement was confirmed by fluorescence in situ hybridization.
Results
We have identified a family segregating a 17p13.3 duplication extending 329.5 kilobases by FISH and array-CGH involving the YWHAE gene, but not PAFAH1B1, affected by a mild dysmorphic phenotype with associated autism and mental retardation. We propose that BHLHA9, YWHAE, and CRK genes contribute to the phenotype of our patient. The small chromosomal duplication was inherited from his mother who was affected by a bipolar and borderline disorder and was alcohol addicted.
Conclusions
We report an additional familial case of small 17p13.3 chromosomal duplication including only BHLHA9, YWHAE, and CRK genes. Our observation and further cases with similar microduplications are expected to be diagnosed, and will help better characterise the clinical spectrum of phenotypes associated with 17p13.3 microduplications.
doi:10.1186/1471-2350-13-93
PMCID: PMC3495055  PMID: 23035971
Familial 17p13.3 duplication syndrome; PAFAH1B1 and YWHAE genes; Array-CGH
2.  Left ventricular hypertrabeculation/noncompaction with epilepsy, other heart defects, minor facial anomalies and new copy number variants 
BMC Medical Genetics  2012;13:60.
Background
Left ventricular hypertrabeculation/noncompaction (LVHT) is a cardiac abnormality of unknown etiology which has been described in children as well as in adults with and without chromosomal aberrations. LVHT has been reported in association with various cardiac and extracardiac abnormalities like epilepsy and facial dysmorphism.
Case presentation
A unique combination of LVHT, atrial septal defect, pulmonary valve stenosis, aortic stenosis, epilepsy and minor facial anomalies is presented in a 5.5 years old girl. Microarray-based genomic hybridization (array-CGH) detected six previously not described copy number variants (CNVs) inherited from a clinically unaffected father and minimally affected mother, thus, most likely, not clinically significant but rare benign variants.
Conclusions
Despite this complex phenotype de novo microdeletions or microduplications were not detected by array CGH. Further investigations, such as whole exome sequencing, could reveal point mutations and small indels as the possible cause.
doi:10.1186/1471-2350-13-60
PMCID: PMC3490829  PMID: 22830313
Cardiomyopathy; Congenital heart disease; Neurology; Pediatrics; Array CGH; Hypertrabeculation; Seizures
3.  1031-1034delTAAC (Leu125Stop): a novel familial UBE3A mutation causing Angelman syndrome in two siblings showing distinct phenotypes 
BMC Medical Genetics  2012;13:124.
Background
More than 50 mutations in the UBE3A gene (E6-AP ubiquitin protein ligase gene) have been found in Angelman syndrome patients with no deletion, no uniparental disomy, and no imprinting defect.
Case Presentation
We here describe a novel UBE3A frameshift mutation in two siblings who have inherited it from their asymptomatic mother. Despite carrying the same UBE3A mutation, the proband shows a more severe phenotype whereas his sister shows a milder phenotype presenting the typical AS features.
Conclusions
We hypothesized that the mutation Leu125Stop causes both severe and milder phenotypes. Potential mechanisms include: i) maybe the proband has an additional problem (genetic or environmental) besides the UBE3A mutation; ii) since the two siblings have different fathers, the UBE3A mutation is interacting with a different genetic variant in the proband that, by itself, does not cause problems but in combination with the UBE3A mutation causes the severe phenotype; iii) this UBE3A mutation alone can cause either typical AS or the severe clinical picture seen in the proband.
doi:10.1186/1471-2350-13-124
PMCID: PMC3543165  PMID: 23256887
Angelman syndrome; UBE3A gene; Imprinting; Novel mutation; Distinct phenotypes; HRM
4.  Frank-ter Haar syndrome associated with sagittal craniosynostosis and raised intracranial pressure 
BMC Medical Genetics  2012;13:104.
Background
Frank-ter Haar syndrome is a rare disorder associated with skeletal, cardiac, ocular and craniofacial features including hypertelorism and brachycephaly. The most common underlying genetic defect in Frank-ter Haar syndrome appears to be a mutation in the SH3PXD2B gene on chromosome 5q35.1. Craniosynostosis, or premature fusion of the calvarial sutures, has not previously been described in Frank-ter Haar syndrome.
Case presentation
We present a family of three affected siblings born to consanguineous parents with clinical features in keeping with a diagnosis of Frank-ter Haar syndrome. All three siblings have a novel mutation caused by the deletion of exon 13 of the SH3PXD2B gene. Two of the three siblings also have non-scaphocephalic sagittal synostosis associated with raised intracranial pressure.
Conclusion
The clinical features of craniosynostosis and raised intracranial pressure in this family with a confirmed diagnosis of Frank-ter Haar syndrome expand the clinical spectrum of the disease. The abnormal cranial proportions in a mouse model of the disease suggests that the association is not coincidental. The possibility of craniosynostosis should be considered in individuals with a suspected diagnosis of Frank-ter Haar syndrome.
doi:10.1186/1471-2350-13-104
PMCID: PMC3532175  PMID: 23140272
Frank-ter Haar syndrome; Craniosynostosis; Sagittal synostosis; Intracranial pressure
5.  Cornelia de Lange syndrome with NIPBL mutation and mosaic Turner syndrome in the same individual 
BMC Medical Genetics  2012;13:43.
Background
Cornelia de Lange syndrome (CdLS) is a dominantly inherited disorder characterized by facial dysmorphism, growth and cognitive impairment, limb malformations and multiple organ involvement. Mutations in NIPBL gene account for about 60% of patients with CdLS. This gene encodes a key regulator of the Cohesin complex, which controls sister chromatid segregation during both mitosis and meiosis. Turner syndrome (TS) results from the partial or complete absence of one of the X chromosomes, usually associated with congenital lymphedema, short stature, and gonadal dysgenesis.
Case presentation
Here we report a four-year-old female with CdLS due to a frameshift mutation in the NIPBL gene (c.1445_1448delGAGA), who also had a tissue-specific mosaic 45,X/46,XX karyotype. The patient showed a severe form of CdLS with craniofacial dysmorphism, pre- and post-natal growth delay, cardiovascular abnormalities, hirsutism and severe psychomotor retardation with behavioural problems. She also presented with minor clinical features consistent with TS, including peripheral lymphedema and webbed neck. The NIPBL mutation was present in the two tissues analysed from different embryonic origins (peripheral blood lymphocytes and oral mucosa epithelial cells). However, the percentage of cells with monosomy X was low and variable in tissues. These findings indicate that, ontogenically, the NIPBL mutation may have appeared before the mosaic monosomy X.
Conclusions
The coexistence in several patients of these two rare disorders raises the issue of whether there is indeed a cause-effect association. The detailed clinical descriptions indicate predominant CdLS phenotype, although additional TS manifestations may appear in adolescence.
doi:10.1186/1471-2350-13-43
PMCID: PMC3458943  PMID: 22676896
Cornelia de Lange syndrome; CdLS; NIPBL; Turner syndrome; TS; Monosomy X mosaicism; Mosaic 45,X/46,XX karyotype
6.  Isolated brachydactyly type E caused by a HOXD13 nonsense mutation: a case report 
BMC Medical Genetics  2012;13:4.
Background
Brachydactyly type E (BDE; MIM#113300) is characterized by shortening of the metacarpal, metatarsal, and often phalangeal bones, and predominantly affects postaxial ray(s) of the limb. BDE may occur as an isolated trait or as part of a syndrome. Isolated BDE is rare and in the majority of cases the molecular pathogenesis has so far not been resolved. Originally, the molecular cause of isolated BDE has been unravelled in 2 families and shown to result from heterozygous missense mutations in the homeodomain of the HOXD13 gene. Since the initial manuscript, one further HOXD13 mutation has been reported only in a single family manifesting isolated BDE.
Case Presentation
In this paper, we report on a Polish family exhibiting isolated BDE caused by a novel nonsense heterozygous HOXD13 mutation. We investigated a Polish female proband and her father, both affected by isolated BDE, in whom we identified a nonsense heterozygous mutation c.820C > T(p.R274X) in the HOXD13 gene. So far, only two missense HOXD13 substitutions (p.S308C and p.I314L), localized within the homeodomain of the HOXD13 transcription factor, as well as a single nonsense mutation (p.E181X) were associated with BDE. Both missense changes were supposed to alter DNA binding affinity of the protein.
Conclusion
The variant p.R274X identified in our proband is the fourth HOXD13 mutation, and the second truncating (nonsense) mutation, reported to result in typical isolated BDE. We refer our clinical and molecular findings to the previously described HOXD13 associated phenotypes and mutations.
doi:10.1186/1471-2350-13-4
PMCID: PMC3278352  PMID: 22233338
brachydactyly type E; BDE; isolated brachydactyly; nonsense mutation; HOXD13
7.  CDKL5 gene status in female patients with epilepsy and Rett-like features: two new mutations in the catalytic domain 
BMC Medical Genetics  2012;13:68.
Background
Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) located in the Xp22 region have been shown to cause a subset of atypical Rett syndrome with infantile spasms or early seizures starting in the first postnatal months.
Methods
We performed mutation screening of CDKL5 in 60 female patients who had been identified as negative for the methyl CpG-binding protein 2 gene (MECP2) mutations, but who had current or past epilepsy, regardless of the age of onset, type, and severity. All the exons in the CDKL5 gene and their neighbouring sequences were examined, and CDKL5 rearrangements were studied by multiplex ligation-dependent probe amplification (MLPA).
Results
Six previously unidentified DNA changes were detected, two of which were disease-causing mutations in the catalytic domain: a frameshift mutation (c.509_510insGT; p.Glu170GlyfsX36) and a complete deletion of exon 10. Both were found in patients with seizures that started in the first month of life.
Conclusions
This study demonstrated the importance of CDKL5 mutations as etiological factors in neurodevelopmental disorders, and indicated that a thorough analysis of the CDKL5 gene sequence and its rearrangements should be considered in females with Rett syndrome-like phenotypes, severe encephalopathy and epilepsy with onset before 5 months of age. This study also confirmed the usefulness of MLPA as a diagnostic screening method for use in clinical practice.
doi:10.1186/1471-2350-13-68
PMCID: PMC3489578  PMID: 22867051
CDKL5; Epilepsy; MECP2; MLPA; Rett syndrome
8.  IGF2/H19 hypomethylation in a patient with very low birthweight, preocious pubarche and insulin resistance 
BMC Medical Genetics  2012;13:42.
Background
Insulin like growth factor 2 (IGF2) is an imprinted gene, which has an important role in fetal growth as established in mice models. IGF2 is downregulated through hypomethylation of a differentially methylated region (DMR) in Silver Russell syndrome (SRS), characterised by growth restriction. We have previously reported that severe pre- and post-natal growth restriction associated with insulin resistance and precocious pubarche in a woman without body asymmetry or other SRS features resulted from a balanced translocation affecting the regulation of her IGF2 gene expression. We hypothesised that severe pre- and post-natal growth restriction associated with insulin resistance and precocious pubarche in the absence of SRS are also caused by downregulation of IGF2 through hypomethylation, gene mutation or structural chromosomal abnormalities.
Methods
We performed routine karyotyping, IGF2 gene sequencing and investigated DNA methylation of the IGF2 differentially methylated region (DMR)0 and H19 DMR using pyrosequencing, in four women selected for very low birth weight (<−3 SDS for gestational age), precocious pubarche, short adult stature (<−2 SDS), and insulin resistance (defined as HOMA-IS < 80%); and compared their methylation results to those of 95 control subjects.
Results
We identified a 20 year old woman with severe hypomethylation at both DMRs. She was the smallest at birth (birthweight SDS,-3.9), and had the shortest adult height (143 cm). The patient was diagnosed with polycystic ovarian syndrome at the age of 15 years, and had impaired fasting glucose in the presence of a low BMI (19.2 kg/m2).
Conclusions
Our case of growth restriction, premature pubarche and insulin resistance in the absence of body asymmetry or other features of SRS adds to the expanding phenotype of IGF2/H19 methylation abnormalities. Further studies are needed to confirm whether growth restriction in association with premature pubarche and insulin resistance is a specific manifestation of reduced IGF2 expression.
doi:10.1186/1471-2350-13-42
PMCID: PMC3459807  PMID: 22646060
Insulin like growth factor 2; Intrauterine growth restriction; Short stature; Insulin resistance
9.  Atypical case of Wolfram syndrome revealed through targeted exome sequencing in a patient with suspected mitochondrial disease 
BMC Medical Genetics  2012;13:3.
Background
Mitochondrial diseases comprise a diverse set of clinical disorders that affect multiple organ systems with varying severity and age of onset. Due to their clinical and genetic heterogeneity, these diseases are difficult to diagnose. We have developed a targeted exome sequencing approach to improve our ability to properly diagnose mitochondrial diseases and apply it here to an individual patient. Our method targets mitochondrial DNA (mtDNA) and the exons of 1,600 nuclear genes involved in mitochondrial biology or Mendelian disorders with multi-system phenotypes, thereby allowing for simultaneous evaluation of multiple disease loci.
Case Presentation
Targeted exome sequencing was performed on a patient initially suspected to have a mitochondrial disorder. The patient presented with diabetes mellitus, diffuse brain atrophy, autonomic neuropathy, optic nerve atrophy, and a severe amnestic syndrome. Further work-up revealed multiple heteroplasmic mtDNA deletions as well as profound thiamine deficiency without a clear nutritional cause. Targeted exome sequencing revealed a homozygous c.1672C > T (p.R558C) missense mutation in exon 8 of WFS1 that has previously been reported in a patient with Wolfram syndrome.
Conclusion
This case demonstrates how clinical application of next-generation sequencing technology can enhance the diagnosis of patients suspected to have rare genetic disorders. Furthermore, the finding of unexplained thiamine deficiency in a patient with Wolfram syndrome suggests a potential link between WFS1 biology and thiamine metabolism that has implications for the clinical management of Wolfram syndrome patients.
doi:10.1186/1471-2350-13-3
PMCID: PMC3281774  PMID: 22226368
10.  Beckwith Wiedemann imprinting defect found in leucocyte but not buccal DNA in a child born small for gestational age 
BMC Medical Genetics  2012;13:99.
Background
Loss of methylation (LOM) at imprinting control region (ICR) 1 or LOM at ICR 2 on chromosome 11p15 in leucocyte DNA is commonly used to diagnose the imprinting disorders Silver Russell syndrome (SRS) characterized by growth restriction or Beckwith Wiedemann syndrome (BWS) characterized by overgrowth, respectively.
Case presentation
A child was normally conceived and born by caesarian section to a healthy 19 year old smoking mother (G2P1) at 38 weeks gestation, with SGA (birthweight SDS −2.44), placenta weight 250g (normal histology), with an umbilical hernia and transient neonatal hypoglycemia but no other features of BWS.
The methylation status at 11p15 region was initially investigated by multiplex ligation dependent probe amplification (MLPA). Subsequently, methylation-specific (ms) PCR was performed to screen for this and other imprinted loci abnormalities at PLAG1 (6q24), IGF2R (6q27), GRB10 (7p12), PEG1/MEST (7q32), DLK1 (14q32), SNRPN (15q11); PEG3 (19q32), NESPAS/GNAS (20q13).
Leucocyte DNA methylation was normal at ICR1 but markedly reduced at ICR2 using both MLPA and ms-PCR, and no other anomalies of imprinting were detected. Buccal DNA methylation was normal at all imprinted sites tested.
Conclusion
This is the first report of an isolated LOM at ICR2 in leucocyte but not buccal DNA in a normally conceived singleton SGA child without overt SRS or BWS.
doi:10.1186/1471-2350-13-99
PMCID: PMC3514203  PMID: 23116464
Insulin like growth factor 2 gene; Small for gestational age; Beckwith Wiedemann syndrome
11.  A missense founder mutation in VLDLR is associated with Dysequilibrium Syndrome without quadrupedal locomotion 
BMC Medical Genetics  2012;13:80.
Background
Dysequilibrium syndrome is a genetically heterogeneous condition that combines autosomal recessive, nonprogressive cerebellar ataxia with mental retardation. The condition has been classified into cerebellar ataxia, mental retardation and disequilibrium syndrome types 1 (CAMRQ1), 2 (CAMRQ2) and 3 (CAMRQ3) and attributed to mutations in VLDLR, CA8 and WDR81 genes, respectively. Quadrupedal locomotion in this syndrome has been reported in association with mutations in all three genes.
Methods
SNP mapping and candidate gene sequencing in one consanguineous Omani family from the United Arab Emirates with cerebellar hypoplasia, moderate mental retardation, delayed ambulation and truncal ataxia was used to identify the mutation. In a second unrelated consanguineous Omani family, massively parallel exonic sequencing was used.
Results
We identified a homozygous missense mutation (c.2117 G > T, p.C706F) in the VLDLR gene in both families on a shared affected haplotype block.This is the first reported homozygous missense mutation in VLDLR and it occurs in a highly conserved residue and predicted to be damaging to protein function.
Conclusions
We have delineated the phenotype associated with dysequilibrium syndrome in two Omani families and identified the first homozygous missense pathogenic mutation in VLDLR gene with likely founder effect in the southeastern part of the Arabian Peninsula.
doi:10.1186/1471-2350-13-80
PMCID: PMC3495048  PMID: 22973972
12.  Identification of 3 novel VHL germ-line mutations in Danish VHL patients 
BMC Medical Genetics  2012;13:54.
Background
von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome in which the patients develop retinal and central nervous system hemangioblastomas, pheochromocytomas and clear-cell renal tumors. The autosomal dominant disease is caused by mutations in the VHL gene.
Methods
VHL mutational analysis was carried out by sequencing of the coding sequence and by multiplex ligation-dependent probe amplification analysis. The functional consequence of the variants was investigated using in silico prediction tools.
Results
A total of 289 probands suspected of having VHL syndrome have been screened for mutations in the VHL gene. Twenty-six different VHL mutations were identified in 36 families including one in-frame duplication, two frame-shift mutations, four nonsense mutations, twelve missense mutations, three intronic mutations and four large genomic rearrangements. Three of these mutations (c.319 C > T, c.342_343dupGGT and c.520_521dupAA) were novel.
Conclusions
In this study we report the VHL germ-line mutations found in Danish families. We found three novel VHL mutations where two were classified as pathogenic and the latter was classified as a variant of unknown significance. Together, our findings contribute to the interpretation of the potential pathogenicity of VHL germ-line mutations.
doi:10.1186/1471-2350-13-54
PMCID: PMC3458949  PMID: 22799452
von Hippel-Lindau disease; VHL; Germ-line mutations; Danish population
13.  A mutation at IVS1 + 5 of the von Hippel-Lindau gene resulting in intron retention in transcripts is not pathogenic in a patient with a tongue cancer?: case report 
BMC Medical Genetics  2012;13:23.
Background
Von Hippel-Lindau disease (VHL) is a dominantly inherited familial cancer syndrome predisposing the patient to a variety of malignant and benign neoplasms, most frequently hemangioblastoma, renal cell carcinoma, pheochromocytoma, and pancreatic tumors. VHL is caused by mutations of the VHL tumor suppressor gene on the short arm of chromosome 3, and clinical manifestations develop if both alleles are inactivated according to the two-hit hypothesis. VHL mutations are more frequent in the coding region and occur occasionally in the splicing region of the gene. Previously, we reported that the loss of heterozygosity (LOH) of the VHL gene is common in squamous cell carcinoma tissues of the tongue.
Case Presentation
We describe a case of squamous cell carcinoma in the tongue caused by a point mutation in the splicing region of the VHL gene and discuss its association with VHL disease. Sequence analysis of DNA extracted from the tumor and peripheral blood of the patient with squamous cell carcinoma revealed a heterozygous germline mutation (c. 340 + 5 G > C) in the splice donor sequence in intron 1 of the VHL gene. RT-PCR analysis of the exon1/intron1 junction in RNA from tumor tissue detected an unspliced transcript. Analysis of LOH using a marker with a heterozygous mutation of nucleotides (G or C) revealed a deletion of the mutant C allele in the carcinoma tissues.
Conclusions
The fifth nucleotide G of the splice donor site of the VHL gene is important for the efficiency of splicing at that site. The development of tongue cancer in this patient was not associated with VHL disease because the mutation occurred in only a single allele of the VHL gene and that allele was deleted in tumor cells.
doi:10.1186/1471-2350-13-23
PMCID: PMC3352036  PMID: 22462637
14.  Deletion of a single-copy DAAM1 gene in congenital heart defect: a case report 
BMC Medical Genetics  2012;13:63.
Background
With an increasing incidence of congenital heart defects (CHDs) in recent years, genotype-phenotype correlation and array-based methods have contributed to the genome-wide analysis and understanding of genetic variations in the CHD population. Here, we report a copy number deletion of chromosomal 14q23.1 in a female fetus with complex congenital heart defects. This is the first description of DAAM1 gene deletion associated with congenital heart anomalies.
Case Presentation
Compared with the control population, one CHD fetus showed a unique copy number deletion of 14q23.1, a region that harbored DAAM1 and KIAA0666 genes.
Conclusions
Results suggest that the copy number deletion on chromosome 14q23.1 may be critical for cardiogenesis. However, the exact relationship and mechanism of how DAAM1 and KIAA0666 deletion contributes to the onset of CHD is yet to be determined.
doi:10.1186/1471-2350-13-63
PMCID: PMC3482563  PMID: 22857009
Congenital heart defect; Copy number deletion; DAAM1 gene
15.  A novel SYBR-based duplex qPCR for the detection of gene dosage: detection of an APC large deletion in a familial adenomatous polyposis patient with an unusual phenotype 
BMC Medical Genetics  2012;13:55.
Background
Familial adenomatous polyposis (FAP) is a hereditary colorectal cancer syndrome caused by a loss of function of the APC gene. Large deletions in APC are a common cause of FAP; despite the existence of a variety of gene dosage detection methodologies, most are labor intensive and time and resource consuming.
Methods
We describe a new duplex qPCR method for gene dosage analysis based on the coamplification of a target and a reference gene in a SYBR Green reaction, followed by a comparison of the ratio between the target and the reference peaks of the melting curve for the test (patient) and control samples. The reliability of the described duplex qPCR was validated for several genes (APC, HPRT1, ATM, PTEN and BRCA1).
Results
Using this novel gene dosage method, we have identified an APC gene deletion in a FAP patient undergoing genetic testing. Comparative genomic hybridization based on microarrays (aCGH) was used to confirm and map the extent of the deletion, revealing a 5.2 MB rearrangement (5q21.3-q22.3) encompassing the entire APC and 19 additional genes.
Conclusion
The novel assay accurately detected losses and gains of one copy of the target sequences, representing a reliable and flexible alternative to other gene dosage techniques. In addition, we described a FAP patient harboring a gross deletion at 5q21.3-q22.3 with an unusual phenotype of the absence of mental impairment and dysmorphic features.
doi:10.1186/1471-2350-13-55
PMCID: PMC3458979  PMID: 22799487
Gene dosage; Quantitative PCR; Familial adenomatous polyposis; APC whole gene deletion
16.  An atypical case of neuronal ceroid lipofuscinosis with co-inheritance of a variably penetrant POLG1 mutation 
BMC Medical Genetics  2012;13:50.
Background
The neuronal ceroid lipofuscinoses (NCLs, or Batten disease) comprise the most common Mendelian form of childhood-onset neurodegeneration, but the functions of the known underlying gene products remain poorly understood. The clinical heterogeneity of these disorders may shed light on genetic interactors that modify disease onset and progression.
Case presentation
We describe a proband with congenital hypotonia and an atypical form of infantile-onset, biopsy-proven NCL. Pathologic and molecular work-up of this patient identified CLN5 mutations as well as a mutation―previously described as incompletely penetrant or a variant of unknown significance―in POLG1, a nuclear gene essential for maintenance of mitochondrial DNA (mtDNA) copy number. The congenital presentation of this patient is far earlier than that described for either CLN5 patients or affected carriers of the POLG1 variant (c.1550 G > T, p.Gly517Val). Assessment of relative mtDNA copy number and mitochondrial membrane potential in the proband and control subjects suggested a pathogenic effect of the POLG1 change as well as a possible functional interaction with CLN5 mutations.
Conclusions
These findings suggest that an incompletely penetrant variant in POLG1 may modify the clinical phenotype in a case of CLN5 and are consistent with emerging evidence of interactions between NCL-related genes and mitochondrial physiology.
doi:10.1186/1471-2350-13-50
PMCID: PMC3443422  PMID: 22727047
Neuronal ceroid lipofuscinosis; CLN5; POLG1; mtDNA depletion; Oxidative phosphorylation
17.  The mGluR5 antagonist AFQ056 does not affect methylation and transcription of the mutant FMR1 gene in vitro 
BMC Medical Genetics  2012;13:13.
Background
Fragile X syndrome (FXS), the leading cause of inherited mental retardation, is due to expansion and methylation of a CGG sequence in the FMR1 gene, which result in its silencing and consequent absence of FMRP protein. This absence causes loss of repression of metabotropic glutamate receptor 5 (mGluR5)-mediated pathways resulting in the behavioral and cognitive impairments associated with FXS. In a randomized, double-blind trial it was recently demonstrated a beneficial effect of AFQ056, a selective inhibitor of metabotrobic glutamate receptor type 5 (mGluR5), on fully methylated FXS patients respect to partially methylated FXS ones.
Methods
To determine whether AFQ056 may have secondary effects on the methylation and transcription of FMR1, here we treated three FXS lymphoblastoid cell lines and one normal control male line. A quantitative RT-PCR was performed to assess transcriptional reactivation of the FMR1 gene. To assess the methylation status of the FMR1 gene promoter it was carried out a bisulphite sequencing analysis.
Results
Both FMR1-mRNA levels and DNA methylation were unmodified with respect to untreated controls.
Conclusions
These results demonstrate that the AFQ056 effect on fully methylated FXS patients is not due to a secondary effect on DNA methylation and consequent transcriptional activation of FMR1.
doi:10.1186/1471-2350-13-13
PMCID: PMC3320553  PMID: 22397687
Fragile X syndrome; AFQ056; mGluR5 inhibitors; DNA methylation; Epigenetic modification
18.  Genome-wide sequencing for the identification of rearrangements associated with Tourette syndrome and obsessive-compulsive disorder 
BMC Medical Genetics  2012;13:123.
Background
Tourette Syndrome (TS) is a neuropsychiatric disorder in children characterized by motor and verbal tics. Although several genes have been suggested in the etiology of TS, the genetic mechanisms remain poorly understood.
Methods
Using cytogenetics and FISH analysis, we identified an apparently balanced t(6,22)(q16.2;p13) in a male patient with TS and obsessive-compulsive disorder (OCD). In order to map the breakpoints and to identify additional submicroscopic rearrangements, we performed whole genome mate-pair sequencing and CGH-array analysis on DNA from the proband.
Results
Sequence and CGH array analysis revealed a 400 kb deletion located 1.3 Mb telomeric of the chromosome 6q breakpoint, which has not been reported in controls. The deletion affects three genes (GPR63, NDUFA4 and KLHL32) and overlaps a region previously found deleted in a girl with autistic features and speech delay. The proband’s mother, also a carrier of the translocation, was diagnosed with OCD and shares the deletion. We also describe a further potentially related rearrangement which, while unmapped in Homo sapiens, was consistent with the chimpanzee genome.
Conclusions
We conclude that genome-wide sequencing at relatively low resolution can be used for the identification of submicroscopic rearrangements. We also show that large rearrangements may escape detection using standard analysis of whole genome sequencing data. Our findings further provide a candidate region for TS and OCD on chromosome 6q16.
doi:10.1186/1471-2350-13-123
PMCID: PMC3556158  PMID: 23253088
Tourette syndrome; Paired end sequencing; Chromosomal translocation; Structural variations
19.  Genotype-phenotype correlation in 22q11.2 deletion syndrome 
BMC Medical Genetics  2012;13:122.
Background
The 22q11.2 deletion syndrome (22q11.2DS) is caused by hemizygous microdeletions on chromosome 22q11.2 with highly variable physical and neuropsychiatric manifestations. We explored the genotype-phenotype relationship in a relatively large 22q11.2DS cohort treated and monitored in our clinic using comprehensive clinical evaluation and detailed molecular characterization of the deletion.
Methods
Molecular analyses in 142 subjects with 22q11.2DS features were performed by FISH and MLPA methods. Participants underwent clinical assessment of physical symptoms and structured psychiatric and cognitive evaluation.
Results
Deletions were found in 110 individuals including one with an atypical nested distal deletion which was missed by the FISH test. Most subjects (88.2%) carried the 3Mb typically deleted region and 11.8% carried 4 types of deletions differing in size and location. No statistically significant genotype-phenotype correlations were found between deletion type and clinical data although some differences in hypocalcemia and cardiovascular anomalies were noted.
Analysis of the patient with the distal nested deletion suggested a redundancy of genes causing the physical and neuropsychiatric phenotype in 22q11.2DS and indicating that the psychiatric and cognitive trajectories may be governed by different genes.
Conclusions
MLPA is a useful and affordable molecular method combining accurate diagnosis and detailed deletion characterization. Variations in deletion type and clinical manifestations impede the detection of significant differences in samples of moderate size, but analysis of individuals with unique deletions may provide insight into the underlying biological mechanisms.
Future genotype-phenotype studies should involve large multicenter collaborations employing uniform clinical standards and high-resolution molecular methods.
doi:10.1186/1471-2350-13-122
PMCID: PMC3548696  PMID: 23245648
Velocardiofacial syndrome (VCFS); Multiplex ligation probe amplification (MLPA); Copy number variation (CNV); Molecular diagnosis; Neuropsychiatric disorders
20.  Novel missense mutation in the RSPO4 gene in congenital hyponychia and evidence for a polymorphic initiation codon (p.M1I) 
BMC Medical Genetics  2012;13:120.
Background
Anonychia/hyponychia congenita is a rare autosomal recessive developmental disorder characterized by the absence (anonychia) or hypoplasia (hyponuchia) of finger- and/or toenails frequently caused by mutations in the R-spondin 4 (RSPO4) gene.
Methods
Three hypo/anonychia consanguineous Pakistani families were ascertained and genotyped using microsatellite markers spanning the RSPO4 locus on chromosome 20p13. Mutation screening of the RSPO4 gene was carried out by direct sequencing of the entire coding region and all intron-exon boundaries.
Results
Mutations in the RSPO4 gene were identified in all families including a novel missense mutation c.178C>T (p.R60W) and two recurrent variants c.353G>A (p.C118Y) and c.3G>A (p.M1I). The c.3G>A variant was identified in unaffected family members and a control sample in a homozygous state.
Conclusions
This study raises to 17 the number of known RSPO4 mutations and further expands the molecular repertoire causing hypo/anonychia. The c.353G>A emerges as a recurrent change with a possible founder effect in the Pakistani population. Our findings suggest that c.3G>A is not sufficient to cause the disorder and could be considered a polymorphism.
doi:10.1186/1471-2350-13-120
PMCID: PMC3532313  PMID: 23234511
Anonychia; Hyponychia; Mutation; RSPO4 gene; Polymorphism
21.  Phosphodiesterase 8B gene polymorphism in women with recurrent miscarriage: A retrospective case control study 
BMC Medical Genetics  2012;13:121.
Background
Recurrent miscarriage affects approximately 1% of all couples. There is a known relation between hypothyroidism and recurrent miscarriage. Phosphodiesterase 8B (PDE8B) is a regulator of cyclic adenosine monophosphate (cAMP) with important influence on human thyroid metabolism. Single nucleotide polymorphism (SNP) rs 4704397 in the PDE8B gene has been shown to be associated with variations in serum Thyroid Stimulating Hormone (TSH) and thyroxine (T4) levels. The aim of this study was to investigate whether there is an association between the SNP rs 4704397 in the PDE8B gene and recurrent miscarriage.
Methods
The study was designed as a retrospective case control study. 188 cases with recurrent miscarriage were included and compared with 391 controls who had delivered at least once and with no history of miscarriage or assisted reproduction.
Results
No difference between cases and controls concerning age was found. Bivariate associations between homozygous A/A (OR 1.57, 95% CI 0.98-2.52) as well as G/G carriers (OR 1.52, 95% CI 1.02-2.25) of SNP rs 4704397 in PDE8B and recurrent miscarriage were verified (test for trend across all 3 genotypes, p = 0.059). After adjustment for known confounders such as age, BMI and smoking the association between homozygous A/A (AOR 1.63, 95% CI 1.01 - 2.64, p = 0.045) and G/G (AOR 1.52, 95% CI 1.02 - 2.27, p = 0.039) carriers of SNP rs 4704397 in PDE8B and recurrent miscarriage remained.
Conclusions
Our findings suggest that there is an association between homozygous A/A as well as homozygous G/G carriers of SNP rs 4704397 in PDE8B and recurrent miscarriage.
doi:10.1186/1471-2350-13-121
PMCID: PMC3556309  PMID: 23237535
Phosphodiesterase 8B; Recurrent miscarriage; Single nucleotide polymorphism; Thyroid
22.  Evaluation of single nucleotide polymorphisms in microRNAs (hsa-miR-196a2 rs11614913 C/T) from Brazilian women with breast cancer 
BMC Medical Genetics  2012;13:119.
Background
Emerging evidence has shown that miRNAs are involved in human carcinogenesis as tumor suppressors or oncogenes. Single nucleotide polymorphisms (SNPs) located in pre-miRNAs may affect the processing and therefore, influence the expression of mature miRNAs. Previous studies generated conflicting results when reporting association between the hsa-miR-196a2 rs11614913 common polymorphism and breast cancer.
Methods
This study evaluated the hsa-miR-196a2 rs11614913 SNP in 388 breast cancer cases and 388 controls in Brazilian women. Polymorphism was determined by real-time PCR; control and experimental groups were compared through statistical analysis using the X2 or Fisher’s exact tests.
Results
The analysis of the SNPs frequencies showed a significant difference between the groups (BC and CT) in regards to genotype distribution (χ2: p = 0.024); the homozygous variant (CC) was more frequent in the CT than in the BC group (p = 0.009). The presence of the hsa-miR-196a2 rs11614913 C/T polymorphism was not associated with histological grades (p = 0.522), axillary lymph node positive status (p = 0.805), or clinical stage (p = 0.670) among the breast cancer patients.
Conclusions
The results of this study indicated that the CC polymorphic genotype is associated with a decreased risk of BC and the presence of the T allele was significantly associated with an increased risk of BC.
doi:10.1186/1471-2350-13-119
PMCID: PMC3563578  PMID: 23228090
Breast; Cancer; Polymorphisms; MicroRNAs
23.  Prediction of lung cancer risk in a Chinese population using a multifactorial genetic model 
BMC Medical Genetics  2012;13:118.
Background
Lung cancer is a complex polygenic disease. Although recent genome-wide association (GWA) studies have identified multiple susceptibility loci for lung cancer, most of these variants have not been validated in a Chinese population. In this study, we investigated whether a genetic risk score combining multiple.
Methods
Five single-nucleotide polymorphisms (SNPs) identified in previous GWA or large cohort studies were genotyped in 5068 Chinese case–control subjects. The genetic risk score (GRS) based on these SNPs was estimated by two approaches: a simple risk alleles count (cGRS) and a weighted (wGRS) method. The area under the receiver operating characteristic (ROC) curve (AUC) in combination with the bootstrap resampling method was used to assess the predictive performance of the genetic risk score for lung cancer.
Results
Four independent SNPs (rs2736100, rs402710, rs4488809 and rs4083914), were found to be associated with a risk of lung cancer. The wGRS based on these four SNPs was a better predictor than cGRS. Using a liability threshold model, we estimated that these four SNPs accounted for only 4.02% of genetic variance in lung cancer. Smoking history contributed significantly to lung cancer (P < 0.001) risk [AUC = 0.619 (0.603-0.634)], and incorporated with wGRS gave an AUC value of 0.639 (0.621-0.652) after adjustment for over-fitting. This model shows promise for assessing lung cancer risk in a Chinese population.
Conclusion
Our results indicate that although genetic variants related to lung cancer only added moderate discriminatory accuracy, it still improved the predictive ability of the assessment model in Chinese population.
doi:10.1186/1471-2350-13-118
PMCID: PMC3573944  PMID: 23228068
Chinese; Cumulative risk; Genetic risk score; Lung cancer; Risk assessment
24.  In-vitro characterization of novel and functional regulatory SNPs in the promoter region of IL2 and IL2R alpha in a Gabonese population 
BMC Medical Genetics  2012;13:117.
Background
The selection pressure imposed by the parasite has a functional consequence on the immune genes, leading to altered immune function in which regulatory T cells (Tregs) induced by parasites during infectious challenges modulate or thwart T effector cell mechanism.
Methods
We identified and investigated regulatory polymorphisms in the immune gene IL2 and its receptor IL2R alpha (also known as CD25) in Gabonese individuals exposed to plentiful parasitic infections.
Results
We identified two reported variants each for IL2 and its receptor IL2R alpha gene loci. Also identified were two novel variants, -83 /-84 CT deletions (ss410961576) for IL2 and -409C/T (ss410961577) for IL2R alpha. We further validated all identified promoter variants for their allelic gene expression using transient transfection assays. Three promoter variants of the IL2 locus revealed no significant expression of the reporter gene. The identified novel variant (ss410961577C/T) of the IL2R alpha revealed a significant higher expression of the reporter gene in comparison to the major allele (P<0.05). In addition, the rs12722616C/T variant of the IL2R alpha locus altered the transcription factor binding site TBP (TATA box binding protein) and C/EBP beta (CCAAT/enhancer binding protein beta) that are believed to regulate the Treg function.
Conclusions
The identification and validation of such regulatory polymorphisms in the immune genes may provide a basis for future studies on parasite susceptibility in a population where T cell functions are compromised.
doi:10.1186/1471-2350-13-117
PMCID: PMC3564939  PMID: 23217119
IL2; IL2R alpha; CD25; Polymorphism; Transfection; Regulatory T cells
25.  Thymic stromal lymphopoietin gene promoter polymorphisms and expression levels in Graves’ disease and Graves’ ophthalmopathy 
BMC Medical Genetics  2012;13:116.
Background
Graves disease (GD) is an organ-specific autoimmune disease characterized by hyperthyroidism, diffuse goiter, autoantibodies against thyroid-specific antigens, and dermopathy. Studies of GD have demonstrated the importance of the Th2 and Th17 immune responses in mediating disease progression. In the present study, we investigated the role of a Th2 cytokine, thymic stromal lymphopoietin (TSLP), in GD and Th17 differentiation.
Methods
In this study, we genotyped 470 patients with GD at 3 single nucleotide polymorphisms (SNPs) in TSLP. In addition, the serum concentrations of TSLP were determined in 432 patients and 272 controls. Ten patients and controls each were further screened using in vitro Th17 differentiation assays. The SNPs were genotyped using ABI TaqMan® SNP genotyping assays. For the Th17 differentiation assays, peripheral blood mononuclear cells (PBMCs) isolated from the patients and controls were placed into Th17 differentiation media, and interleukin 17 expression levels were determined.
Results
Haplotype analysis indicated that patients with the Ht3 (TCC) haplotype have a 3.28-fold higher risk of developing GD (p = 0.007), whereas those with the Ht5 (TCG) haplotype had a 0.03-fold, reduced risk of developing GD (p = 1 × 10−14). SNP rs3806933 (p = 0.007) was associated with female Graves ophthalmopathy (GO). TSLP expression levels were higher in GD patients than in control subjects, and TLSP was also shown to promote the differentiation of Th17 cells in GD patients.
Conclusions
These results suggest that polymorphisms in TSLP may be used as genetic markers for the diagnosis and prognosis of GD. Furthermore, TLSP may be a target for treating GD.
doi:10.1186/1471-2350-13-116
PMCID: PMC3582428  PMID: 23194010
Graves’ disease; Graves’ ophthalmopathy; Thymic stromal lymphopoietin; Th17

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