Patients and methods: We have studied two additional families with partial phenotypes and atypical 7q11.23 deletions. Deletions were precisely defined at the nucleotide level and expression levels of some affected and flanking genes were assessed in lymphoblastoid cell lines.

Results: Affected individuals presented variable cardiovascular and connective tissue manifestations, subtle craniofacial features, normal visuospatial construction abilities with low average IQ and no endocrine abnormalities. The deletion in family NW1 encompassed 817 kb with 11 genes (CLDN3-GTF2IRD1), and 610 kb with 14 genes (VPS37D-RFC2) in family NW2. All deleted genes in typical and atypical deletions revealed low expression levels in lymphoblastoid cell lines, except for GTF2IRD1. CLIP2 was also underexpressed in all patients despite being outside the deletion in NW2, while no other flanking non-deleted gene showed significantly different expression compared to controls.

Conclusions: Along with previously reported cases, clinical-molecular correlations in these two families further corroborate that functional hemizygosity for the GTF2I +/- GTF2IRD1 genes is the main cause of the neurocognitive profile and some aspects of the gestalt phenotype of WBS.

In 2008, loss-of-function mutations in the pericentrin gene (PCNT) have been identified in 28 patients, including 3 SCKL and 25 MOPDII cases [6, 7]. This gene encodes a centrosomal protein which plays a key role in the organization of mitotic spindles.

The aim of our study was to analyze PCNT in a large series of SCKL-MOPD II cases to further define the clinical spectrum associated with PCNT mutations. Among 18 consanguineous families (13 SCKL and 5 MOPDII) and 6 isolated cases (3 SCKL and 3 MOPD II), we identified thirteen distinct mutations in 5/16 SCKL and 8/8 MOPDII including five stop mutations, five frameshift mutations, two splice site mutations and one apparent missense mutation affecting the last base of exon 19. Moreover, we demonstrated that this latter mutation leads to an abnormal splicing with a predicted premature termination of translation. The clinical analysis of the 5 SCKL cases with PCNT mutations showed that they all presented minor skeletal changes and clinical features compatible with MOPDII diagnosis. We therefore conclude that, despite variable severity, MOPDII is a genetically homogeneous condition due to loss-of function of pericentrin.

Objective: This study aims to determine the identity and frequency of FUS gene mutations in a large cohort of Italian patients enriched in sporadic cases (SALS).

Methods: We screened exons 5, 6, 14, and 15 of FUS gene for mutations in 1009 Italian ALS patients (45 FALS and 964 SALS). The genetic analysis was extended to the entire coding sequence of FUS in all the FALS and in 293 of the SALS patients.

Results: We identified 7 missense mutations (p.G191S, p.R216C, p.G225V, p.G230C, p.R234C, p.G507D, and p.R521C) in 9 patients (7 SALS and 2 FALS) and none in 500 healthy Italian controls. All mutations are novel with the exception of the p.R521C that we identified in one SALS and one FALS case. Both patients showed a similar unusual presentation with proximal, mostly symmetrical, upper limb weakness, with neck and axial involvement. With the exception of p.G507D and p.R521C, the mutations identified in SALS individuals are all localized in the glycine-rich region encoded by exon 6. In addition, we detected 8 different in-frame deletions in two poly-glycine motifs whose frequency was not significantly different in patients and controls.

Conclusions Our results show that FUS missense mutations are present in 0.7% of Italian SALS cases and confirm the previous mutational frequency reported in FALS (4.4%). An unusual proximal and axial clinical presentation seems associated to the presence of the p.R521C mutation.

Myosin binding protein C (MyBPC) is essential for the structure of the sarcomeres in striated muscle. There is one cardiac specific isoform and two skeletal muscle specific isoforms. Mutations in MYBPC3 encoding the cardiac isoform cause cardiomyopathy.

L1 syndrome is an X-linked recessive disorder for which we aimed to: develop a comprehensive mutation analysis system with a high rate of detection, develop a tool to predict the chance of detecting a mutation in the L1CAM gene, and look for genotype-phenotype correlations.

In the last decade, Hermansky-Pudlak syndrome (HPS) has arisen as an instructive disorder for cell biologists to study the biogenesis of lysosome-related organelles (LROs). Of the eight human HPS subtypes, only subtypes 1 through 5 are well described. Here, we extensively characterize the HPS-6 subtype, caused by defects in HPS6, a subunit of the biogenesis of lysosome-related organelles complex-2 (BLOC-2).

Our report highlights three features of SUFU-related tumors. These are mainly medulloblastomas with extensive nodularity or classic desmoplastic/nodular medulloblastomas. These tumors mostly appear during the first three years of life. The penetrance of the mutation may be low.

The present study investigated whether single nucleotide polymorphisms (SNPs) in human urate transporter 1 (hURAT1) gene are associated with primary hyperuicemia (HUA) in Han Chinese people.

Radiotherapy-induced DNA double strand breaks (DSB) are critical cytotoxic lesions. Inherited defects in DSB DNA repair pathways lead to hypersensitivity to ionising radiation, immunodeficiency and increased cancer incidence. A patient with xeroderma pigmentosum complementation group C, with a scalp angiosarcoma exhibited dramatic clinical radiosensitivity following radiotherapy, resulting in death. A fibroblast cell line from non-affected skin (XP14BRneo17) was hypersensitive to ionising radiation and defective in DNA double strand break repair.

Bardet-Biedl syndrome (BBS) is primarily an autosomal recessive disorder characterized by rod-cone dystrophy, obesity, hypogonadism, post-axial polydactyly, renal cysts, and other anomalies of the kidney and urinary tract. To date, mutations in 12 BBS genes as well as in MKS1 and CEP290 have been identified as causing BBS. The vast genetic heterogeneity of BBS renders molecular genetic diagnosis difficult in terms of both the time and cost required to screen all 204 coding exons. Here, we report the use of genome-wide homozygosity mapping as a tool to identify homozygous segments at known BBS loci in BBS individuals from inbred and outbred background. In a worldwide cohort of 45 families, we identified, via direct exon sequencing, causative homozygous mutations in 20 families. Eleven of these mutations were novel, thereby increasing the number of known BBS mutations by 5% (11/218). Thus, in the presence of extreme genetic locus heterogeneity, homozygosity mapping provides a valuable approach to the molecular genetic diagnosis of BBS and will facilitate the discovery of novel pathogenic mutations.

Introduction: Fabry disease (FD) may present with left ventricular hypertrophy, renal insufficiency or stroke. Several studies investigated FD prevalence in populations expressing these symptoms. We conducted a systematic review to calculate the overall prevalence of FD in these cohorts.

Methods: We searched online databases for studies on screening for FD. We recorded study population selection, screening methods and outcome of screening.

Results: We identified 20 studies, 10 of which included both male and female patients. In all (N=19) studies with male and almost all (N=10) with female patients aGal A activity was used as screening method. In males on dialysis (10 studies) overall FD prevalence was 0.33% (95% CI 0.20-0.47) and in females (6 studies) 0.10% (95%CI 0-0.19). Combined prevalence of FD in patients with renal transplant was 0.38% in males (95% CI 0.07-0.69) and 0% in females. In patients with LVH, selection of study-population and differences in the method of screening hampered the calculation of an overall prevalence (ranging from 0.9-3.9% in males and 1.1-11.8% in females). In premature strokes (N=2 studies) overall FD prevalence was 4.2% (95CI 2.4-6.0) in males and 2.1% (95CI 0.5-3.7)) in females.

Discussion: Prevalence of FD in dialysis patients is 0.33% for males and 0.10% for females. Prevalence of FD in LVH is at least 1% for both genders. In females most studies were performed with aGal A activity measurements as screening tool, although this method fails to detect 1/3 of female patients with FD, underestimating the overall prevalence in females.

Background: Recurrent microdeletions and microduplications of ~555kb at 16p11.2 confer susceptibility to autism spectrum disorder (ASD) in up to 1% of ASD patients. No physical or behavioral features have been identified that distinguish these individuals as having a distinct ASD subtype, but clinical data are limited.

Methods: We report five autistic probands identified by microarray analysis with copy number variation (CNV) of 16p11.2 (three deletions, two duplications). Each patient was assessed for ASD and dysmorphic features. We also describe a deletion-positive 26-month old female who has developmental delay (DD) and autistic features.

Results: Proband 1 (female with ASD, de novo deletion) is not dysmorphic. Proband 2 (male with autism, de novo deletion) and proband 3 and his brother (males with autism, inherited deletions) are dysmorphic, but the two probands do not resemble one another. Proband 3’s mother has mild mental retardation (MR), minor dysmorphism and meets the criteria for ASD. Proband 4 (dysmorphic autistic male, de novo duplication) had a congenital diaphragmatic hernia. Proband 5 (non-dysmorphic ASD female with a duplication) has two apparently healthy duplication-positive relatives. Probands 1 and 2 have deletion-negative siblings with ASD and Asperger syndrome, respectively. The 6th proband (a female with DD and an inherited duplication) is dysmorphic, but has oligohydramnios sequence.

Conclusions: The phenotypic spectrum associated with CNV at 16p11.2 includes ASD, MR/DD and/or possibly other primary psychiatric disorders. Compared with the microduplications, the reciprocal microdeletions are more likely to be penetrant and to be associated with non-specific major or minor dysmorphism. There are deletion-positive ASD probands with a less severe phenotype than deletion-negative ASD siblings underscoring the significant phenotypic heterogeneity.

Silver-Russell syndrome (SRS) is a heterogeneous disorder associated with intrauterine and postnatal growth restriction, body asymmetry, a relative macrocephaly, a characteristic triangular face and further dysmorphisms. In about 50% of patients genetic/epigenetic alterations can be detected: >38% of patients show a hypomethylation of the IGF2/H19 imprinting region in 11p15, additional 10% carry a maternal uniparental disomy of chromosome 7. In single cases, cytogenetic aberrations can be detected. Nevertheless, there still remain 50% of SRS patients without known genetic/epigenetic alterations. To find out whether submicroscopic imbalances contribute to the aetiology of SRS we screened 20 idiopathic SRS patients with the Affymetrix GeneChip® Human Mapping 500 K array set.

Apart from known apathogenic copy number variations (CNVs) we identified one patient with a 12q14 microdeletion. The 12q14 microdeletion syndrome is characterised by dwarfism but it additionally includes mental retardation and osteopoikilosis. The deletion in our patient is smaller than those in the 12q14 microdeletion carriers but it also affects the LEMD3 and the HMGA2 genes. LEMD3 haploinsufficiency and point mutations have been previously associated with osteopoikilosis but radiographs of our patient at the age of 16 years did not reveal any hint for osteopoikilosis lesions. Haploinsufficiency of HMGA2 is probably responsible for aberrant growth in 12q14 microdeletion syndrome. However, we excluded a general role of HMGA2 mutations for SRS by sequencing of 20 idiopathic patients.

In conclusion, our results exclude a common cryptic chromosomal imbalance in idiopathic SRS patients but show that chromosomal aberrations are relevant in this disease. Thus molecular karyotyping is indicated in SRS and should be included in the diagnostic algorithm.

Background: Ostium secundum atrial septal defects (ASDII) account for approximately 10% of all congenital heart defects (CHD) and mutations in cardiac transcription factors, including TBX20, were identified as an underlying cause for ASDII. However, very little is known about disease penetrance in families and functional consequences of inherited TBX20 mutations.

Methods: The coding region of TBX20 was directly sequenced in 170 ASDII patients. Functional consequences of one novel mutation were investigated by surface plasmon resonance, CD spectropolarymetry, fluorescence spectrophotometry, luciferase assay and chromatin immunoprecipitation.

Results: We found a novel mutation in a highly conserved residue in the T-box DNA-binding domain (I121M) segregating with CHD in a three generation kindred. Four mutation carriers revealed cardiac phenotypes in terms of cribriform ASDII, large patent foramen ovale or cardiac valve defects. Interestingly, tertiary hydrophobic interactions within the mutant TBX20 T-box were significantly altered leading to a more dynamic structure of the protein. Moreover, Tbx20-I121M resulted in a significantly enhanced transcriptional activity, which was further increased in the presence of co-transcription factors GATA4/5 and NKX2-5. Occupancy of DNA binding sites on target genes was also increased.

Conclusions: We suggest that TBX20-I121M adopts a more fluid tertiary structure leading to enhanced interactions with cofactors and more stable transcriptional complexes on target DNA sequences. Our data, combined with that of others, suggest that human ASDII may be related to loss- as well as gain-of-function TBX20 mutations.

Having identified teratogenic factors, primary prevention of congenital defects is possible by the implementation of specific measures in pregnant women or those planning pregnancy. Our current understanding of the epigenetic processes acting during reproductive events raises new possibilities to prevent both heritable and sporadic congenital anomalies. Cell differentiation during embryonic-foetal development involves different epigenetic processes which, if altered, may affect either somatic or germ cells. Epigenetic alterations can occur in somatic cells at different stages of life, from fecundation to adulthood, and when germ cells are affected, such changes can even be passed on to future generations.

This review summarizes the main epigenetic processes that influence gene expression and cell specification at different stages of development. The experimental and epidemiological evidence of environmental agents that cause epigenetic alterations is evaluated, as well as their effects in males and females. As a result, new avenues for primary prevention are proposed.

Objective: To estimate heritability of metabolic syndrome traits among healthy younger adults in human population in China, and examine potential sex-difference in heritability and parental effect on metabolic syndrome.

Methods: Using offspring-parent regression, we estimated heritability (h2) of metabolic syndrome traits based on 452 child-parent triads identified from a population-based random survey on metabolic syndrome among people over age of 15 years in Guangzhou, China.

Results: BMI, Cholesterol, HDL-C, LDL-C, FPG and Waist-C were more heritable (h2, 0.42 to 0.545), whereas SBP, DBP, and TG were less heritable (h2, 0.14 to 0.28). Sons had marked increases in heritability for all traits over daughters, particularly Cholesterol (0.653 vs 0.356), FPG (0.602vs 0.313), LDL-C(0.521 vs 0.329) and TG (0.395 vs 0.187) over daughters. Offspring-mother seemed to have a higher heritability in every traits except FPG (0.67 vs 0.794) than offspring-father, most notably for BDP (0.308 vs 0.122), SBP (0.288 vs 0.146), TG (0.387 vs 0.239) and Waist-C (0.581 vs 0.354).

Conclusion: We estimated the heritability of metabolic syndrome traits in human population based on unique population-based offspring-parent sample from China, and found important evidence that maternal and paternal effect on these traits are different and the sex-difference in heritability is marked.

Background: Craniosynostosis can be caused by both genetic and environmental factors, the relative contributions of which vary between patients. Genetic testing identifies a pathogenic mutation or chromosomal abnormality in ~20% of cases, but it is likely that further causative mutations remain to be discovered. We aimed to identify a shared signature of genetically determined craniosynostosis by comparing the expression patterns in 3 monogenic syndromes with a control group of patients with non-syndromic sagittal synostosis.

Methods: We cultured fibroblasts from 10 individuals each with Apert syndrome (FGFR2 substitution S252W), Muenke syndrome (FGFR3 substitution P250R), Saethre-Chotzen syndrome (various mutations in TWIST1) and non-syndromic sagittal synostosis (no mutation detected). The relative expression of ~47,000 transcripts was quantified on Affymetrix arrays.

Results: We identified 435, 45 and 47 transcripts in the Apert, Muenke and Saethre-Chotzen groups, respectively, that differed significantly from the controls. Forty-six of these transcripts were shared between two or more syndromes and, in all but one instance, showed the same direction of altered expression level compared with controls. Pathway analysis showed overrepresentation of the shared transcripts in core modules involving cell-to-cell communication and signal transduction. Individual samples from the Apert syndrome cases could be reliably distinguished from non-syndromic samples based on the gene expression profile, but this was not possible for samples from Muenke and Saethre-Chotzen syndrome patients.

Conclusions: We have identified common modules of altered gene expression shared by genetically distinct forms of craniosynostosis. Although the expression profiles cannot currently be used to classify individual patients, this may be overcome by using more sensitive assays and sampling additional tissues.

IIntrachromosomal triplications are rare chromosomal rearrangements. In most triplication cases the phenotype is similar to, but more severe than in patients with a duplication of the same region. The Williams Beuren syndrome (WBS) region on 7q11.23, is prone to chromosomal rearrangements. A common deletion causes the well characterised Williams Beuren syndrome. The reciprocal duplication has been described in 27 families only, and is associated with a variable phenotype, including speech delay with (mild) mental retardation, autism and mild dysmorphic features. As the duplication of the WBS region is sometimes found in unaffected parents, initially some doubts have been raised about the pathogenicity of the duplication. We here describe the first triplication of a large part of the WBS region, detected with array CGH and confirmed by MLPA and FISH. The phenotypic features include mental retardation, a severe expressive language delay, behavioural problems and dysmorphisms. These features are remarkably similar, but seem more severe, compared to features seen in duplication patients. Therefore, our findings support the idea that a amplification of the WBS region is a disease causing event, although the penetrance might be incomplete.

Background: Silver-Russell syndrome (SRS, OMIM# 180860) features fetal and postnatal growth restriction and variable dysmorphisms. Genetic and epigenetic aberrations on chromosomes 7 and 11 are commonly found in SRS. However, a large fraction of SRS remain with unknown genetic etiology.

Methods: We studied 22 patients diagnosed with SRS (ten with H19 hypomethylation and twelve of unknown molecular etiology) and their parents with the Affymetrix 250K Sty microarray. Several analytical approaches were used to identify genomic aberrations such as copy number changes (CNCs), loss of heterozygosity (LOH), and uniparental disomy (UPD). Selected CNCs were verified with quantitative real-time PCR.

Results: The largest unambiguous CNCs were found in previously molecularly unexplained SRS patients with relatively mild phenotypes: a heterozygous deletion of chromosome 15q26.3 including the IGF1R gene (2.6 Mb), an atypical, distal 22q11.2 deletion (1.1 Mb), and a pseudoautosomal region duplication (2.7 Mb) in a male patient. We also identified LOH regions of potential relevance to the SRS phenotype. Importantly, we did not identify any duplications or UPD of chromosomes 7 or 11.

Conclusion: In summary, we found unexpected submicroscopic genomic events with pathogenic potential in three molecularly unexplained patients with mild SRS. Our findings emphasize that SRS is heterogeneous in genetic etiology beyond the major groups of H19 hypomethylation and matUPD7 and that unbiased genome-scale screens may reveal novel genotype-phenotype correlations.

Background: Epilepsy and Mental Retardation Limited to Females (EFMR) is an intriguing X-linked disorder affecting heterozygous females and sparing hemizygous males. Mutations in the protocadherin 19 (PCDH19) gene have been identified in seven unrelated families with EFMR.

Methods and Results: Here, we assessed the frequency of PCDH19 mutations in individuals with clinical features which overlap those of EFMR. We analysed 185 females from three cohorts: 42 with Rett syndrome (RS) who were negative for MECP2 and CDKL5 mutations, 57 with autism spectrum disorders (ASD) and 86 with epilepsy with or without intellectual disability (ID). No mutations were identified in the RS and ASD cohorts suggesting that despite sharing similar clinical characteristics with EFMR, PCDH19 mutations are not generally associated with these disorders. Among the 86 females with epilepsy (of whom 51 had seizure onset before 3 years), with or without ID, we identified two (2.3%) missense changes. One (c.1671C>G, p.N557K), reported previously without clinical data, was found in two affected sisters, the first EFMR family without a multigenerational family history of affected females [1]. The second, reported here, is a novel de novo missense change identified in a sporadic female. The change, p.S276P, is predicted to result in functional disturbance of PCDH19 as it affects a highly conserved residue adjacent to the adhesion interface of EC3 of PCDH19.

Conclusions: This de novo PCDH19 mutation in a sporadic female highlights that mutational analysis should be considered in isolated instances of girls with infantile onset seizures and developmental delay, in addition to those with the characteristic family history of EFMR.

Smith-Magenis syndrome (SMS) is caused by del(17)p11.2, including the retinoic acid induced 1 gene (RAI1), or mutation of RAI1. Haploinsufficiency of RAI1 results in developmental delay, mental retardation, sleep disturbance, self-abusive behaviors, and most features commonly seen in SMS. Fifty-two subjects were referred for molecular analysis of RAI1 due to the presence of an SMS-like phenotype in each case. For this cohort, deletion and mutation analyses of RAI1 were negative; thus, the clinical diagnosis of SMS could not be confirmed and suggested that at least one other locus was responsible for the phenotype(s) observed. Here, we present whole-genome array comparative genomic hybridization and detailed phenotypic data of these 52 subjects. Specifically, this SMS-like cohort exhibits developmental delays, sleep disturbance, self-abusive behaviors, motor dysfunction, and hyperactivity of the same type and prevalence as that of SMS. From this study, we have discovered at least 5 new loci that likely contribute to the SMS-like phenotype, including CNVs that were found in more than one subject. Genes in these regions function in development, neurological integrity, and morphology, all of which are affected in SMS. In addition, as a result of the phenotypic overlap between SMS and the SMS-like cases, these data may provide some insight into the function of RAI1, including the pathways in which it may be involved and the genes it may regulate. These data will improve diagnosis, understanding, and potentially treatment of these complex behavior and mental retardation syndromes.

Objectives: To determine if the anatomical severity of oral clefting affects familial recurrence in a large population based sample. To provide reliable recurrence risk estimates for oral cleft for first-, second-, and third-degree relatives.

Design: Population based cohort study.

Setting: Denmark.

Participants: 6,776 individuals affected with an oral cleft born from 1952 to 2005 and 54,229 relatives.

Main outcome measures: Recurrence risk estimates for oral cleft for first-, second-, and third-degree relatives and stratification by severity, specificity, parent of origin effect, and family size for first-degree relatives.

Results: For cleft lip and palate probands we observed recurrence risks for first-, second-, and third degree relatives of respectively 3.5% (95% confidence interval 3.1% to 4.0%), 0.8 % (0.6% to 1.0%), and 0.6% (0.4% to 0.8%). Individuals affected by the most severe oral cleft had a significantly higher recurrence risk among both offspring and siblings, e.g. the recurrence risk for siblings of a proband with isolated bilateral cleft lip with cleft palate was 4.6% (3.2 to 6.1) versus 2.5% (1.8 to 3.2) for a proband born with a unilateral defect.

Conclusions: Anatomical severity does have an effect on recurrence in first-degree relatives and the type of cleft is predictive of the recurrence type. Highly reliable estimates of recurrence have been provided for first cousins in addition to more accurate estimates for first and second degree relatives. These results and the majority of prior data continue to support a multifactorial threshold model of inheritance.

Mutations affecting over 2,000 of the 20,000 or so genes in the human genome have been linked so far to specific inherited diseases, most of which are rare and have been poorly understood. Many of the genes involved encode components of intracellular signalling pathways that regulate processes such as the growth, proliferation, differentiation, and survival or programmed death of cells during development and the maintenance of tissues and organs. Mutations that change function of genes encoding signalling proteins thereby cause disorders ranging from birth defects to cancer. For Mendelian disorders, the essentially causal relationship between mutation and disease may present direct opportunities to therapeutically manipulate intracellular signalling. Here, we review recent examples of the use of small-molecule drugs to target components of signalling networks in single gene disorders. We also consider the limitations of these approaches and the difficulties in their clinical development as therapies for rare genetic diseases.

Background: Nonsyndromic isolated cleft lip with or without cleft palate (NCL/P) is a common congenital anomaly in humans, the aetiology of which is complex and associated with both genetic and environmental factors. It has been reported that maternal nutritional factors are likely to play a major role in development of NCL/P in the embryo.

Objective: As the mechanism by which folic acid and choline supplementation prevents NCL/P is poorly understood, we investigated the relationship between 16 polymorphic variants of 12 genes encoding enzymes involved in the metabolism of these two nutrients and the risk of facial clefts.

Results: We found that individuals with the AA genotype of the BHMT rs3733890 polymorphism have a significantly lower risk of orofacial clefts (OR=0.1450; 95%CI: 0.0420-0.4995; p=0.0005; p_corr=0,008). We also demonstrate that the rs7639752 polymorphism of the PCYT1A gene increases the risk of NCL/P nearly twofold in the Polish population (OR=1.891; 95%CI: 1.151-3.107; p=0.011), but this association would not withstand correction for multiple testing (p_corr=0,176). The genetic variations in CBS, MTHFD1, MTHFR, MTR, MTRR, TCN2, BHMT2, CHDH, CHKA and PEMT were not separately correlated with NCL/P risk. However, the Multifactor Dimensionality Reduction (MDR) analysis showed a significant epistatic interaction between MTHFR (rs1801133), MTR (rs1805087) and PEMT (rs4646406) in NCL/P susceptibility.

Conclusion: This study demonstrates that choline metabolism may play an important role in the aetiology of NCL/P. Polymorphic variants of BHMT and PCYT1A and interactions between genes of choline and folate metabolism might influence the risk of NCL/P in the Polish population.

Background: The lethal group of short-rib polydactyly (SRP) includes type I (Saldino-Noonan; MIM 263530), type II (Majewski; MIM 263520), type III (Verma-Naumoff; MIM 263510), and type IV (Beemer-Langer; MIM 269860). Jeune and Ellis-van Creveld (EVC) dysplasias also used to be classified in the group of SRP. Recently, mutations in a gene encoding a protein involved in intraflagellar transport (IFT), IFT80, have been identified in 3/39 patients with Jeune dysplasia, but no extraskeletal manifestation.

Methods: Due to clinical and radiological similarities between Jeune dysplasia and the other lethal types of SRP we decided to investigate IFT80 in a cohort of foetuses with the lethal forms of SRP (Majewski, Verma-Naumoff, and Beemer-Langer) and cases antenatally diagnosed of Jeune dysplasia. Fifteen foetuses were ascertained to this study. We adopted a double molecular approach. For consanguineous families and for those with recurrent sibs we first performed a haplotype analysis around the gene locus and for the others we directly sequenced all the coding exons of IFT80.

Results: Using the haplotype approach for two families, we excluded the IFT80 region as a candidate for them. By direct sequencing of IFT80 in the other 13 cases we found a G-to-C transversion within exon 8 (G241R) in only one SRP case closely related to the type III phenotype.

Conclusions: Our findings demonstrate that mutations in IFT80 can also be responsible for a lethal form of SRP and provides the molecular basis for the Jeune-Verma-Naumoff dysplasia spectrum.

Background: Congenital chromosome abnormalities are relatively common in our species and among structural abnormalities the most common class is balanced reciprocal translocations. Determining the parental origin of de novo balanced translocations may provide insights into how and when they arise. While there is a general paternal bias in the origin of non-recurrent unbalanced rearrangements, there are few data on parental origin of non-recurrent balanced rearrangements.

Methods: The parental origin of a series of de novo balanced reciprocal translocations was determined using DNA from flow sorted derivative chromosomes and linkage analysis.

Results: Of 27 translocations, we found 26 to be of paternal origin and only one of maternal origin. We also found the paternally derived translocations to be associated with a significantly increased paternal age (p<0.008).

Conclusion: Our results suggest there is a very marked paternal bias in the origin of all non-recurrent reciprocal translocations and that they may arise during one of the numerous mitotic divisions that occur in the spermatogonial germ cells prior to meiosis.

Background: Malformations of cortical development are not rare and cause a wide spectrum of neurological diseases based on the affected region in the cerebral cortex. A significant proportion of these malformations could have a genetic basis. However, genetic studies are limited because most cases are sporadic and mendelian forms are rare.

Methods: In order to identify new genetic causes in patients presenting defects of cortical organization, we have performed array-based comparative genomic hybridization (array-CGH) in a cohort of 100 sporadic cases with various types of cortical malformations in search for inframicroscopic chromosomal rearrangements.

Results: In one patient presenting with periventricular nodular heterotopias and marked corpus callosum hypoplasia, we have identified a small (400 kilobases) 17p13.3 deletion involving the YWHAE gene. We show that YWHAE is the only brain-expressed gene in the deleted region and that the other genes in the interval are unlikely to contribute to the brain malformation phenotype of this patient.

Conclusion: Most 17p13.3 deletions reported to date are large, such as the deletions causing Miller-Dieker syndrome, and involve several genes implicated in various steps of brain development. Haploinsufficiency of the mouse ortholog of YWHAE causes a defect of neuronal migration. However, the human counterpart of this phenotype was not known. The case described here represents the smallest reported deletion involving the YWHAE gene and could represent the human counterpart of the abnormal cortical organization phenotype presented by the Ywhae heterozygous knock-out mouse.

Women with a germline mutation in one of the MMR-genes MLH1, MSH2 or MSH6 reportedly have 4-12% lifetime risk of ovarian cancer, but there is limited knowledge on survival. Prophylactic bilateral salpingo-oophorectomy (PBSO) has been suggested for prophylaxis.

The purpose of this retrospective multicentre study was to describe survival in carriers of pathogenic mutations in one of the MMR-genes, and who had contracted ovarian cancer.

Women who had ovarian cancer, and who tested positive for or were obligate carriers of an MMR-mutation, were included from eleven European centres for hereditary cancer. Most women had not attended for gynaecological screening. Crude and disease-specific survival was calculated by the Kaplan-Meier algorithm.

Among the 144 women included, 81.5% had FIGO stage 1 or 2 at diagnosis. Ten-year ovarian cancer specific survival independent of staging was 80.6%, compared to less than 40% that is reported both in population based series and in BRCA-mutation carriers. Disease specific 30 years survival for ovarian cancer was 71.5%, and for all HNPCC/Lynch syndrome related cancers including ovarian cancer 47.3%.

In the series examined, infiltrating ovarian cancer in Lynch syndrome had a better prognosis than infiltrating ovarian cancer in BRCA1/2 mutation carriers or in the general population. Lifetime risk of ovarian cancer of about 10% and a risk of dying of ovarian cancer of 20% gave a lifetime risk of dying of ovarian cancer of about 2% in female MMR-mutation carriers.

Background: Recent candidate and genome wide association studies have identified variants altering susceptibility to breast cancer. We aimed to establish the relevance of these variants to breast cancer risk in familial breast cancer cases both with and without BRCA1 or BRCA2 (BRCA1/2) mutations.

Methods: A cohort of unrelated individuals affected from breast cancer due to the presence of either BRCA1 (121) or BRCA2 mutations (109) and females with familial breast cancer, not due to BRCA1/2 mutations (722) were genotyped using Taqman SNP Genotyping Assays. Allele frequencies were compared with an ethnically and gender-matched group (436).

Results: A synonymous variant (Ser51) in TOX3 (previously TNRC9) was associated with an increased risk of breast cancer (OR=p<0.001) in BRCA2 mutation carriers. We replicated the associations for FGFR2 (p=0.046), TOX3 (p<0.001), MAP3K1 (p=0.03), CASP8 (p=0.02) and the chromosome 8-associated SNP (p=0.004) in individuals without BRCA1/2 mutations. Additionally, homozygote carriers of MAP3K1 variants were shown to have a significantly lower Manchester Score (p=0.003), while individuals carrying one or two copies of the FGFR2 variant had a higher Manchester Score (p=0.01).

Conclusion: Our study confirms that susceptibility variants in FGFR2, TOX3, MAP3K1, and on chromosome 8q are all associated with increased risk of cancer in individuals with a family history of breast cancer, whereas CASP8 is protective in this context. The level of risk is dependent upon the strength of the family history and the presence of a BRCA1/2 mutation and contributes to the understanding of the use of these variants in clinical risk prediction.

Deletions in the 17p13.3 region are associated with abnormal neuronal migration. Point mutations or deletion copy number variants of the PAFAH1B1 gene in this genomic region cause lissencephaly whereas extended deletions involving both PAFAH1B1 and YWHAE result in Miller-Dieker syndrome characterized by facial dysmorphisms and a more severe grade of lissencephaly. The phenotypic consequences of YWHAE deletion without deletion of PAFAH1B1 have not been studied systematically. We performed a detailed clinical and molecular characterization of five patients with deletions involving YWHAE but not PAFAH1B1, two with deletion including PAFAH1B1 but not YWHAE and one with deletion of YWHAE and mosaic for deletion of PAFAH1B1. Three deletions were terminal whereas five were interstitial. Patients with deletions including YWHAE but not PAFAH1B1 presented with significant growth restriction, cognitive impairment, shared craniofacial features, and variable structural abnormalities of the brain. Growth restriction was not observed in one patient with deletion of YWHAE and TUSC5, implying that other genes in the region may have a role in regulation of growth with CRK being the most likely candidate. Using array based comparative genomic hybridization and long range PCR, we have delineated the breakpoints of these non-recurrent deletions and show that the interstitial genomic rearrangements are likely generated by diverse mechanisms, including the recently described Fork Stalling and Template Switching (FoSTeS)/ Microhomology Mediated Break Induced Replication (MMBIR).

Objectives: Mucolipidoses II and III alpha/beta (ML II and ML III) are lysosomal disorders in which the essential mannose-6-phosphate recognition marker is not synthesized onto lysosomal hydrolases and other glycoproteins. The disorders are caused by mutations in GNPTAB, which encodes two of three subunits of the heterohexameric enzyme, N-acetylglucosamine-1-phosphotransferase. Clinical, biochemical, and molecular findings in 61 probands (63 patients) are presented in order to provide a broad perspective of these mucolipidoses.

Methods: GNPTAB was sequenced in all probands and/or parents. Activity of several lysosomal enzymes was measured in plasma, and GlcNac-1-phosphotransferase was assayed in leukocytes. Thirty-six patients were studied in detail, allowing extensive clinical data to be abstracted.

Results: ML II correlates with near total absence of phosphotransferase activity resulting from homozygosity or compound heterozygosity for frameshift or nonsense mutations. Craniofacial and orthopedic manifestations are evident at birth, skeletal findings become more obvious within the first year, and growth is severely impaired. Speech, ambulation, and cognitive function are impaired. ML III retains a low level of phosphotransferase activity due to at least one missense or splice site mutation. The phenotype is milder with minimal delays in milestones, the appearance of facial coarsening by early school age, and slowing of growth after age four years.

Conclusions: Fifty-one pathogenic changes in GNPTAB are presented, including 42 novel mutations. Ample clinical information improves criteria for delineation of ML II and ML III. Phenotype-genotype correlations suggested in more general terms in earlier reports on smaller groups of patients are specified and extended.

Over the last few years, array-CGH has remarkably improved the ability to detect cryptic unbalanced rearrangements in patients presenting with syndromic mental retardation. Using whole genome oligonucleotide array-CGH, we detected 5q14.3 microdeletions ranging from 216 kb to 8.8 Mb in 5 unrelated patients showing phenotypic similarities, namely severe mental retardation with absent speech, hypotonia and stereotypic movements. Most of the patients presented also with facial dysmorphic features, epilepsy and/or cerebral malformations. The minimal common deleted region of these 5q14 microdeletions encompassed only MEF2C, known to act in brain as a neurogenesis effector which regulates excitatory synapse number. In a patient presenting a similar phenotype, we subsequently identified a MEF2C nonsense mutation. Taken together, these results strongly suggest that haploinsufficiency of MEF2C is responsible for severe mental retardation with stereotypic movements, seizures and/or cerebral malformations.

Background: Leukoencephalopathy with brain stem and spinal cord involvement and high brain lactate (LBSL) was first defined by characteristic MRI and spectroscopic findings. The clinical features include childhood or juvenile-onset slowly progressive ataxia, spasticity, and dorsal column dysfunction, occasionally accompanied by learning difficulties. Mutations in DARS2, encoding mitochondrial aspartyl-tRNA synthetase, were recently shown to cause LBSL. The signs and symptoms show some overlap with the most common leukoencephalopathy of young adults, multiple sclerosis (MS).

Objective: To clarify the molecular background of LBSL patients in Finland, and to look for DARS2 mutations in a group of MS patients.

Methods: Clinical evaluation of LBSL patients, DARS2 sequencing and haplotype analysis, and carrier frequency determination in Finland.

Results: All eight LBSL patients were compound heterozygotes for DARS2 mutations: all carried R76SfsX5 change, seven had M134_K165del and one had C152F change. Axonal neuropathy was found in five of the eight patients. The carrier frequencies of the R76SfsX5 and M134_K165del mutations were 1:95 and 1:380, respectively. All patients shared common European haplotypes, suggestive of common European LBSL ancestors. No enrichment of the two common DARS2 mutations was found in 321 MS patients.

Conclusion: All LBSL patients were compound heterozygotes, which suggests that DARS2 mutation homozygosity may be lethal or manifest as a different phenotype. We show here that despite identical mutations the clinical picture was quite variable in the patients. Axonal neuropathy was an important feature of LBSL. DARS2 mutations cause childhood-to-adolescence-onset leukoencephalopathy, but they do not seem to be associated with MS.

Background: Dravet syndrome is a severe infantile epileptic encephalopathy caused in approximately 80% of cases by mutations in the voltage-gated sodium channel subunit gene SCN1A. The majority of these mutations are de novo. The parental origin of de novo mutations varies widely among genetic disorders and the aim of this study was to determine this for Dravet syndrome.

Methods: Ninety-one patients with de novo SCN1A mutations and their parents were genotyped for single-nucleotide polymorphisms (SNPs) in the region surrounding their mutation. Allele-specific PCR based on informative SNPs was used to separately amplify and sequence the paternal and maternal alleles to determine in which parental chromosome the mutation arose.

Results: We established the parental origin of SCN1A mutations in 44 patients for whom both parents were available and SNPs were informative. The mutations were of paternal origin in 33 cases and of maternal origin in the remaining 11 cases. De novo mutation of SCN1A most commonly, but not exclusively, originates from the paternal chromosome. The average age of parents originating mutations did not differ from that of the general population.

Conclusions: The greater frequency of paternally derived mutations in SCN1A is likely to be due to the greater chance of mutational events during the increased number of mitoses which occur during spermatogenesis compared to oogenesis and greater susceptibility to mutagenesis of the methylated DNA characteristic of sperm cells.

Peripheral arterial disease (PAD) is associated with significant morbidity and mortality, and has a higher prevalence in African Americans than Caucasians. Ankle arm index (AAI) is the ratio of systolic blood pressure in the leg to that in the arm, and, when low, is a marker of PAD. We used an admixture mapping approach to search for genetic loci associated with low AAI. Using data from 1040 African-American participants in the observational, population-based Health, Aging, and Body Composition Study who were genotyped at 1322 single nucleotide polymorphisms(SNPs) that are informative for African versus European ancestry and span the entire genome, we estimated genetic ancestry in each chromosomal region and then tested the association between AAI and genetic ancestry at each locus. We found a region of chromosome 11 that reaches its peak between 80 and 82 Mb associated with low AAI (p<0.001 for rs12289502 and rs9665943, both within this region). 753 African-American participants in the observational, population-based Cardiovascular Health Study were genotyped at rs9665943 to test the reproducibility of this association, and this association was also statistically significant (odds ratio(OR) for homozygous African genotype 1.59 (95% confidence interval (CI) 1.12-2.27)). Another candidate SNP (rs1042602) in the same genomic region was tested in both populations, and was also found to be significantly associated with low AAI in both populations (OR for homozygous African genotype 1.89 (95% CI 1.29-2.76)). This study identifies a novel region of chromosome 11 representing an area with a potential candidate gene associated with PAD in African Americans.

Background: The 10q24 chromosomal region has previously been implicated in Split Hand Foot Malformation (SHFM). SHFM3 was mapped to a large interval on chromosome 10q. The corresponding Dactylaplasia mouse model was linked to the syntenic locus on chromosome 19. It was shown that the two existing Dac alleles result from MusD-insertions upstream of or within Dactylin (Fbxw4). However, all efforts to find the underlying cause for the human SHFM3 have failed on the anaysis of all the genes within the linkage region. Intriguingly a submicroscopic duplication within the critical locus on chromosome 10q24 was associated with the phenotype.

Methods and Results: As a part of screening for genomic rearrangements in cases with unexplained syndromic limb defects, a cohort of patients was analyzed by array CGH (Comperative Genomic Hybridization). A 10q24 microduplication was detected in 6 individuals with distal limb deficiencies associated with micrognathia, hearing problems and renal hypoplasia. In addition, in a family with two affected siblings, a somatic/gonadal mosaicism for the microduplication was detected in the apparently healthy mother. Using a high resolution oligoarray further delineation of the duplication size was performed.

Conclusions: The detected 10q24 genomic imbalance in our syndromic patients has a similar size to the duplication in the previously reported individuals with an isolated form of SHFM, thus extending the clinical spectrum of SHFM3. These findings clearly demonstrate the importance of array CGH in the detection of the etiology of complex, clinically heterogeneous entities.

Background: Primary open angle glaucoma (POAG) is a progressive optic neuropathy characterised by the selective loss of retinal ganglion cells (RGCs), pathological optic disc cupping and visual field defects. The OPA1 gene encodes for an inner mitochondrial membrane protein crucial for normal mitochondrial function and pathogenic mutations cause autosomal dominant optic atrophy (DOA) by specifically targeting RGCs. This raises the distinct possibility that more subtle genetic variations in OPA1 could alter the risk of developing glaucoma.

Methods: We studied 137 POAG patients, 67 patients with high tension glaucoma (HTG), 70 patients with normal tension glaucoma (NTG), and 75 controls from the North East of England. Three single nucleotide polymorphisms (SNPs) in intron 8 (IVS8+4c>t and IVS8+32t>c) and exon 4 (c.473A>G) of the OPA1 gene were genotyped in our study group. In addition, the entire OPA1 coding region was sequenced in 24 individuals with the CT/TT compound genotype using standard BigDyeTM chemistries.

Results: There was no difference in either allele or genotype frequency for the IVS8+32t>c SNP between patients and controls, but there was a significant association between the T allele at IVS8+4c>t and the risk of developing NTG (Odds ratio (OR) = 2.04, 95% confidence interval (CI) = 1.10-3.81, P = 0.004), but not HTG. Logistic regression analysis also confirmed a strong association between the CT/TT compound genotype at IVS8+4 and IVS8+32 with NTG (OR = 29.75, 95% CI = 3.83-231.21, P = 0.001).

Conclusions: The CT/TT compound genotype at IVS8+4 and IVS8+32 is a strong genetic risk determinant for NTG but not HTG.

Background: Cytomegalovirus (CMV) infection is the most frequent infectious disease following organ transplantation. Strategies to prevent this infection remain matter of debate, and discovering genetic risk factors might assist in adapting preventive strategies. By inhibiting IFN production, programmed death 1 (PD-1) has a crucial role in anti-CMV immune response. A SNP within intron 4 of the gene (rs11568821), called PD-1.3, has recently been reported to be clinically relevant in several immune disorders. However, its association with CMV infection has never been reported.

Methods: In this study, we investigated the risk of CMV infection according to PD-1.3 genotype in 469 kidney graft recipients transplanted between 1995 and 2005.

Results: We found that the A allele was associated with the risk of CMV infection in seropositive patients who did not receive CMV prophylaxis (OR=2.60, p=0.006). Multivariate analysis including other risk factors for CMV infection showed that this allele was independently associated with CMV infection (OR=2.54, p=0.010). Interestingly, combined analysis of PD-1.3 with the IL12B 3’UTR SNPs (previously shown to be associated with CMV infection) revealed that patients with the PD-1.3 A allele had a much higher risk of CMV infection compared to those having neither risk allele (OR=3.76, p=0.0003).

Conclusion: This study identified a new genetic risk factor for CMV infection after kidney transplantation and suggests that an adjustment of CMV prophylaxis based on genetic markers would merit further investigation.

Background: Mutations and deletions of the homeobox transcription factor gene SHOX are known to cause short stature. We have analyzed SHOX enhancer regions in a large cohort of short stature patients to study the importance of regulatory regions in developmentally relevant genes like SHOX.

Methods: We tested for the presence of copy number variations in the pseudoautosomal region of the sex chromosomes in 735 individuals with idiopathic short stature and compared the results to 58 cases with Leri-Weill syndrome and 100 normal height controls, using FISH, SNP, microsatellites and MLPA analysis.

Results: A total of 31/735 (4.2%) microdeletions were identified in the pseudoautosomal region in patients with idiopathic short stature; eight of these microdeletions (8/31; 26%) involved only enhancer sequences residing a considerable distance away from the gene. In 58 Leri-Weill syndrome patients, a total of 29 microdeletions were identified; almost half of these (13/29; 45%) involve enhancer sequences and leave the SHOX gene intact. These deletions were absent in 100 control persons.

Conclusion: We conclude that enhancer deletions in the SHOX gene region are a relatively frequent cause of growth failure in patients with idiopathic short stature and Leri-Weill syndrome. Our data highlight the growing recognition that regulatory sequences are of crucial importance in the genome when diagnosing and understanding the etiology of disease.

Background: Hearing loss with enlarged vestibular aqueduct (EVA) can be inherited as an autosomal recessive trait caused by bi-allelic mutations of SLC26A4. However, many EVA patients have non-diagnostic SLC26A4 genotypes with only one or no detectable mutant alleles.

Methods and results: In this study, we were unable to detect occult SLC26A4 mutations in EVA patients with non-diagnostic genotypes by custom comparative genomic hybridization (CGH) microarray analysis or by sequence analysis of conserved non-coding regions. We sought to compare the segregation of EVA among 71 families with two (M2), one (M1) or no (M0) detectable mutant alleles of SLC26A4. The segregation ratios of EVA in the M1 and M2 groups were similar, but the segregation ratio for M1 was significantly higher than in the M0 group. Haplotype analyses of SLC26A4-linked STR markers in M0 and M1 families revealed discordant segregation of EVA with these markers in eight of 24 M0 families.

Conclusion: Our results support the hypothesis of a second, undetected SLC26A4 mutation that accounts for EVA in the M1 patients, in contrast to non-genetic factors, complex inheritance, or etiologic heterogeneity in the M0 group of patients. These results will be helpful for counseling EVA families with non-diagnostic SLC26A4 genotypes.

Background: Rett syndrome is a severe neurodevelopmental disorder representing one of the most common genetic causes of mental retardation in girls. The classic form is caused by MECP2 mutations. In two patients affected by the congenital variant of Rett we have recently identified mutations in the FOXG1 gene encoding a brain-specific transcriptional repressor, essential for early development of the telencephalon.

Methods: Sixty MECP2/CDKL5 mutation-negative European Rett patients (classic and variants), 43 patients with encephalopathy with early-onset seizures and 4 atypical Rett patients were analyzed for mutations in FOXG1.

Results: Mutations have been identified in 4 patients, independently classified as congenital Rett variants from France, Spain and Latvia.

Conclusions: Clinical data have been compared with the two previously reported patients with mutations in FOXG1. In all cases hypotonia, irresponsiveness and irritability were present in the neonatal period. At birth head circumference was normal while a deceleration of growth was recognised soon afterwards, leading to severe microcephaly. Motor development was severely impaired and voluntary hand use was absent. In contrast with classic Rett, patients showed poor eye contact. Typical stereotypic hand movements with hand-washing and hand-mouthing activities were present continuously. Some patients showed abnormal movements of the tongue and jerky movements of the limbs. Brain MRI showed corpus callosum hypoplasia in most cases, while epilepsy was a variable sign. Scoliosis was present and severe in the older patients. Neurovegetative symptoms typical of Rett were frequently present.

Background: Roberts syndrome (RBS) and SC phocomelia are caused by mutations in ESCO2, which codes for an acetyltransferase involved in the regulation of sister chromatid cohesion. From 26 mutations described to date only one missense mutation has been reported and all others are predicted to be truncating mutations. Genotype–phenotype analysis has been hampered by limited numbers of patients with clinical information available.

Methods: We provide unpublished clinical data for 31 individuals with proven ESCO2 mutations and combined this series with previously reported clinical and mutation data on 18 cases. We analyzed genotype-phenotype correlations and functional effects of two novel ESCO2 mutations. In situ hybridization on human embryos at Carnegie stages 14, 17, and 21 was performed to study ESCO2 expression during development.

Results and conclusions: Using the cohort of 49 patients, we delineate the clinical criteria for RBS to include: growth retardation, symmetric mesomelic shortening of the limbs in which the upper limbs are more frequently and severely affected than the lower limbs and characteristic facies with microcephaly. The severity of malformations of the facies correlates with the severity of limb reduction. We found that the occurrence of corneal opacities may be associated with specific mutations. We describe two new mutations, both in the ESCO2 acetyltransferase domain and show their acetylation effects in vitro. In situ hybridization on human embryos showed ESCO2 expression in brain, face, limb, kidney and gonads, which corresponds with the structures affected in RBS.

Objective: To identify genetic causes of COACH syndrome.

Background: COACH syndrome is a rare autosomal recessive disorder characterized by Cerebellar vermis hypoplasia, Oligophrenia (developmental delay/mental retardation), Ataxia, Coloboma, and Hepatic fibrosis. The vermis hypoplasia falls in a spectrum of mid-hindbrain malformation called the molar tooth sign (MTS), making COACH a Joubert syndrome-related disorder (JSRD).

Subjects and Methods: In a cohort of 251 families with JSRD, 26 subjects in 23 families met criteria for COACH syndrome, defined as JSRD plus clinically apparent liver disease. Diagnostic criteria for JSRD were clinical findings (intellectual impairment, hypotonia, ataxia) plus supportive brain imaging findings (MTS or cerebellar vermis hypoplasia). MKS3/TMEM67 was sequenced in all subjects for whom DNA was available. In COACH subjects without MKS3 mutations, CC2D2A, RPGRIP1L and CEP290 were also sequenced.

Results: 19/23 families (83%) with COACH syndrome carried MKS3 mutations, compared to 2/209 (1%) with JSRD but no liver disease. Two other families with COACH carried CC2D2A mutations, one family carried RPGRIP1L mutations and one lacked mutations in MKS3, CC2D2A, RPGRIP1L and CEP290. Liver biopsies from three subjects, each with mutations in one of the three genes, revealed changes within the congenital hepatic fibrosis/ductal plate malformation spectrum. In JSRD with and without liver disease, MKS3 mutations account for 21/232 families (9%).

Conclusions: Mutations in MKS3 are responsible for the majority of COACH syndrome, with minor contributions from CC2D2A and RPGRIP1L; therefore, MKS3 should be the first gene tested in patients with JSRD plus liver disease and/or coloboma, followed by CC2D2A and RPGRIP1L.

Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant syndrome characterized by a predisposition to early-onset colorectal, endometrial and other cancers. The tumours typically exhibit microsatellite instability due to defective mismatch repair. HNPCC is classically caused by heterozygous loss-of-function mutations within the mismatch repair genes MLH1, MSH2, MSH6 and PMS2, but no pathogenic mutations are identified in a third of cases. In recent years, constitutional epimutations of the MLH1 gene, characterized by soma-wide allele-specific promoter methylation and transcriptional silencing, have been identified in a handful of mutation-negative HNPCC cases. In contrast to genetic mutations, MLH1 epimutations are reversible between generations and thus display non-Mendelian inheritance. This review focuses on the aetiological role of constitutional MLH1 epimutations in the development of HNPCC-related cancers. The molecular characteristics, clinical ramifications and potential mechanism underlying this defect are discussed. Recommendations for the selection of cases warranting screening for MLH1 epimutations are proffered.

Background: The FOXG1 gene has been recently implicated in the congenital form of Rett syndrome (RTT). It encodes the Forkhead box protein G1, a winged-helix transcriptional repressor with expression restricted to testis and brain where it is critical for forebrain development. So far, only two point mutations in FOXG1 have been reported in females affected by the congenital form of RTT.

Aim: To assess the implication of FOXG1 in the molecular aetiology of classical RTT syndrome and related disorders.

Methods: We screened the entire multi-exon coding sequence of FOXG1 for point mutations and large rearrangements in a cohort of 35 MECP2/CDKL5 mutation-negative female individuals including 31 classical and 4 congenital forms of RTT.

Results: We identified two different de novo heterozygous FOXG1 truncating mutations. The individual with the p.Trp308X mutation presented with a severe RTT-like neurodevelopment disorder, whereas the p.Tyr400X allele was associated with a classical clinical RTT presentation.

Conclusions: These new cases give additional support to the genetic heterogeneity in RTT, and help to delineate the clinical spectrum in the FOXG1-related phenotypes. FOXG1 screening should be considered in the molecular diagnosis of RTT.

HHT is an autosomal dominant disease with an estimated prevalence of at least 1/5000 which can frequently be complicated by the presence of clinically significant arteriovenous malformations in the brain, lung, gastrointestinal tract and liver. HHT is under-diagnosed and families may be unaware of the available screening and treatment, leading to unnecessary stroke and life-threatening hemorrhage in children and adults. The goal of this international HHT guidelines process was to develop evidence-informed consensus guidelines regarding the diagnosis of HHT and the prevention of HHT-related complications and treatment of symptomatic disease. The overall guidelines process was developed using the AGREE framework, using a systematic search strategy and literature retrieval with incorporation of expert evidence in a structured consensus process where published literature was lacking. The Guidelines Working Group included experts (clinical and genetic) from eleven countries, in all aspects of HHT, guidelines methodologists, health care workers, health care administrators, HHT clinic staff, medical trainees, patient advocacy representatives and patients with HHT. The Working Group determined clinically relevant questions during the pre-conference process. The literature search was conducted using the OVID MEDLINE database, from 1966 to October 2006. The Working Group subsequently convened at the Guidelines Conference to partake in a structured consensus process using the evidence tables generated from the systematic searches. The outcome of the conference was the generation of 33 recommendations for the diagnosis and management of HHT, with at least 80% agreement amongst the expert panel for 30 of the 33 recommendations.

Hirschsprung disease (HSCR) is a developmental disorder due to a defect of neural crest neuroblasts migration process. It is considered as the paradigm of complex disorders, with many loci contributing to the manifestation of the disease. Although HSCR commonly appears as a sporadic trait, approximately 20% of HSCR cases are familial, with complex patterns of inheritance. Here we report a multiplex HSCR family with an additive model of inherence, in which the contribution of 3 genes (RET, NTRK3, EDN3) leads to HSCR phenotype. Our findings suggest that both RET and NTRK3 mutations acting together would be necessary and sufficient for the appearance of the disease, while the EDN3 mutation would act as a phenotype modifier factor in the context of this family as 2 different HSCR phenotypes are seen among the affected members: a short segment form, and a total colonic aganglionosis. The present results therefore support the complex additive model of inheritance previously proposed for Hirschsprung disease.

Background: We report a child with autism and mild microcephaly who was found to have a de novo 3.3 Mb microdeletion on chromosome 1p34.2p34.3. Here, we test the hypothesis that this microdeletion contains one or more genes that underlie the autism phenotype in this child and in other children with autism spectrum disorders.

Methods: To search for submicroscopic chromosomal rearrangements in the child, we performed array comparative genomic hybridization (aCGH) using a 19K whole-genome human BAC array and the Illumina 610-Quad BeadChip microarray. Ingenuity Pathway Analysis (IPA) was used to construct functional biological networks to identify candidate autism genes. To identify putative functional variants in candidate genes, we performed mutation screening using PCR-based Sanger sequencing in 512 unrelated autism patients and 462 control subjects.

Results: We identified a de novo 3.3 Mb deletion containing ~43 genes in chromosome 1p34.2p34.3 that we confirmed using fluorescence in situ hybridization (FISH). Literature review and bioinformatics analyses identified RIMS3 (Regulating Synaptic Membrane Exocytosis 3) as the most promising autism candidate gene. Mutation screening of this gene in autism patients identified five inherited coding variants, including one (p.E177A) that segregated with the autism phenotype in a sibship, was predicted to be deleterious, and was absent in 1,161 controls.

Conclusions: Our case report and mutation screening data suggest that RIMS3 is an autism causative or contributory gene. Functional studies of RIMS3 variants such as p.E177A should provide additional insight into the role of synaptic proteins in the pathophysiology of autism.

Background: Leigh syndrome is an early-onset progressive neurodegenerative disorder with developmental and motor skills regression. Characteristic MRI abnormalities consist of focal bilateral lesions in the basal ganglia and/or the brainstem. The main cause is a deficiency in oxidative phosphorylation (OXPHOS) due to mutations in one of the mtDNA or nuclear OXPHOS genes.

Methods and results: Here we report on a mutation in a new complex I assembly gene C20orf7 in a consanguineous Moroccan family with Leigh syndrome. The family consists of 11 children, three of which are affected. Marker analysis revealed a homozygous region of 11.5Mb on chromosome 20, containing 111 genes. Eight possible mitochondrial candidate genes were sequenced. Patients were homozygous for an unclassified variant (p.P193L) in the cardiolipin synthase gene (CRLS1). As this variant was present in 10% of the normal population and enzyme activity was only reduced to 50%, this could not explain the clinical phenotype in our family. Patients were also homozygous for an amino acid substitution (p.L159F) in C20orf7. Parents were heterozygous and unaffected sibs heterozygous or homozygous wild type. The mutation affects the predicted S-adenosylmethionine (SAM)-dependent methyltransferase domain of C20orf7, possibly involved in methylation of NDUFB3 during the assembly process. Blue native gel electrophoresis showed an altered complex I assembly with only 30-40% of mature complex I present in patients and 70-90% in carriers.

Conclusions: We conclude that a new cause of Leigh syndrome can be a defect in early complex I assembly due to C20orf7 mutations.

Background: Selection for genetic testing of BRCA1/BRCA2 is an important area of healthcare. Whilst testing costs for mutational analysis are falling, costs in North America remains in excess of US $3,000 (UK price can be £690). Guidelines in most countries use a 10-20% threshold of detecting a mutation in BRCA1/2 combined within a family before mutational analysis is considered. A number of computer based models have been developed. However, use of these models can be time consuming and difficult to use. The Manchester scoring system was developed in 2003 to simplify the selection process without losing accuracy.

Methods: In order to increase accuracy of prediction we have incorporated breast pathology of the index case into the Manchester scoring system based on 2156 samples from unrelated non-Jewish patients fully tested for BRCA1/2, and adapted the scores accordingly.

Results/Discussion: Data from breast pathology allowed adjustment of BRCA1 and combined BRCA1/2 scores alone. There was a lack of pathological homogeneity for BRCA2, therefore specific pathological correlates could not be identified. Upward adjustments in BRCA1 mutation prediction scores were made for grade 3 ductal cancers, ER and triple negative tumours. Downward adjustments in the score were made for Grade 1 tumours, lobular cancer, DCIS and ER/HER2 positivity. Application of the updated scoring system led to 4/9 more mutations in BRCA1 being identified at the 10/20% threshold respectively. Furthermore, there were 65/58 fewer cases meeting the 10/20% threshold for testing. Moreover, the adjusted score significantly improved the trade-off between sensitivity and specificity for BRCA1/2 prediction.

Background: Two recent genome-wide association studies identified the liver-expressed transmembrane protein adiponutrin to be associated with liver-related phenotypes such as nonalcoholic fatty liver disease and liver function enzymes. These associations were not uniformly reported for various ethnicities. The aim of this study was to investigate a common nonsynonymous variant within adiponutrin (rs738409, exon 3) with parameters of liver function in three independent West-Eurasian study populations including a total of 4290 participants.

Methods: The study was performed in 1) the population-based Bruneck Study (n=783), 2) the SAPHIR Study from Austria based on a healthy working population (n=1705), and the Utah Obesity Case-Control Study including a group of 1019 severely obese individuals (average BMI 46.0 kg/m2) and 783 controls from the same geographical region of Utah. Liver enzymes measured were alanine-aminotransferase (ALT), aspartate-aminotransferase (AST) and gamma-glutamyl transferase (GGT).

Results and discussion: We found a strong recessive association of this polymorphism with age- and gender-adjusted ALT and AST levels: being homozygous for the minor allele resulted in a highly significant increase of ALT levels of 3.53 U/L (p=1.86x10-9) and of AST levels of 2.07 U/L (p=9.58x10-6), respectively. The associations were consistently found in all three study populations. In conclusion, the highly significant associations of this transversion polymorphism within the adiponutrin gene with increased ALT and AST levels support a role for adiponutrin as a susceptibility gene for hepatic dysfunction.

Background: Male carriers of the FMR1 premutation are at risk of developing the fragile X-associated tremor/ataxia syndrome (FXTAS), a newly recognized and largely under-diagnosed late onset neurodegenerative disorder. Patients affected with FXTAS primarily present with cerebellar ataxia and intention tremor. Cognitive decline has also been associated with the premutation but the lack of data on its penetrance is a growing concern for clinicians who provide genetic counseling.

Methods: The Mattis Dementia Rating Scale (MDRS) was administered in a double-blind fashion to 74 males aged 50 years or more recruited from fragile X families (35 premutation carriers and 39 intrafamilial controls) regardless of their clinical manifestation. Based on previous publications, marked cognitive impairment was defined by a score ≤ 123 on the MDRS.

Results: Both logistic and survival models confirmed that in addition to age and education level, premutation size plays a significant (p<0.01 and p<0.03 for logistic and survival model, respectively) role in cognitive impairment. The estimated penetrance of marked cognitive impairment in our sample (adjusted for the mean age: 63.4 years and mean education level: 9.7 years) for midsize/large (70-200 CGG) and small (55-69 CGG) premutation alleles was 33.3% (RR: 6.5; p = 0.01) and 5.9% (RR: 1.15; p = 0.9) respectively. Penetrance in the control group was 5.1%.

Conclusions: Male carriers of midsize to large premutation alleles had a 6-fold increased risk of developing cognitive decline and the risk increases with allele size. In addition, it was observed that cognitive impairment may precede motor symptoms. These data provide guidance for genetic counseling although larger samples are required to refine these estimates.

Background: Genome-wide screening of patients with mental retardation using Array Comparative Genomic Hybridization (array-CGH) has identified several novel imbalances. With this genotype-first approach, the 2q22.3q23.3 deletion was recently described as a novel microdeletion syndrome. We report two unrelated patients with a de novo interstitial deletion mapping in this genomic region and presenting similar "pseudo-Angelman" phenotypes, including severe psychomotor retardation, speech impairment, epilepsy, microcephaly, ataxia and behavioural disabilities.

Methods: The microdeletions were identified by array-CGH using oligonucleotide and BAC-arrays, and further confirmed by Fluorescence In Situ Hybridization (FISH) and semi-quantitative PCR.

Results: The boundaries and sizes of the deletions in the two patients were different but an overlapping region of about 250 kb was defined, which mapped to 2q23.1 and included two genes: MBD5 and EPC2. The SIP1 gene associated with the Mowat Wilson syndrome was not included in the deleted genomic region.

Discussion: Haploinsufficiency of one of the deleted genes (MBD5 or EPC2) could be responsible for the common clinical features observed in the 2q23.1 microdeletion syndrome and this hypothesis needs further investigation.

Background: Targeted delivery of the angiogenic factor, VEGF, to motor neurons prolongs survival in rodent models of ALS, while mice expressing reduced VEGF levels develop motor neuron degeneration reminiscent of ALS, raising the question whether VEGF contributes to the pathogenesis of ALS. An initial association study reported that VEGF haplotypes conferred an increased susceptibility to ALS in humans, but later studies challenged this initial finding.

Methods and Findings: A meta-analysis was undertaken to critically reappraise whether any of the three common VEGF gene variations (-2578C/A, -1154G/A and -634G/C) increase the risk of ALS. Over 7,000 individuals from 8 European and 3 American populations were included in the analysis. Pooled odds ratios were calculated using fixed and random effect models and 4 potential sources of heterogeneity (location of disease onset, gender, age at disease onset and disease duration) were assessed. After correction, none of the genotypes or haplotypes was significantly associated with ALS. Subgroup analyses by gender revealed, however, that the -2578AA genotype, which lowers VEGF expression, increased the risk of ALS in males (OR=1.46 males versus females; CI=1.19-1.80; p=7.8 10E-5), even after correction for publication bias and multiple testing.

Conclusion: This meta-analysis does not support the original conclusion that VEGF haplotypes increase the risk of ALS in humans, but the significant association of the low-VEGF -2578AA genotype with an increased susceptibility to ALS in male subjects reappraises the link between reduced VEGF levels and ALS, as originally revealed by the fortuitous mouse genetic studies.

Many formal tributes have been made in print enumerating Bob Gorlin’s major contributions to Medicine and Dentistry; listing his academic appointments, his many honours and his extensive list of publications. However, what can’t be enumerated are the thousands and thousands of patients’ lives he benefited either in his own practice or by giving his opinion to other physicians. Also too numerous to list are the thousands of doctors whose skills have been enhanced by knowing or listening to Bob. In this tribute I will try to capture how Bob enriched and influenced my life and the lives of many of my colleagues in Europe. I am aware however that I am only taking about one of his ‘constituencies’, the Clinical Geneticists, and that he had others; dentistry, oral pathology, craniofacial development and dermatology.

"In human genetics, we think Bob belongs to us, but the dentists, the pathologists, the dermatologists, the oncologists, the reconstructive surgeons, and the craniofacial specialists all think he belongs to them too." So extolled his longtime colleague and friend Judy Hall in her citation for Bob Gorlin’s Award for Excellence in Human Genetics Education presented at the 2004 Annual Meeting of the American Society of Human Genetics (ASHG) in Toronto.1 It is almost impossible to describe in words Bob’s tremendous influence in all aspects of medicine, and to so many people, his largesse, and his unselective unconditional generosity. Yet, this single citation says it all. No wonder his influence is so far-reaching in all walks of medicine and life, and no wonder he has so many admirers the world around.

Aims: In UK patients with clinically-defined definite familial hypercholesterolaemia (FH) to determine the relative frequency of mutations in three different genes (LDLR, APOB, PCSK9), and to examine their impact in development of coronary heart disease (CHD).

Subject and Methods: 409 FH patients (158 with CHD) were studied. The LDLR was partially screened by SSCP (exons 3, 4, 6-10 and 14) and using a commercial kit for gross deletions/rearrangements. APOB (p.R3500Q) and PCSK9 (p.D374Y) were detected by specific assays. Coding exons of PCSK9 were screened by SSCP.

Results: Mutations were detected in 253 (61.9%) patients; 236 (57.7%) in LDLR; 10 (2.4%) carried APOB p.Q3500 and 7 (1.7%) PCSK9 p.Y374. No additional mutations were identified in PCSK9. After adjusting for age, sex, smoking and systolic blood pressure, compared to those with no detectable mutation, the odds ratio of having CHD in those with an LDLR mutation was 1.84 (95% CI 1.10-3.06), for APOB 3.40 (0.71-16.36), and for PCSK9 19.96 (1.88-211.5), (p=0.0003 overall). The high risk in LDLR and PCSK9 p.Y374-carrying subjects was partly explained by their higher pre-treatment cholesterol levels (LDLR, PCSK9, no-mutation, 10.29±1.85mmol/l, 13.12±1.69mmol/l, 9.85±1.90mmol/l, p=0.0002). The post-statin treatment lipid profile in PCSK9 p.Y374 carriers was worse than subjects with no identified mutation (LDL-C, 6.77±1.82mmol/l versus 4.19±1.26mmol/l, p=0.0001, HDL-C 1.09±0.27mmol/l versus 1.36±0.36mmol/l, p=0.03).

Conclusions: The higher CHD risk in patients carrying PCSK9 p.Y347 or a detected LDLR mutation supports the usefulness of DNA testing in the diagnosis and management of FH patients. Mutations in PCSK9 appear uncommon in UK FH patients.