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Atrial fibrillation (AF) is the most common cardiac arrhythmia affecting 1-2% of the general population. A number of studies have demonstrated that AF, and in particular lone AF, has a substantial genetic component. Monogenic mutations in lone and familial AF, although rare, have been recognized for many years. Presently, mutations in 25 genes have been associated with AF. However, the complexity of monogenic AF is illustrated by the recent finding that both gain- and loss-of-function mutations in the same gene can cause AF. Genome-wide association studies (GWAS) have indicated that common single-nucleotide polymorphisms (SNPs) have a role in the development of AF. Following the first GWAS discovering the association between PITX2 and AF, several new GWAS reports have identified SNPs associated with susceptibility of AF. To date, nine SNPs have been associated with AF. The exact biological pathways involving these SNPs and the development of AF are now starting to be elucidated. Since the first GWAS, the number of papers concerning the genetic basis of AF has increased drastically and the majority of these papers are for the first time included in a review. In this review, we discuss the genetic basis of AF and the role of both common and rare genetic variants in the susceptibility of developing AF. Furthermore, all rare variants reported to be associated with AF were systematically searched for in the Exome Sequencing Project Exome Variant Server. [ABSTRACT FROM AUTHOR]

National and ethnic mutation databases provide comprehensive information about genetic variations reported in a population or an ethnic group. In this paper, we present the Moroccan Genetic Disease Database (MGDD), a catalogue of genetic data related to diseases identified in the Moroccan population. We used the PubMed, Web of Science and Google Scholar databases to identify available articles published until April 2013. The Database is designed and implemented on a three-tier model using Mysql relational database and the PHP programming language. To date, the database contains 425 mutations and 208 polymorphisms found in 301 genes and 259 diseases. Most Mendelian diseases in the Moroccan population follow autosomal recessive mode of inheritance (74.17%) and affect endocrine, nutritional and metabolic physiology. The MGDD database provides reference information for researchers, clinicians and health professionals through a user-friendly Web interface. Its content should be useful to improve researches in human molecular genetics, disease diagnoses and design of association studies. MGDD can be publicly accessed at http://mgdd.pasteur.ma. [ABSTRACT FROM AUTHOR]

The availability of the human genome sequence has allowed identification of disease-causing mutations in many Mendelian disorders, and detection of significant associations of nucleotide polymorphisms to complex diseases and traits. Despite these progresses, finding the causative variations for most of the common diseases remains a complex task. Several studies have shown gene expression analyses provide a quite unbiased way to investigate complex traits and common disorders' pathogenesis. Therefore, whole-transcriptome analysis is increasingly acquiring a key role in the knowledge of mechanisms responsible for complex diseases. Hybridization- and tag-based technologies have elucidated the involvement of multiple genes and pathways in pathological conditions, providing insights into the expression of thousand of coding and noncoding RNAs, such as microRNAs. However, the introduction of Next-Generation Sequencing, particularly of RNA-Seq, has overcome some drawbacks of previously used technologies. Identifying, in a single experiment, potentially novel genes/exons and splice isoforms, RNA editing, fusion transcripts and allele-specific expression are some of its advantages. RNA-Seq has been fruitfully applied to study cancer and host-pathogens interactions, and it is taking first steps for studying neurodegenerative diseases (ND) as well as neuropsychiatric diseases. In addition, it is emerging as a very powerful tool to study quantitative trait loci associated with gene expression in complex diseases. This paper provides an overview on gene expression profiling of complex diseases, with emphasis on RNA-Seq, its advantages over conventional technologies for studying cancer and ND, and for linking nucleotide variations to gene expression changes, also discussing its limitations. [ABSTRACT FROM AUTHOR]

Hirschsprung's disease (HSCR), a congenital complex disorder of intestinal innervation, is often associated with other inherited syndromes. Identifying genes involved in syndromic HSCR cases will not only help understanding the specific underlying diseases, but it will also give an insight into the development of the most frequent isolated HSCR. The association between hydrocephalus and HSCR is not surprising as a large number of patients have been reported to show the same clinical association, most of them showing mutations in the L1CAM gene, encoding a neural adhesion molecule often involved in isolated X-linked hydrocephalus. L1 defects are believed to be necessary but not sufficient for the occurrence of the intestinal phenotype in syndromic cases. In this paper, we have carried out the molecular characterization of a patient affected with Hirschsprung's disease and X-linked hydrocephalus, with a de novo reciprocal balanced translocation t(3;17)(p12;q21). In particular, we have taken advantage of this chromosomal defect to gain access to the predisposing background possibly leading to Hirschsprung's disease. Detailed analysis of the RET and L1CAM genes, and molecular characterization of MYO18A and TIAF1, the genes involved in the balanced translocation, allowed us to identify, besides the L1 mutation c.2265delC, different additional factors related to RET-dependent and -independent pathways which may have contributed to the genesis of enteric phenotype in the present patient.European Journal of Human Genetics (2009) 17, 483–490; doi:10.1038/ejhg.2008.191; published online 29 October 2008 [ABSTRACT FROM AUTHOR]

Familial forms of retinoblastoma, an embryonic neoplasm of retinal origin, are caused by constitutional mutations of the RB1 gene. In this paper, we describe a family with retinoblastoma affecting two brothers with no previous family history of cancer. Complete RB1 mutational screening including point mutation and large rearrangement screening failed to demonstrate any mutation. The whole coding sequence was therefore investigated at the cDNA level, demonstrating a 103 bp intronic insertion between exons 23 and 24, leading to subsequent frameshift and premature termination of translation. This intronic exonisation was caused by a deep intronic mutation in intron 23 generating a cryptic 3′ splice site. This is the first report of a deep intronic mutation in RB1 and is a proof of concept that some undetected RB1 mutations should be investigated at the cDNA level, particularly in hereditary forms of retinoblastoma.European Journal of Human Genetics (2007) 15, 473–477. doi:10.1038/sj.ejhg.5201787; published online 14 February 2007 [ABSTRACT FROM AUTHOR]

Two recent papers suggest distorted sex and transmission ratios associated with BRCA1 mutations. If real, these would provide novel insights into the normal biological function of this gene and have implications for genetic epidemiologic methods used to estimate penetrance. We addressed these observations in two settings: offspring of 283 mutation carriers and 471 mutation negative subjects from BRCA1/2 mutation-positive families with multiple cases of breast and ovarian cancer (NCI families); and relatives of 115 BRCA1/2 mutation carriers from the Washington Ashkenazi Study (WAS). The male:female ratio was below one in both BRCA1 (0.85, 95% CI 0.7-1.1 in NCI families; 0.90, 95% CI 0.6-1.4 in WAS) and BRCA2 families (0.77, 95% CI 0.5-1.3 and 0.80, 95% CI 0.5-1.2, in the NCI and WAS study groups, respectively). None of the sex ratios deviated significantly from one, and there was no significant difference between BRCA1 and BRCA2 families. The reduced sex ratio was due largely to the offspring of males, a distortion that is probably an artifact of ascertainment biases. Among adult daughters without breast or ovarian cancer born to mutation carriers, as expected, fewer than 50% were mutation carriers (39% in BRCA1 families and 44% in BRCA2 families). It is difficult, due to ascertainment biases, to draw firm conclusions regarding sex ratios in studies of a sex-limited phenotype. Nonetheless, these observations do not support the idea that BRCA1 mutation carriers have a lower ratio of male offspring than BRCA2 mutation carriers.European Journal of Human Genetics (2004) 12, 663-667. doi:10.1038/sj.ejhg.5201210 Published online 28 April 2004 [ABSTRACT FROM AUTHOR]

Many genetic disorders demonstrate mutations that can be traced to a founder, sometimes a person who can be identified. These founder mutations have generated considerable interest, because they facilitate studies of prevalence and penetrance and can be used to quantify the degree of homogeneity within a population. This paper reports on founder mutations among the Dutch and relates their occurrence to the history and demography of the Netherlands. International migration, regional and religious endogamy, and rapid population growth played key roles in shaping the Dutch population. In the first millenniums BC and AD, the Netherlands were invaded by Celts, Romans, Huns, and Germans. In more recent times, large numbers of Huguenots and Germans migrated into the Netherlands. Population growth within the Netherlands was slow until the 19th century, when a period of rapid population growth started. Today, the Dutch population numbers 16 million inhabitants. Several different classes of founder mutations have been identified among the Dutch. Some mutations occur among people who represent genetic isolates within this country. These include mutations for benign familial cholestasis, diabetes mellitus, type I, infantile neuronal ceroid lipofuscinosis, L-DOPA responsive dystonia, and triphalangeal thumb. Although not related to a specific isolate, other founder mutations were identified only within the Netherlands, including those predisposing for hereditary breast-ovarian cancer, familial hypercholesterolemia, frontotemporal dementia, hereditary paragangliomas, juvenile neuronal ceroid lipofuscinosis, malignant melanoma, protein C deficiency, and San Filippo disease. Many of these show a regional distribution, suggesting dissemination from a founder. Some mutations that occur among the Dutch are shared with other European populations and others have been transmitted by Dutch émigrés to their descendents in North America and South Africa. The occurrence of short chromosomal regions that have remained identical by descent has resulted in relatively limited genetic heterogeneity for many genetic conditions among the Dutch. These observations demonstrate the opportunity for gene discovery for other diseases and traits in the Netherlands.European Journal of Human Genetics (2004) 12, 591-600. doi:10.1038/sj.ejhg.5201151 Published online 10 March 2004 [ABSTRACT FROM AUTHOR]

Autosomal dominant polycystic kidney disease (ADPKD) exhibits a genetically heterogeneous transmission involving at least three different genes. PKD1 gene linked mutations are responsible for the disease in about 85% of ADPKD cases. The search for mutations is a very important step in understanding the molecular mechanisms underlying ADPKD. We undertook this study using denaturing gradient gel electrophoresis (DGGE), after a stage of long range PCR, to scan for mutations in the duplicated region of the PKD1 gene in French ADPKD families. This allowed us to identify eight novel mutations and several polymorphisms: among the mutations, three are nonsense mutations, two are deletions in the coding sequence leading to frameshift mutations, one is a splice mutation and two are highly probable missense mutations. In this paper, we also provide a review of the mutations reported so far which are widespread throughout the gene. Although no clear hot spot for mutation is apparent, we will focus on some clustering observed. [ABSTRACT FROM AUTHOR]

Hirschsprung disease (HSCR, aganglionic megacolon) is a complex and heterogeneous disease with an incidence of 1 in 5000 live births. Despite the multifactorial determination of HSCR in the vast majority of cases, there is a monogenic subgroup for which private rare RET coding sequence mutations with high penetrance are found (45% of HSCR familial cases). An asymmetrical parental origin is observed for RET coding sequence mutations with a higher maternal inheritance. A parent-of-origin effect is usually assumed. Here we show that a differential reproductive rate for males and females also leads to an asymmetrical parental origin, which was never considered as a possible explanation till now. In the case of HSCR, we show a positive association between penetrance of the mutation and parental transmission asymmetry: no parental transmission asymmetry is observed in sporadic RET CDS mutation carrier cases for which penetrance of the mutation is low, whereas a parental transmission asymmetry is observed in affected sib-pairs for which penetrance of the mutation is higher. This allows us to conclude that the explanation for this parental asymmetry is that more severe mutations have resulted in a differential reproductive rate between male and female carriers. [ABSTRACT FROM AUTHOR]

Mutations in the gene Indian Hedgehog (IHH) that cause Brachydactyly A-1 (BDA1) have been restricted to a specific region of the N-terminal active fragment of Indian Hedgehog involving codons 95, 100, 131, and 154. We describe two novel mutations in codons 128 and 130, not previously implicated in BDA1. Furthermore, we identified an independent mutation at codon 131 and we also describe a New Zealand family, which carries the ‘Farabee’ founder mutation and haplotype. All of the BDA1 mutations occur in a restricted area of the N-terminal active fragment of the IHH and are in contrast to those mutations causing an autosomal recessive acrocapitofemoral dysplasia, whose mutations are located at the distal N- and C-terminal regions of IHH-N and are physically separated from the BDA1-causing mutations. The identification of multiple independent mutations in codons 95, 100, and now in 131, implicate a discrete function for this region of the protein. Finally, we present a clinical review of all reported and confirmed cases of BDA1, highlighting features of the disorder, which add to the spectrum of the IHH mutations.European Journal of Human Genetics (2009) 17, 1112–1120; doi:10.1038/ejhg.2009.18; published online 11 March 2009 [ABSTRACT FROM AUTHOR]

The article focuses on cancer genetics. The largest mutation analysis series yet reported for tuberous sclerosis (TSC), on page 731 of this issue, provides insights into the number of genes that underlie this syndrome and sheds light onto several other important questions. In this new study, the Halley-van den Ouweland lab report on mutation analysis of 490 samples from definite or suspect TSC cases summarizing several years of work from their clinical diagnostic laboratory. A combination of detection techniques were used, with a mutation detection rate of 74 percent overall, and 85 percent in patients with definite TSC. This level of mutation detection is strikingly similar to two previous comprehensive studies on somewhat smaller numbers of patients.

A response from the author of the article regarding the inclusion of mutations with proper frequency below 1% in the formula wit a gene frequency rising to 1.95% from 1.91 is presented.

A correction to the article "Expansion of Mutation Spectrum, Determination of Mutation Cluster Regions and Predictive Structural Classification of SPAST Mutations in Hereditary Spastic Paraplegia" that was published in the 2008 issue is presented.

European Journal of Human Genetics (2002) 10, 887–888. doi:10.1038/sj.ejhg.5200904CORRECTION TO:: European Journal of Human Genetics 2002 10, 631–637. doi:10.1038/sj.ejhg.5200853 [ABSTRACT FROM AUTHOR]

A correction to the name of Hana Allen Lango, one of the authors of the article "Biallelic RFX6 mutations can cause childhood as well as neonatal onset diabetes mellitus," published online in the August 12, 2015 issue, which should have read as Hana Lango Allen, is presented.

CACNA1A loss-of-function mutations classically present as episodic ataxia type 2 (EA2), with brief episodes of ataxia and nystagmus, or with progressive spinocerebellar ataxia (SCA6). A minority of patients carrying CACNA1A mutations develops epilepsy. Non-motor symptoms associated with these mutations are often overlooked. In this study, we report 16 affected individuals from four unrelated families presenting with a spectrum of cognitive impairment including intellectual deficiency, executive dysfunction, ADHD and/or autism, as well as childhood-onset epileptic encephalopathy with refractory absence epilepsy, febrile seizures, downbeat nystagmus and episodic ataxia. Sequencing revealed one CACNA1A gene deletion, two deleterious CACNA1A point mutations including one known stop-gain and one new frameshift variant and a new splice-site variant. This report illustrates the phenotypic heterogeneity of CACNA1A loss-of-function mutations and stresses the cognitive and epileptic manifestations caused by the loss of CaV2.1 channels function, presumably affecting cerebellar, cortical and limbic networks. [ABSTRACT FROM AUTHOR]

Genetic diagnosis of steroid-resistant nephrotic syndrome (SRNS) using Sanger sequencing is complicated by the high genetic heterogeneity and phenotypic variability of this disease. We aimed to improve the genetic diagnosis of SRNS by simultaneously sequencing 26 glomerular genes using massive parallel sequencing and to study whether mutations in multiple genes increase disease severity. High-throughput mutation analysis was performed in 50 SRNS and/or focal segmental glomerulosclerosis (FSGS) patients, a validation cohort of 25 patients with known pathogenic mutations, and a discovery cohort of 25 uncharacterized patients with probable genetic etiology. In the validation cohort, we identified the 42 previously known pathogenic mutations across NPHS1, NPHS2, WT1, TRPC6, and INF2 genes. In the discovery cohort, disease-causing mutations in SRNS/FSGS genes were found in nine patients. We detected three patients with mutations in an SRNS/FSGS gene and COL4A3. Two of them were familial cases and presented a more severe phenotype than family members with mutation in only one gene. In conclusion, our results show that massive parallel sequencing is feasible and robust for genetic diagnosis of SRNS/FSGS. Our results indicate that patients carrying mutations in an SRNS/FSGS gene and also in COL4A3 gene have increased disease severity. [ABSTRACT FROM AUTHOR]

Osteogenesis imperfecta (OI) is a rare hereditary bone fragility disorder, caused by collagen I mutations in 90% of cases. There are no comprehensive genotype-phenotype studies on >100 families outside North America, and no population-based studies determining the genetic epidemiology of OI. Here, detailed clinical phenotypes were recorded, and the COL1A1 and COL1A2 genes were analyzed in 164 Swedish OI families (223 individuals). Averages for bone mineral density (BMD), height and yearly fracture rate were calculated and related to OI and mutation type. N-terminal helical mutations in both the α1- and α2-chains were associated with the absence of dentinogenesis imperfecta (P<0.0001 vs 0.0049), while only those in the α1-chain were associated with blue sclera (P=0.0110). Comparing glycine with serine substitutions, α1-alterations were associated with more severe phenotype (P=0.0031). Individuals with type I OI caused by qualitative vs quantitative mutations were shorter (P<0.0001), but did not differ considering fractures or BMD. The children in this cohort were estimated to represent >95% of the complete Swedish pediatric OI population. The prevalence of OI types I, III, and IV was 5.16, 0.89, and 1.35/100 000, respectively (7.40/100 000 overall), corresponding to what has been estimated but not unequivocally proven in any population. Collagen I mutation analysis was performed in the family of 97% of known cases, with causative mutations found in 87%. Qualitative mutations caused 32% of OI type I. The data reported here may be helpful to predict phenotype, and describes for the first time the genetic epidemiology in >95% of an entire OI population. [ABSTRACT FROM AUTHOR]

In this study, we evaluated the diagnostic value of the Dutch Clinical Genetic Center (CGC) referral guidelines for BRCA1/2 mutation testing in 903 early breast cancer patients, unselected for family history, diagnosed in a cancer hospital before the age of 50 years in 1974-2002; most prevalent Dutch pathogenic BRCA1/2 mutations had been analyzed on coded DNA in a research setting. Forty-nine (5.4%) of the patients were proven to be BRCA1/2 mutation carriers. We found that 78% and 69% of BRCA1 and BRCA2 mutation carriers identified met the criteria for referral to the CGC based on age, family history and synchronous multiple tumors; reflected by a combined sensitivity of 75.5% and specificity of 63.2%. More than half of the BRCA1 mutation carriers, that is, 58% had a triple-negative tumor. The highest AUC was obtained by shifting the age at diagnosis threshold criterion from 40 to 35 years and by adding a 'triple-negative breast cancer' criterion with an age threshold of 45 years; the specificity increased to 71.2%, whereas the sensitivity remained the same; that is, a referral of fewer patients will lead to the identification of at least the same number of BRCA1/2 mutation carriers. Two-thirds of the BRCA1/2 mutation carriers identified in this research setting had been referred for counseling and testing. Our results indicate that, awaiting a possibly more extended mutation screening of all breast cancer patients, the triple-negative status of a breast cancer should be added to the CGC referral criteria. [ABSTRACT FROM AUTHOR]

So far very few patients with sequence variants in the closely related tectonic genes TCTN1-3 have been described. By multi-gene panel next-generation sequencing (NGS) in patients with Joubert syndrome, we identified two more patients and summarize what is currently known about the phenotypes associated with sequence variants in these genes. In a boy aged 12 years with intellectual disability and the classical molar tooth sign on MRI, a homozygous splice-site sequence variant in TCTN3 leading to in-frame skipping of exon 7 was detected. A previously described non-truncating sequence variant in TCTN3 was also associated with Joubert syndrome, whereas four truncating sequence variants were detected in patients with Meckel-Gruber or Mohr-Majewski syndrome. The second patient, a boy aged 7 years with severe psychomotor retardation, was found to carry a homozygous canonic splice-site sequence variant in TCTN2. So far, only three sequence variants associated with Joubert syndrome and two with Meckel-Gruber syndrome have been described in this gene. Reviewing the clinical data on patients with sequence variants in the tectonic genes TCTN1-3 reveals that all of them have a neurological phenotype with vermis hypoplasia or occipital encephalocele associated with severe intellectual disability in the surviving patients. In contrast, other features frequently seen in patients with ciliopathies such as nephronophthisis, liver fibrosis, retinal dystrophy or coloboma have not been reported. Our patients emphasize the usefulness and efficacy of a comprehensive NGS panel approach. A concise genetic diagnosis may help to prevent unnecessary investigations and improve the clinical management of these patients. [ABSTRACT FROM AUTHOR]

In recent years, antisense therapy has emerged as an increasingly important therapeutic approach to tackle several genetic disorders, including inborn errors of metabolism. Intronic mutations activating cryptic splice sites are particularly amenable to antisense therapy, as the canonical splice sites remain intact, thus retaining the potential for restoring constitutive splicing. Mutational analysis of Portuguese galactosemic patients revealed the intronic variation c.820+13A>G as the second most prevalent mutation, strongly suggesting its pathogenicity. The aim of this study was to functionally characterize this intronic variation, to elucidate its pathogenic molecular mechanism(s) and, ultimately, to correct it by antisense therapy. Minigene splicing assays in two distinct cell lines and patients' transcript analyses showed that the mutation activates a cryptic donor splice site, inducing an aberrant splicing of the GALT pre-mRNA, which in turn leads to a frameshift with inclusion of a premature stop codon (p.D274Gfs*17). Functional-structural studies of the recombinant wild-type and truncated GALT showed that the latter is devoid of enzymatic activity and prone to aggregation. Finally, two locked nucleic acid oligonucleotides, designed to specifically recognize the mutation, successfully restored the constitutive splicing, thus establishing a proof of concept for the application of antisense therapy as an alternative strategy for the clearly insufficient dietary treatment in classic galactosemia. [ABSTRACT FROM AUTHOR]

Baraitser-Winter, Fryns-Aftimos and cerebrofrontofacial syndrome types 1 and 3 have recently been associated with heterozygous gain-of-function mutations in one of the two ubiquitous cytoplasmic actin-encoding genes ACTB and ACTG1 that encode β- and γ-actins. We present detailed phenotypic descriptions and neuroimaging on 36 patients analyzed by our group and six cases from the literature with a molecularly proven actinopathy (9 ACTG1 and 33 ACTB). The major clinical anomalies are striking dysmorphic facial features with hypertelorism, broad nose with large tip and prominent root, congenital non-myopathic ptosis, ridged metopic suture and arched eyebrows. Iris or retinal coloboma is present in many cases, as is sensorineural deafness. Cleft lip and palate, hallux duplex, congenital heart defects and renal tract anomalies are seen in some cases. Microcephaly may develop with time. Nearly all patients with ACTG1 mutations, and around 60% of those with ACTB mutations have some degree of pachygyria with anteroposterior severity gradient, rarely lissencephaly or neuronal heterotopia. Reduction of shoulder girdle muscle bulk and progressive joint stiffness is common. Early muscular involvement, occasionally with congenital arthrogryposis, may be present. Progressive, severe dystonia was seen in one family. Intellectual disability and epilepsy are variable in severity and largely correlate with CNS anomalies. One patient developed acute lymphocytic leukemia, and another a cutaneous lymphoma, indicating that actinopathies may be cancer-predisposing disorders. Considering the multifaceted role of actins in cell physiology, we hypothesize that some clinical manifestations may be partially mutation specific. Baraitser-Winter cerebrofrontofacial syndrome is our suggested designation for this clinical entity. [ABSTRACT FROM AUTHOR]

Mutations in the low-density lipoprotein receptor (LDLR) gene cause familial hypercholesterolemia (FH), a disorder characterized by coronary heart disease (CHD) at young age. We aimed to apply an extreme sampling method to enhance the statistical power to identify novel genetic risk variants for CHD in individuals with FH. We selected cases and controls with an extreme contrast in CHD risk from 17 000 FH patients from the Netherlands, whose functional LDLR mutation was unequivocally established. The genome-wide association (GWA) study was performed on 249 very young FH cases with CHD and 217 old FH controls without CHD (above 65 years for males and 70 years of age for females) using the Illumina HumanHap550K chip. In the next stage, two independent samples (one from the Netherlands and one from Italy, Norway, Spain, and the United Kingdom) of FH patients were used as replication samples. In the initial GWA analysis, we identified 29 independent single nucleotide polymorphisms (SNPs) with suggestive associations with premature CHD (P<1 × 10−4). We examined the association of these SNPs with CHD risk in the replication samples. After Bonferroni correction, none of the SNPs either replicated or reached genome-wide significance after combining the discovery and replication samples. Therefore, we conclude that the genetics of CHD risk in FH is complex and even applying an 'extreme genetics' approach we did not identify new genetic risk variants. Most likely, this method is not as effective in leveraging effect size as anticipated, and may, therefore, not lead to significant gains in statistical power. [ABSTRACT FROM AUTHOR]

Mutations in SUCLA2, encoding the ß-subunit of succinyl-CoA synthetase of Krebs cycle, are one cause of mitochondrial DNA depletion syndrome. Patients have been reported to have severe progressive childhood-onset encephalomyopathy, and methylmalonic aciduria, often leading to death in childhood. We studied two families, with children manifesting with slowly progressive mitochondrial encephalomyopathy, hearing impairment and transient methylmalonic aciduria, without mtDNA depletion. The other family also showed dominant inheritance of bilateral retinoblastoma, which coexisted with mitochondrial encephalomyopathy in one patient. We found a variant in SUCLA2 leading to Asp333Gly change, homozygous in one patient and compound heterozygous in one. The latter patient also carried a deletion of 13q14 of the other allele, discovered with molecular karyotyping. The deletion spanned both SUCLA2 and RB1 gene regions, leading to manifestation of both mitochondrial disease and retinoblastoma. We made a homology model for human succinyl-CoA synthetase and used it for structure-function analysis of all reported pathogenic mutations in SUCLA2. On the basis of our model, all previously described mutations were predicted to result in decreased amounts of incorrectly assembled protein or disruption of ADP phosphorylation, explaining the severe early lethal manifestations. However, the Asp333Gly change was predicted to reduce the activity of the otherwise functional enzyme. On the basis of our findings, SUCLA2 mutations should be analyzed in patients with slowly progressive encephalomyopathy, even in the absence of methylmalonic aciduria or mitochondrial DNA depletion. In addition, an encephalomyopathy in a patient with retinoblastoma suggests mutations affecting SUCLA2. [ABSTRACT FROM AUTHOR]

Shprintzen-Goldberg syndrome (SGS) is a rare, systemic connective tissue disorder characterized by craniofacial, skeletal, and cardiovascular manifestations that show a significant overlap with the features observed in the Marfan (MFS) and Loeys-Dietz syndrome (LDS). A distinguishing observation in SGS patients is the presence of intellectual disability, although not all patients in this series present this finding. Recently, SGS was shown to be due to mutations in the SKI gene, encoding the oncoprotein SKI, a repressor of TGFβ activity. Here, we report eight recurrent and three novel SKI mutations in eleven SGS patients. All were heterozygous missense mutations located in the R-SMAD binding domain, except for one novel in-frame deletion affecting the DHD domain. Adding our new findings to the existing data clearly reveals a mutational hotspot, with 73% (24 out of 33) of the hitherto described unrelated patients having mutations in a stretch of five SKI residues (from p.(Ser31) to p.(Pro35)). This implicates that the initial molecular testing could be focused on mutation analysis of the first half of exon 1 of SKI. As the majority of the known mutations are located in the R-SMAD binding domain of SKI, our study further emphasizes the importance of TGFβ signaling in the pathogenesis of SGS. [ABSTRACT FROM AUTHOR]

Defects in complex II of the mitochondrial respiratory chain are a rare cause of mitochondrial disorders. Underlying autosomal-recessive genetic defects are found in most of the 'SDHx' genes encoding complex II (SDHA, SDHB, SDHC, and SDHD) and its assembly factors. Interestingly, SDHx genes also function as tumor suppressor genes in hereditary paragangliomas, pheochromocytomas, and gastrointestinal stromal tumors. In these cases, the affected patients are carrier of a heterozygeous SDHx germline mutation. Until now, mutations in SDHx associated with mitochondrial disease have not been reported in association with hereditary tumors and vice versa. Here, we characterize four patients with isolated complex II deficiency caused by mutations in SDHA presenting with multisystem mitochondrial disease including Leigh syndrome (LS) and/or leukodystrophy. Molecular genetic analysis revealed three novel mutations in SDHA. Two mutations (c.64-2A>G and c.1065-3C>A) affect mRNA splicing and result in loss of protein expression. These are the first mutations described affecting SDHA splicing. For the third new mutation, c.565T>G, we show that it severely affects enzyme activity. Its pathogenicity was confirmed by lentiviral complementation experiments on the fibroblasts of patients carrying this mutation. It is of special interest that one of our LS patients harbored the c.91C>T (p.Arg31*) mutation that was previously only reported in association with paragangliomas and pheochromocytomas, tightening the gap between these two rare disorders. As tumor screening is recommended for SDHx mutation carriers, this should also be considered for patients with mitochondrial disorders and their family members. [ABSTRACT FROM AUTHOR]

We present a study of five children from three unrelated Irish Traveller families presenting with primary ciliary dyskinesia (PCD). As previously characterized disorders in the Irish Traveller population are caused by common homozygous mutations, we hypothesised that all three PCD families shared the same recessive mutation. However, exome sequencing showed that there was no pathogenic homozygous mutation common to all families. This finding was supported by histology, which showed that each family has a different type of ciliary defect; transposition defect (family A), nude epithelium (family B) and absence of inner and outer dynein arms (family C). Therefore, each family was analysed independently using homozygosity mapping and exome sequencing. The affected siblings in family A share a novel 1 bp duplication in RSPH4A (NM_001161664.1:c.166dup; p.Arg56Profs*11), a radial-spoke head protein involved in ciliary movement. In family B, we identified three candidate genes (CCNO, KCNN3 and CDKN1C), with a 5-bp duplication in CCNO (NM_021147.3:c.258_262dup; p.Gln88Argfs*8) being the most likely cause of ciliary aplasia. This is the first study to implicate CCNO, a DNA repair gene reported to be involved in multiciliogenesis, in PCD. In family C, we identified a ∼3.5-kb deletion in DYX1C1, a neuronal migration gene previously associated with PCD. This is the first report of a disorder in the relatively small Irish Traveller population to be caused by >1 disease gene. Our study identified at least three different PCD genes in the Irish Traveller population, highlighting that one cannot always assume genetic homogeneity, even in small consanguineous populations. [ABSTRACT FROM AUTHOR]

The recently proposed adaptor protein 4 (AP-4) deficiency syndrome comprises a group of congenital neurological disorders characterized by severe intellectual disability (ID), delayed or absent speech, hereditary spastic paraplegia, and growth retardation. AP-4 is a heterotetrameric protein complex with important functions in vesicle trafficking. Mutations in genes affecting different subunits of AP-4, including AP4B1, AP4E1, AP4S1, and AP4M1, have been reported in patients with the AP-4 deficiency phenotype. We describe two siblings from a non-consanguineous couple who presented with severe ID, absent speech, microcephaly, growth retardation, and progressive spastic tetraplegia. Whole-exome sequencing in the two patients identified the novel homozygous 2-bp deletion c.1160_1161delCA (p.(Thr387Argfs*30)) in AP4B1. Sanger sequencing confirmed the mutation in the siblings and revealed it in the heterozygous state in both parents. The AP4B1-associated phenotype has previously been assigned to spastic paraplegia-47. Identification of a novel AP4B1 alteration in two patients with clinical manifestations highly similar to other individuals with mutations affecting one of the four AP-4 subunits further supports the observation that loss of AP-4 assembly or functionality underlies the common clinical features in these patients and underscores the existence of the clinically recognizable AP-4 deficiency syndrome. [ABSTRACT FROM AUTHOR]

The heritable cardiovascular disorder long QT syndrome (LQTS), characterized by prolongation of the QT interval on electrocardiogram, carries a high risk of sudden cardiac death. We sought to add new data to the existing knowledge of genetic mutations contributing to LQTS to both expand our understanding of its genetic basis and assess the value of genetic testing in clinical decision-making. Direct sequencing of the five major contributing genes, KCNQ1, KCNH2, SCN5A, KCNE1, and KCNE2, was performed in a cohort of 115 non-related LQTS patients. Pathogenicity of the variants was analyzed using family segregation, allele frequency from public databases, conservation analysis, and Condel and Provean in silico predictors. Phenotype-genotype correlations were analyzed statistically. Sequencing identified 36 previously described and 18 novel mutations. In 51.3% of the index cases, mutations were found, mostly in KCNQ1, KCNH2, and SCN5A; 5.2% of cases had multiple mutations. Pathogenicity analysis revealed 39 mutations as likely pathogenic, 12 as VUS, and 3 as non-pathogenic. Clinical analysis revealed that 75.6% of patients with QTc≥500 ms were genetically confirmed. Our results support the use of genetic testing of KCNQ1, KCNH2, and SCN5A as part of the diagnosis of LQTS and to help identify relatives at risk of SCD. Further, the genetic tools appear more valuable as disease severity increases. However, the identification of genetic variations in the clinical investigation of single patients using bioinformatic tools can produce erroneous conclusions regarding pathogenicity. Therefore segregation studies are key to determining causality. [ABSTRACT FROM AUTHOR]

Alexander disease (AxD) is an astrogliopathy that primarily affects the white matter of the central nervous system (CNS). AxD is caused by mutations in a gene encoding GFAP (glial fibrillary acidic protein). The GFAP mutations in AxD have been reported to act in a gain-of-function manner partly because the identified mutations generate practically full-length GFAP. We found a novel nonsense mutation (c.1000 G>T, p.(Glu312Ter); also termed p.(E312*)) within a rod domain of GFAP in a 67-year-old Korean man with a history of memory impairment and leukoencephalopathy. This mutation, GFAP p.(E312*), removes part of the 2B rod domain and the whole tail domain from the GFAP. We characterized GFAP p.(E312*) using western blotting, in vitro assembly and sedimentation assay, and transient transfection of human adrenal cortex carcinoma SW13 (Vim+) cells with plasmids encoding GFAP p.(E312*). The GFAP p.(E312*) protein, either alone or in combination with wild-type GFAP, elicited self-aggregation. In addition, the assembled GFAP p.(E312*) aggregated into paracrystal-like structures, and GFAP p.(E312*) elicited more GFAP aggregation than wild-type GFAP in the human adrenal cortex carcinoma SW13 (Vim+) cells. Our findings are the first report, to the best of our knowledge, on this novel nonsense mutation of GFAP that is associated with AxD and paracrystal formation. [ABSTRACT FROM AUTHOR]

Pathogenic mitochondrial DNA (mtDNA) mutations leading to mitochondrial dysfunction can cause a variety of chronic diseases in central nervous system (CNS). However, the role of mtDNA mutations in sporadic Creutzfeldt-Jakob disease (sCJD) has still been unknown. In this study, we comparatively analyzed complete mtDNA sequences of 31 Chinese sCJD patients and 32 controls. Using MITOMASTER and PhyloTree, we characterized 520 variants in sCJD patients and 507 variants in control by haplogroup and allele frequencies. We classified the mtDNAs into 40 sub-haplogroups of 5 haplogroups, most of them being Asian-specific haplogroups. Haplogroup U, an European-specific haplogroups mtDNA, was found only in sCJD. The analysis to control region (CR) revealed a 31% increase in the frequency of mtDNA CR mutations in sCJD versus controls. In functional elements of the mtDNA CR, six CR mutations were in conserved sequence blocks I (CSBI) in sCJD, while only one in control (P<0.05). More mutants in transfer ribonucleic acid-Leu (tRNA-Leu) were detected in sCJD. The frequencies of two synonymous amino-acid changes, m.11467A>G, p.(=) in NADH dehydrogenase subunit 4 (ND4) and m.12372G>A, p.(=) in NADH dehydrogenase subunit 5 (ND5), in sCJD patients were higher than that of controls. Our study, for the first time, screened the variations of mtDNA of Chinese sCJD patients and identified some potential disease-related mutations for further investigations. [ABSTRACT FROM AUTHOR]

Split-hand/foot malformation (SHFM) is a congenital limb deformity due to the absence or dysplasia of central rays of the autopod. Six SHFM loci have already been identified. Here we describe a Chinese family with autosomal-dominant SHFM1 that has previously been mapped to 7q21.2-21.3. The two affected family members, mother and son, showed deep median clefts between toes, ectrodactyly and syndactyly; the mother also showed triphalangeal thumbs. Exome sequencing and variant screening of candidate genes in the six loci known to be responsible for SHFM revealed a novel heterozygous mutation, c.558G>T (p.(Gln186His)), in distal-less homeobox 5 (DLX5). As DLX5 encodes a transcription factor capable of transactivating MYC, we also tested whether the mutation could affect DLX5 transcription acitivity. Results from luciferase reporter assay revealed that a mutation in DLX5 compromised its transcriptional activity. This is the first report of a mutation in DLX5 leading to autosomal-dominant SHFM1. [ABSTRACT FROM AUTHOR]

C12orf65 participates in the process of mitochondrial translation and has been shown to be associated with a spectrum of phenotypes, including early onset optic atrophy, progressive encephalomyopathy, peripheral neuropathy, and spastic paraparesis.We used whole-genome homozygosity mapping as well as exome sequencing and targeted gene sequencing to identify novel C12orf65 disease-causing mutations in seven affected individuals originating from two consanguineous families. In four family members affected with childhood-onset optic atrophy accompanied by slowly progressive peripheral neuropathy and spastic paraparesis, we identified a homozygous frame shift mutation c.413_417 delAACAA, which predicts a truncated protein lacking the C-terminal portion. In the second family, we studied three affected individuals who presented with early onset optic atrophy, peripheral neuropathy, and spastic gait in addition to moderate intellectual disability. Muscle biopsy in two of the patients revealed decreased activities of the mitochondrial respiratory chain complexes I and IV. In these patients, we identified a homozygous splice mutation, g.21043 T>A (c.282+2 T>A) which leads to skipping of exon 2. Our study broadens the phenotypic spectrum of C12orf65 defects and highlights the triad of optic atrophy, axonal neuropathy and spastic paraparesis as its key clinical features. In addition, a clear genotype-phenotype correlation is anticipated in which deleterious mutations which disrupt the GGQ-containing domain in the first coding exon are expected to result in a more severe phenotype, whereas down-stream C-terminal mutations may result in a more favorable phenotype, typically lacking cognitive impairment. [ABSTRACT FROM AUTHOR]

Restrictive dermopathy (RD) is a rare and extremely severe congenital genodermatosis, characterized by a tight rigid skin with erosions at flexure sites, multiple joint contractures, low bone density and pulmonary insufficiency generally leading to death in the perinatal period. RD is caused in most patients by compound heterozygous or homozygous ZMPSTE24 null mutations. This gene encodes a metalloprotease specifically involved in lamin A post-translational processing. Here, we report a total of 16 families for whom diagnosis and molecular defects were clearly established. Among them, we report seven new ZMPSTE24 mutations, identified in classical RD or Mandibulo-acral dysplasia (MAD) affected patients. We also report nine families with one or two affected children carrying the common, homozygous thymine insertion in exon 9 and demonstrate the lack of a founder effect. In addition, we describe several new ZMPSTE24 variants identified in unaffected controls or in patients affected with non-classical progeroid syndromes. In addition, this mutation update includes a comprehensive search of the literature on previously described ZMPSTE24 mutations and associated phenotypes. Our comprehensive analysis of the molecular pathology supported the general rule: complete loss-of-function of ZMPSTE24 leads to RD, whereas other less severe phenotypes are associated with at least one haploinsufficient allele. [ABSTRACT FROM AUTHOR]

Fanconi anaemia (FA) is characterized by progressive bone marrow failure, congenital anomalies, and predisposition to malignancy. In a minority of cases, FA results from biallelic FANCD1/BRCA2 mutations that are associated with early-onset leukaemia and solid tumours. Here, we describe the clinical and molecular features of a remarkable family presenting with multiple primary colorectal cancers (CRCs) without detectable mutations in genes involved in the Mendelian predisposition to CRCs. We unexpectedly identified, despite the absence of clinical cardinal features of FA, a biallelic mutation of the FANCD1/BRCA2 corresponding to a frameshift alteration (c.1845_1846delCT, p.Asn615Lysfs*6) and a missense mutation (c.7802A>G, p.Tyr2601Cys). The diagnosis of FA was confirmed by the chromosomal analysis of lymphocytes. Reverse transcriptase (RT)-PCR analysis revealed that the c.7802A>G BRCA2 variation was in fact a splicing mutation that creates an aberrant splicing donor site and results partly into an aberrant transcript encoding a truncated protein (p.Tyr2601Trpfs*46). The atypical FA phenotype observed within this family was probably explained by the residual amount of BRCA2 with the point mutation c.7802A>G in the patients harbouring the biallelic FANCD1/BRCA2 mutations. Although this report is based in a single family, it suggests that CRCs may be part of the tumour spectrum associated with FANCD1/BRCA2 biallelic mutations and that the presence of such mutations should be considered in families with CRCs, even in the absence of cardinal features of FA. [ABSTRACT FROM AUTHOR]

Borrone Dermato-Cardio-Skeletal (BDCS) syndrome is a severe progressive autosomal recessive disorder characterized by coarse facies, thick skin, acne conglobata, dysmorphic facies, vertebral abnormalities and mitral valve prolapse. We identified a consanguineous kindred with a child clinically diagnosed with BDCS. Linkage analysis of this family (BDCS1) identified five regions homozygous by descent with a maximum LOD score of 1.75. Linkage analysis of the family that originally defined BDCS (BDCS3) identified an overlapping linkage peak at chromosome 5q35.1. Sequence analysis identified two different homozygous mutations in BDCS1 and BDCS3, affecting the gene encoding the protein SH3 and PX domains 2B (SH3PXD2B), which localizes to 5q35.1. Western blot analysis of patient fibroblasts derived from affected individuals in both families demonstrated complete loss of SH3PXD2B. Homozygosity mapping and sequence analysis in a second published BDCS family (BDCS2) excluded SH3PXD2B. SH3PXD2B is required for the formation of functional podosomes, and loss-of-function mutations in SH3PXD2B have recently been shown to underlie 7 of 13 families with Frank-Ter Haar syndrome (FTHS). FTHS and BDCS share some overlapping clinical features; therefore, our results demonstrate that a proportion of BDCS and FTHS cases are allelic. Mutations in other gene(s) functioning in podosome formation and regulation are likely to underlie the SH3PXD2B-mutation-negative BDSC/FTHS patients. [ABSTRACT FROM AUTHOR]

Acromesomelic chondrodysplasias (ACDs) are characterized by disproportionate shortening of the appendicular skeleton, predominantly affecting the middle (forearms and forelegs) and distal segments (hands and feet). Here, we present two consanguineous families with missense (c.157T>C, p.(C53R)) or nonsense (c.657G>A, p.(W219*)) mutations in BMPR1B. Homozygous affected individuals show clinical and radiographic findings consistent with ACD-type Grebe. Functional analysis of the missense mutation C53R revealed that the mutated receptor was partially located at the cell membrane. In contrast to the wild-type receptor, C53R mutation hindered the activation of the receptor by its ligand GDF5, as shown by reporter gene assay. Further, overexpression of the C53R mutation in an in vitro chondrogenesis assay showed no effect on cell differentiation, indicating a loss of function. The nonsense mutation (c.657G>A, p.(W219*)) introduces a premature stop codon, which is predicted to be subject to nonsense-mediated mRNA decay, causing reduced protein translation of the mutant allele. A loss-of-function effect of both mutations causing recessive ACD-type Grebe is further supported by the mild brachydactyly or even non-penetrance of these mutations observed in the heterozygous parents. In contrast, dominant-negative BMPR1B mutations described previously are associated with autosomal-dominant brachydactyly-type A2. [ABSTRACT FROM AUTHOR]

The ABCB4 gene encodes for MDR3, a protein that translocates phosphatidylcholine from the inner to the outer leaflet of the hepatocanalicular membrane; its deficiency favors the formation of 'toxic bile'. Several forms of hepatobiliary diseases have been associated with ABCB4 mutations, but the detrimental effects of most mutations on the encoded protein needs to be clarified. Among subjects with cholangiopathies who were screened for mutations in ABCB4 by direct sequencing, we identified the new mutation p.(L481R) in three brothers. According to our model of tertiary structure, this mutation affects the Q-loop, whereas the p.(Y403H) mutation, that we already described in two other families, involves the A-loop. This study was aimed at analyzing the functional relevance of these two ABCB4 mutations: MDR3 expression and lipid content in the culture supernatant were evaluated in cell lines stably transfected with the ABCB4 wild-type clone and corresponding mutants. No differences of expression were observed between wild-type and mutant gene products. Instead, both mutations caused a reduction of phosphatidylcholine secretion compared with the wild-type transfected cell lines. On the contrary, cholesterol (Chol) release, after 1 and 3 mM sodium taurocholate stimulation, was higher in the mutant-transfected cell lines than that in the wild-type and was particularly enhanced in cells transfected with the p.Y403H-construct.In summary, our data show that both mutations do not seem to affect protein expression, but are able to reduce the efflux of phosphatidylcholine associated with increase of Chol, thereby promoting the formation of toxic bile. [ABSTRACT FROM AUTHOR]

To meet challenges in terms of throughput and turnaround time, many diagnostic laboratories are shifting from Sanger sequencing to higher throughput next-generation sequencing (NGS) platforms. Bearing in mind that the performance and quality criteria expected from NGS in diagnostic or research settings are strikingly different, we have developed an Ion Torrent's PGM-based routine diagnostic procedure for BRCA1/2 sequencing. The procedure was first tested on a training set of 62 control samples, and then blindly validated on 77 samples in parallel with our routine technique. The training set was composed of difficult cases, for example, insertions and/or deletions of various sizes, large-scale rearrangements and, obviously, mutations occurring in homopolymer regions. We also compared two bioinformatic solutions in this diagnostic context, an in-house academic pipeline and the commercially available NextGene software (Softgenetics). NextGene analysis provided higher sensitivity, as four previously undetected single-nucleotide variations were found. Regarding specificity, an average of 1.5 confirmatory Sanger sequencings per patient was needed for complete BRCA1/2 screening. Large-scale rearrangements were identified by two distinct analyses, that is, bioinformatics and fragment analysis with electrophoresis profile comparison. Turnaround time was enhanced, as a series of 30 patients were sequenced by one technician, making the results available for the clinician in 10 working days following blood sampling. BRCA1/2 genes are a good model, representative of the difficulties commonly encountered in diagnostic settings, which is why we believe our findings are of interest for the whole community, and the pipeline described can be adapted by any user of PGM for diagnostic purposes. [ABSTRACT FROM AUTHOR]

Charcot-Marie-Tooth disease (CMT) is a group of hereditary peripheral neuropathies. The dominantly inherited axonal CMT2 displays striking genetic heterogeneity, with 17 presently known disease genes. The large number of candidate genes, combined with lack of genotype-phenotype correlations, has made genetic diagnosis in CMT2 time-consuming and costly. In Finland, 25% of dominant CMT2 is explained by either a GDAP1 founder mutation or private MFN2 mutations but the rest of the families have remained without molecular diagnosis. Whole-exome and genome sequencing are powerful techniques to find disease mutations for CMT patients but they require large amounts of sequencing to confidently exclude heterozygous variants in all candidate genes, and they generate a vast amount of irrelevant data for diagnostic needs. Here we tested a targeted next-generation sequencing approach to screen the CMT2 genes. In total, 15 unrelated patients from dominant CMT2 families from Finland, in whom MFN2 and GDAP1 mutations had been excluded, participated in the study. The targeted approach produced sufficient sequence coverage for 95% of the 309 targeted exons, the rest we excluded by Sanger sequencing. Unexpectedly, the screen revealed a disease mutation only in one family, in the HSPB1 gene. Thus, new disease genes underlie CMT2 in the remaining families, indicating further genetic heterogeneity. We conclude that targeted next-generation sequencing is an efficient tool for genetic screening in CMT2 that also aids in the selection of patients for genome-wide approaches. [ABSTRACT FROM AUTHOR]

Although several syndromes include abnormalities of both the ventral body wall and external genitalia, the developmental bases of this correlation are largely unknown. Naturally occurring mutations in Aristaless-like 4 (Alx4, Strong's luxoid: Alx4Lst) have ventral body wall and pelvic girdle abnormalities. We sought to determine whether the development of the genital tubercle (GT) and its derivatives, the external genitalia, is affected by this mutation. We thus performed genetic and tissue labeling analyses in mutant mice. Alx4Lst/Lst mutants displayed hypoplasia of the dorsal GT and reduced expression of Fibronectin. We analyzed cell migration during GT formation by tissue labeling experiments and discovered that the cells located in the proximal segment of the umbilical cord (infra-umbilical mesenchyme) migrate toward the dorsal part of the GT. The Alx4Lst/Lst mutants also displayed augmented expression of Hh signal-related genes. Hence, we analyzed a series of combinatorial mutants for Alx4, Sonic hedgehog (Shh) and GLI-Kruppel family member 3 (Gli3). These phenotype-genotype analyses suggested a genetic interaction between Alx4 and Hh signaling during GT formation. Moreover, Hh gain-of-function mutants phenocopied some of these phenotypes. These observations reveal novel information regarding the pathogenic mechanisms of syndromic lower ventral body malformations, which are largely unknown. [ABSTRACT FROM AUTHOR]

Mitochondrial disorders are caused by defects in mitochondrial or nuclear DNA. Although the existence of large deletions in mitochondrial DNA (mtDNA) is well known, deletions affecting whole genes are not commonly described in patients with mitochondrial disorders. Based on the results of whole-genome analyses, copy number variations (CNVs) occur frequently in the human genome and may overlap with many genes associated with clinical phenotypes. We report the discovery of two large heterozygous CNVs on 22q13.33 in two patients with mitochondrial disorders. The first patient harboured a novel point mutation c.667G>A (p.D223N) in the SCO2 gene in combination with a paternally inherited 87-kb deletion. As hypertrophic cardiomyopathy (HCMP) was not documented in the patient, this observation prompted us to compare his clinical features with all 44 reported SCO2 patients in the literature. Surprisingly, the review shows that HCMP was present in only about 50% of the SCO2 patients with non-neonatal onset. In the second patient, who had mitochondrial neurogastrointestinal encephalopathy (MNGIE), a maternally inherited 175-kb deletion and the paternally inherited point mutation c.261G>T (p.E87D) in the TYMP gene were identified. [ABSTRACT FROM AUTHOR]

Hereditary retinal dystrophies (RD) constitute a group of blinding diseases that are characterized by clinical variability and pronounced genetic heterogeneity. The different forms of RD can be caused by mutations in >100 genes, including >1600 exons. Consequently, next generation sequencing (NGS) technologies are among the most promising approaches to identify mutations in RD. So far, NGS is not routinely used in gene diagnostics. We developed a diagnostic NGS pipeline to identify mutations in 170 genetically and clinically unselected RD patients. NGS was applied to 105 RD-associated genes. Underrepresented regions were examined by Sanger sequencing. The NGS approach was successfully established using cases with known sequence alterations. Depending on the initial clinical diagnosis, we identified likely causative mutations in 55% of retinitis pigmentosa and 80% of Bardet-Biedl or Usher syndrome cases. Seventy-one novel mutations in 40 genes were newly associated with RD. The genes USH2A, EYS, ABCA4, and RHO were more frequently affected than others. Occasionally, cases carried mutations in more than one RD-associated gene. In addition, we found possible dominant de-novo mutations in cases with sporadic RD, which implies consequences for counseling of patients and families. NGS-based mutation analyses are reliable and cost-efficient approaches in gene diagnostics of genetically heterogeneous diseases like RD. [ABSTRACT FROM AUTHOR]

Mutations of genes encoding the subunits of the succinate dehydrogenase (SDH) complex were described in KIT/PDGFRA wild-type GIST separately in different reports. In this study, we simultaneously sequenced the genome of all subunits, SDHA, SDHB, SDHC, and SDHD in a larger series of KIT/PDGFRA wild-type GIST in order to evaluate the frequency of the mutations and explore their biological role. SDHA, SDHB, SDHC, and SDHD were sequenced on the available samples obtained from 34 KIT/PDGFRA wild-type GISTs. Of these, in 10 cases, both tumor and peripheral blood (PB) were available, in 19 cases only tumor, and in 5 cases only PB. Overall, 9 of the 34 patients with KIT/PDGFRA wild-type GIST carried mutations in one of the four subunits of the SDH complex (six patients in SDHA, two in SDHB, one in SDHC). WB and immunohistochemistry analysis showed that patients with KIT/PDGFRA wild-type GIST who harbored SDHA mutations exhibited a significant downregulation of both SDHA and SDHB protein expression, with respect to the other GIST lacking SDH mutations and to KIT/PDGFRA-mutated GIST. Clinically, four out of six patients with SDHA mutations presented with metastatic disease at diagnosis with a very slow, indolent course. Patients with KIT/PDGFRA wild-type GIST may harbor germline and/or de novo mutations of SDH complex with prevalence for mutations within SDHA, which is associated with a downregulation of SDHA and SDHB protein expression. The presence of germline mutations may suggest that these patients should be followed up for the risk of development of other cancers. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) may appear to be familial or sporadic, with recognised dominant and recessive inheritance in a proportion of cases. Sporadic ALS may be caused by rare homozygous recessive mutations. We studied patients and controls from the UK and a multinational pooled analysis of GWAS data on homozygosity in ALS to determine any potential recessive variant leading to the disease. Six-hundred and twenty ALS and 5169 controls were studied in the UK cohort. A total of 7646 homozygosity segments with length >2 Mb were identified, and 3568 rare segments remained after filtering 'common' segments. The mean total of the autosomal genome with homozygosity segments was longer in ALS than in controls (unfiltered segments, P=0.05). Two-thousand and seventeen ALS and 6918 controls were studied in the pooled analysis. There were more regions of homozygosity segments per case (P=1 × 10−5), a greater proportion of cases harboured homozygosity (P=2 × 10−5), a longer average length of segment (P=1 × 10−5), a longer total genome coverage (P=1 × 10−5), and a higher rate of these segments overlapped with RefSeq gene regions (P=1 × 10−5), in ALS patients than controls. Positive associations were found in three regions. The most significant was in the chromosome 21 SOD1 region, and also chromosome 1 2.9-4.8 Mb, and chromosome 5 in the 65 Mb region. There are more than twenty potential genes in these regions. These findings point to further possible rare recessive genetic causes of ALS, which are not identified as common variants in GWAS. [ABSTRACT FROM AUTHOR]

Frontotemporal lobar degeneration (FTLD) is a progressive neurodegenerative disease with an age at onset generally below 65 years. Mutations in progranulin (GRN) have been reported to be able to cause FTLD through haploinsufficiency. We have sequenced GRN in 121 patients with FTLD and detected six different mutations in eight patients: p.Gly35Glufs*19, p.Asn118Phefs*4, p.Val200Glyfs*18, p.Tyr294*, p.Cys404* and p.Cys416Leufs*30. Serum was available for five of the mutations, where the serum-GRN levels were found to be >50% reduced compared with FTLD patients without GRN mutations. Moreover, the p.Cys416Leufs*30 mutation segregated in an affected family with different dementia diagnoses. The mutation frequency of GRN mutation was 6.6% in our FTLD cohort. [ABSTRACT FROM AUTHOR]

Autosomal dominant sensorineural hearing loss (ADSNHL) is extremely genetically heterogeneous, making it difficult to molecularly diagnose. We identified a multiplex (n=28 affected) family from the genetic isolate of Newfoundland, Canada with variable SNHL and used a targeted sequencing approach based on population-specific alleles in WFS1, TMPRSS3 and PCDH15; recurrent mutations in GJB2 and GJB6; and frequently mutated exons of KCNQ4, COCH and TECTA. We identified a novel, in-frame deletion (c.806_808delCCT: p.S269del) in the voltage-gated potassium channel KCNQ4 (DFNA2), which in silico modeling predicts to disrupt multimerization of KCNQ4 subunits. Surprisingly, 10/23 deaf relatives are non-carriers of p.S269del. Further molecular characterization of the DFNA2 locus in deletion carriers ruled out the possibility of a pathogenic mutation other than p.S269del at the DFNA2A/B locus and linkage analysis showed significant linkage to DFNA2 (maximum LOD=3.3). Further support of genetic heterogeneity in family 2071 was revealed by comparisons of audio profiles between p.S269del carriers and non-carriers suggesting additional and as yet unknown etiologies. We discuss the serious implications that genetic heterogeneity, in this case observed within a single family, has on molecular diagnostics and genetic counseling. [ABSTRACT FROM AUTHOR]

In recent studies on prenatal testing for Noonan syndrome (NS) in fetuses with an increased nuchal translucency (NT) and a normal karyotype, mutations have been reported in 9-16% of cases. In this study, DNA of 75 fetuses with a normal karyotype and abnormal ultrasound findings was tested in a diagnostic setting for mutations in (a subset of) the four most commonly mutated NS genes. A de novo mutation in either PTPN11, KRAS or RAF1 was detected in 13 fetuses (17.3%). Ultrasound findings were increased NT, distended jugular lymphatic sacs (JLS), hydrothorax, renal anomalies, polyhydramnios, cystic hygroma, cardiac anomalies, hydrops fetalis and ascites. A second group, consisting of anonymized DNA of 60 other fetuses with sonographic abnormalities, was tested for mutations in 10 NS genes. In this group, five possible pathogenic mutations have been identified (in PTPN11 (n=2), RAF1, BRAF and MAP2K1 (each n=1)). We recommend prenatal testing of PTPN11, KRAS and RAF1 in pregnancies with an increased NT and at least one of the following additional features: polyhydramnios, hydrops fetalis, renal anomalies, distended JLS, hydrothorax, cardiac anomalies, cystic hygroma and ascites. If possible, mutation analysis of BRAF and MAP2K1 should be considered. [ABSTRACT FROM AUTHOR]

We report a novel pathogenic mutation of the mitochondrial transfer RNA (tRNA) gene for tryptophan in a patient with isolated myopathy and persistently elevated creatine kinase. Muscle studies revealed ragged red fibres and decreased activity of respiratory chain complex I and cytochrome c oxidase (COX). Sequencing of the 22 mitochondrial tRNA genes revealed a mutation m.5522G>A, which alters a conserved base pairing in the D-stem of the tRNA for tryptophan. The mutation was heteroplasmic with a mutational load between 88 and 99% in COX-negative fibres. This case contributes to the genetic heterogeneity of mitochondrial diseases caused by mutations in mitochondrial tRNA genes. [ABSTRACT FROM AUTHOR]

Meckel-Gruber syndrome (MKS, OMIM #249000) is a multiple congenital malformation syndrome that represents the severe end of the ciliopathy phenotypic spectrum. Despite the relatively common occurrence of this syndrome among Arabs, little is known about its genetic architecture in this population. This is a series of 18 Arab families with MKS, who were evaluated clinically and studied using autozygome-guided mutation analysis and exome sequencing. We show that autozygome-guided candidate gene analysis identified the underlying mutation in the majority (n=12, 71%). Exome sequencing revealed a likely pathogenic mutation in three novel candidate MKS disease genes. These include C5orf42, Ellis-van-Creveld disease gene EVC2 and SEC8 (also known as EXOC4), which encodes an exocyst protein with an established role in ciliogenesis. This is the largest and most comprehensive genomic study on MKS in Arabs and the results, in addition to revealing genetic and allelic heterogeneity, suggest that previously reported disease genes and the novel candidates uncovered by this study account for the overwhelming majority of MKS patients in our population. [ABSTRACT FROM AUTHOR]