Taking the next step forward - Diagnosing inherited infantile cholestatic disorders with next generation sequencing.

Identifying rare genetic forms of infantile cholestasis is challenging due to their similar clinical presentation and their diverse etiology. After exclusion of common non-genetic causes a huge list of rare differential diagnosis remains to be solved. More than 90 genes are associated with monogenic forms of infantile cholestasis, thus preventing routine genetic workup by Sanger sequencing. Here we demonstrate a next generation sequencing approach to discover the underlying cause in clinically well characterized patients in whom common causes of infantile cholestasis have been excluded. After validation of the analytical sensitivity massive parallel sequencing was performed for 93 genes in six prospectively studied patients. Six novel mutations (PKHD1: p.Thr777Met, p.Tyr2260Cys; ABCB11: p.Val1112Phe, c.611+1G > A, p.Gly628Trpfs*3 and NPC1: p.Glu391Lys) and two known pathogenic mutations were detected proving our multi gene panel for infantile cholestasis to be a sensitive and specific method overcoming the complexity of the phenotype-based, candidate gene approach. Three exemplary clinical cases of infants with cholestasis are presented and discussed in the context of their genetic and histopathological findings (autosomal recessive polycystic kidney disease, atypical PFIC and Niemann-Pick syndrome type C1). These case reports highlight the critical impact of integrating clinical, histopathological and genetic data during the process of multi gene panel testing to ultimately pinpoint rare genetic diagnoses.

Craniofrontonasal syndrome in a male due to chromosomal mosaicism involving EFNB1: further insights into a genetic paradox.

Craniofrontonasal syndrome (CFNS) is an X-linked disorder caused by inactivating mutations in the gene for ephrin-B1 (EFNB1). Paradoxically it shows a more severe phenotype in females than in males. As a result of X inactivation cell populations with and without EFNB1 expression are found in EFNB1+/- females. This is thought to initiate a process termed cellular interference which may be responsible for the phenotype in females. We present a boy with severe clinical features of CFNS. In ∼42% of his blood cells we found a supernumerary ring X chromosome containing EFNB1 but lacking XIST. Mosaicism for cell populations with different levels of EFNB1 expression can explain the severe phenotype of this patient. In vitro experiments in Xenopus tissue showed that cells overexpress ephrinB1 cluster and sort out from wild-type cells. Our report provides further evidence that cellular interference contributes to the paradoxical inheritance pattern of CFNS.

Clinical and mutation data in 12 patients with the clinical diagnosis of Nager syndrome.

Nager syndrome (MIM #154400) is the best-known preaxial acrofacial dysostosis, mainly characterized by craniofacial and preaxial limb anomalies. The craniofacial abnormalities mainly consist of downslanting palpebral fissures, malar hypoplasia, micrognathia, external ear anomalies, and cleft palate. The preaxial limb defects are characterized by radial and thumb hypoplasia or aplasia, duplication of thumbs and proximal radioulnar synostosis. Haploinsufficiency of SF3B4 (MIM *605593), which encodes SAP49, a component of the pre-mRNA spliceosomal complex, has recently been identified as the underlying cause of Nager syndrome. In our study, we performed exome sequencing in two and Sanger sequencing of SF3B4 in further ten previously unreported patients with the clinical diagnosis of Nager syndrome, including one familial case. We identified heterozygous SF3B4 mutations in seven out of twelve patients. Four of the seven mutations were shown to be de novo; in three individuals, DNA of both parents was not available. No familial mutations were discovered. Three mutations were nonsense, three were frameshift mutations and one T > C transition destroyed the translation start signal. In three of four SF3B4 negative families, EFTUD2 was analyzed, but no pathogenic variants were identified. Our results indicate that the SF3B4 gene is mutated in about half of the patients with the clinical diagnosis of Nager syndrome and further support genetic heterogeneity for this condition.

Mutations in the homeodomain of the human SIX3 gene cause holoprosencephaly.

Holoprosencephaly (HPE) is a common, severe malformation of the brain that involves separation of the central nervous system into left and right halves. Mild HPE can consist of signs such as a single central incisor, hypotelorism, microcephaly, or other craniofacial findings that can be present with or without associated brain malformations. The aetiology of HPE is extremely heterogeneous, with the proposed participation of a minimum of 12 HPE-associated genetic loci as well as the causal involvement of specific teratogens acting at the earliest stages of neurulation. The HPE2 locus was recently characterized as a 1-Mb interval on human chromosome 2p21 that contained a gene associated with HPE. A minimal critical region was defined by a set of six overlapping deletions and three clustered translocations in HPE patients. We describe here the isolation and characterization of the human homeobox-containing SIX3 gene from the HPE2 minimal critical region (MCR). We show that at least 2 of the HPE-associated translocation breakpoints in 2p21 are less than 200 kb from the 5' end of SIX3. Mutational analysis has identified four different mutations in the homeodomain of SIX3 that are predicted to interfere with transcriptional activation and are associated with HPE. We propose that SIX3 is the HPE2 gene, essential for the development of the anterior neural plate and eye in humans.

Genetic assessment of cortical malformations.

Malformations of cortical development comprise a clinically and etiologically heterogeneous group of distinct structural abnormalities of the cerebral cortex, commonly identified during MR imaging of patients with seizure disorders and/or developmental delay. MR imaging is crucial for further classification and together with additional clinical information and family history guiding specific genetic testing, which today is an integral part of the interdisciplinary diagnostic work-up and allows identification of an underlying genetic alteration in a significant subset of patients. Results of genetic testing may provide important prognostic information and subsequently support prospective therapeutic decisions. Furthermore, genetic forms of cortical malformations may be associated with a significantly increased recurrence risk for further siblings or other relatives and require genetic counselling of the family on individual risks and the options of prenatal or even preimplantation genetic diagnosis.

Clinical spectrum of SIX3-associated mutations in holoprosencephaly: correlation between genotype, phenotype and function.

BACKGROUND: Holoprosencephaly (HPE) is the most common structural malformation of the human forebrain. There are several important HPE mutational target genes, including the transcription factor SIX3, which encodes an early regulator of Shh, Wnt, Bmp and Nodal signalling expressed in the developing forebrain and eyes of all vertebrates. OBJECTIVE: To characterise genetic and clinical findings in patients with SIX3 mutations. METHODS: Patients with HPE and their family members were tested for mutations in HPE-associated genes and the genetic and clinical findings, including those for additional cases found in the literature, were analysed. The results were correlated with a mutation-specific functional assay in zebrafish. RESULTS: In a cohort of patients (n = 800) with HPE, SIX3 mutations were found in 4.7% of probands and additional cases were found through testing of relatives. In total, 138 cases of HPE were identified, 59 of whom had not previously been clinically presented. Mutations in SIX3 result in more severe HPE than in other cases of non-chromosomal, non-syndromic HPE. An over-representation of severe HPE was found in patients whose mutations confer greater loss of function, as measured by the functional zebrafish assay. The gender ratio in this combined set of patients was 1.5:1 (F:M) and maternal inheritance was almost twice as common as paternal. About 14% of SIX3 mutations in probands occur de novo. There is a wide intrafamilial clinical range of features and classical penetrance is estimated to be at least 62%. CONCLUSIONS: Our data suggest that SIX3 mutations result in relatively severe HPE and that there is a genotype-phenotype correlation, as shown by functional studies using animal models.

Frequency and phenotype of SPG11 and SPG15 in complicated hereditary spastic paraplegia.

BACKGROUND: Hereditary spastic paraplegias (HSP) are clinically and genetically highly heterogeneous. Recently, two novel genes, SPG11 (spatacsin) and SPG15 (spastizin), associated with autosomal recessive HSP, were identified. Clinically, both are characterised by complicated HSP and a rather similar phenotype consisting of early onset spastic paraplegia, cognitive deficits, thin corpus callosum (TCC), peripheral neuropathy and mild cerebellar ataxia. OBJECTIVE: To compare the frequency of SPG11 and SPG15 in patients with early onset complicated HSP and to further characterise the phenotype of SPG11 and SPG15. RESULTS: A sample of 36 index patients with early onset complicated HSP and a family history compatible with autosomal recessive inheritance was collected and screened for mutations in SPG11 and SPG15. Overall frequency of SPG11 was 14% (5/36) but was considerably higher in patients with TCC (42%). One patient with mental retardation and thinning of the corpus callosum was compound heterozygous for two novel SPG15 mutations. Additionally, several new polymorphisms and sequence variants of unknown significance have been identified in the SPG15 gene. CONCLUSIONS: TCC seems to be the best phenotypic predictor for SPG11 as well as SPG15. No clinical features could discriminate between SPG11 and SPG15. Therefore, priority of genetic testing should be driven by mutation frequency that appears to be substantially higher in SPG11 than in SPG15.

Andermann syndrome can be a phenocopy of hereditary motor and sensory neuropathy--report of a discordant sibship with a compound heterozygous mutation of the KCC3 gene.

Andermann syndrome is a rare autosomal recessive disorder characterized by agenesis of the corpus callosum (ACC), progressive motor-sensory neuropathy, mental retardation and facial features. We report on two siblings with the clinical picture of a demyelinating hereditary motor and sensory neuropathy (HMSN), where only the presence of ACC in the younger brother pointed to the diagnosis of Andermann syndrome. Mutation analysis of the KCC3 (SLC12A6) gene showed a compound heterozygous mutation; a maternal missense mutation c.1616G>A (p.G539D) and a paternal splice mutation c.1118+1G>A in both siblings. We hypothesize that mutations of the KCC3 gene may result in non-syndromic childhood onset HMSN.

POMT1-associated walker-warburg syndrome: a disorder of dendritic development of neocortical neurons.

We have analyzed the morphology and dendritic development of neocortical neurons in a 2.5-month-old infant with Walker-Warburg syndrome homozygotic for a novel POMT1 gene mutation, by Golgi methods. We found that pyramidal neurons frequently displayed abnormal (oblique, horizontal, or inverted) orientation. A novel finding of this study is that members of the same population of pyramidal neurons display different stages of development of their dendritic arborizations: some neurons had poorly developed dendrites and thus resembled pyramidal neurons of the late fetal cortex; for some neurons, the level of differentiation corresponded to that in the newborn cortex; finally, some neurons had quite elaborate dendritic trees as expected for the cortex of 2.5-month-old infant. In addition, apical dendrites of many pyramidal neurons were conspiciously bent to one side, irrespective to the general orientation of the pyramidal neuron. These findings suggest that Walker-Warburg lissencephaly is characterized by two hitherto unnoticed pathogenetic changes in the cerebral cortex: (a) heterochronic decoupling of dendritic maturation within the same neuronal population (with some members significantly lagging behind the normal maturational schedule) and (b) anisotropically distorted shaping of dendritic trees, probably caused by patchy displacement of molecular guidance cues for dendrites in the malformed cortex.

Forty-eight new cases with infertility due to balanced chromosomal rearrangements: detailed molecular cytogenetic analysis of the 90 involved breakpoints.

A molecular cytogenetic study was performed on 48 infertile patients who were identified as carriers of balanced translocations (40 cases), inversions (6 cases) or insertions (2 cases) by means of banding cytogenetics. Cases with a Robertsonian translocation or pericentric inversion 2 or 9 were not included. In summary, 100 break-events occurred in these patients, and 90 different chromosomal regions were involved. Thus, this study confirmed the presence of abnormal karyotypes in a subgroup of patients seeking infertility treatment. Breaks were demonstrated to appear preferentially in GTG-light bands in these patients. Furthermore, the observed breakpoints were associated with genomic regions prone to instability due to the presence of segmental duplications. Nonetheless, further detailed molecular analysis will be necessary in the future to characterize the mechanisms and genetic basis for this phenomenon.

A filamin A splice mutation resulting in a syndrome of facial dysmorphism, periventricular nodular heterotopia, and severe constipation reminiscent of cerebro-fronto-facial syndrome.

BACKGROUND: Mutations of the filamin A locus (FLNA) on Xq28 have been established in girls with periventricular nodular heterotopia and in patients with otopalatodigital and overlapping phenotypes, the pathogenesis of these phenotypes being thought to be quite distinct. To date only six male cases of periventricular nodular heterotopia (PVNH) have been reported and these almost invariably associated with severe neurological signs. METHODS AND RESULTS: We report a new phenotype of male PVNH, with relatively normal development, no epilepsy or other neurological abnormality, severe constipation, and facial dysmorphism and without a discernible skeletal phenotype. This phenotype is associated with a splice site mutation in FLNA c.1923C>T, resulting in the generation of both normal and aberrant mRNA. CONCLUSIONS: We postulate that the patient retains enough FLNA function to avoid the usual lethality associated with loss of function mutations in males and suggest that the severe constipation may be a clue to the molecular aetiology of other X linked conditions associated with severe constipation.

Clinical spectrum of muscle-eye-brain disease: from the typical presentation to severe autistic features.

Muscle-eye-brain disease (MEB) is an autosomal recessive congenital muscular dystrophy with ocular abnormalities and type II lissencephaly. MEB is caused by mutations in the protein O-linked mannose beta1,2-N-acetylglucosaminyltransferase (POMGnT1) gene on chromosome 1q33. POMGnT1 is a glycosylation enzyme that participates in the synthesis of O-mannosyl glycan. The disease is characterized by altered glycosylation of alpha-dystroglycan. The clinical spectrum of MEB phenotype and POMGnT1 mutations are significantly expanded. We would like to present two cases with MEB disease with POMGnT1 mutations, whose clinical picture shows heterogeneity. The patient with R442H mutation had the classical form of the disease although the one with IVS17-2A-->G homozygous mutation had severe autistic features as the dominating presenting sign. These two cases represent different spectrums of one disorder. To the best of our knowledge, autistic features and stereotypical movements have not been included thus far as a part of broad and heterogeneous MEB spectrum.

First Report of a Single Exon Deletion in TCOF1 Causing Treacher Collins Syndrome.

Treacher Collins syndrome (TCS) is a rare craniofacial disorder characterized by facial anomalies and ear defects. TCS is caused by mutations in the TCOF1 gene and follows autosomal dominant inheritance. Recently, mutations in the POLR1D and POLR1C genes have also been identified to cause TCS. However, in a subset of patients no causative mutation could be found yet. Inter- and intrafamilial phenotypic variability is high as is the variety of mainly family-specific mutations identified throughout TCOF1. No obvious correlation between pheno- and genotype could be observed. The majority of described point mutations, small insertions and deletions comprising only a few nucleotides within TCOF1 lead to a premature termination codon. We investigated a cohort of 112 patients with a tentative clinical diagnosis of TCS by multiplex ligation-dependent probe amplification (MLPA) to search for larger deletions not detectable with other methods used. All patients were selected after negative screening for mutations in TCOF1, POLR1D and POLR1C. In 1 patient with an unequivocal clinical diagnosis of TCS, we identified a 3.367 kb deletion. This deletion abolishes exon 3 and is the first described single exon deletion within TCOF1. On RNA level we observed loss of this exon which supposedly leads to haploinsufficiency of TREACLE, the nucleolar phosphoprotein encoded by TCOF1.

Two Adult Patients with Ellis-van Creveld Syndrome Extending the Clinical Spectrum.

Ellis-van Creveld (EvC) syndrome is a rare autosomal recessive malformation syndrome with the main features cardiac defects, postaxial hexadactyly, mesomelic shortening of the limbs, short ribs, dysplastic nails and teeth, oral frenula and various other abnormalities while mental function is normal. We describe 2 adult EvC patients with the cardinal skeletal features of mesomelic short stature and severe, progressive genu valgum deformity, resulting from loss of function mutations in the EVC genes. While the genu valgum was the predominating and disabling feature in patient 1, patient 2 showed acroosteolyses in the distal phalanges and a symmetrical synostosis of metacarpals in his hands. Moreover, patient 2 developed synostoses in the additional fingers in adolescence which had not been present at the age of 12 years, suggesting a further progression of skeletal disease. Joint fusion of phalanges so far has not been reported in EvC syndrome. Our data further expand the phenotypic spectrum of EvC related skeletal malformations and contribute important new information on the clinical course of EvC syndrome with increasing age.

Correlation of enzyme activity and clinical phenotype in POMT1-associated dystroglycanopathies.

BACKGROUND: Mutations in protein O-mannosyltransferases (POMTs) cause a heterogeneous group of muscular dystrophies with abnormal glycosylation of alpha-dystroglycan (dystroglycanopathies). The wide spectrum of clinical severities ranges from Walker-Warburg syndrome (WWS), associated with brain and eye abnormalities, to mild forms of limb girdle muscular dystrophy (LGMD). OBJECTIVE: The aim of this study was to elucidate the impact of mutations in POMT1 on the clinical phenotype. METHODS: We examined 2 patients with POMT1-associated alpha-dystroglycanopathy, 1 displaying a LGMD2K and 1 with a WWS phenotype. Using dermal fibroblasts, we analyzed the influence of the POMT1 mutations on the glycosylation status of alpha-dystroglycan, protein O-mannosyltransferase activity, and the stability of the mutant POMT1 protein. RESULTS: We report on novel compound heterozygous mutations in POMT1 (p.L171A and p.A589VfsX38) that result in LGMD2K. We further demonstrate that a homozygous splice site mutation of a recently identified WWS patient results in POMT1 p.del77-93. Using dermal fibroblasts, we show that mannosyltransferase activity is reduced in the patients and that stability of POMT1 mutant proteins p.A589VfsX38 and p.del77-93 is significantly decreased. CONCLUSIONS: Our results suggest that dermal fibroblasts can be applied to facilitate the diagnostic analysis of dystroglycanopathy patients as well as to study the pathogenic mechanism of POMT mutations. Characterization of the POMT1 substrate protein alpha-dystroglycan and POMT in vitro mannosyltransferase activity shows that the severity of the clinical phenotype of the patients analyzed is inversely correlated with POMT activity.

Disturbed Wnt Signalling due to a Mutation in CCDC88C Causes an Autosomal Recessive Non-Syndromic Hydrocephalus with Medial Diverticulum.

The etiology of non-syndromic hydrocephalus is poorly understood. Via positional cloning in a consanguineous family with autosomal recessive hydrocephalus we have now identified a homozygous splice site mutation in the CCDC88C gene as a novel cause of a complex hydrocephalic brain malformation. The only living patient showed normal psychomotor development at the age of 3 years and 3 months and her deceased aunt, who was assumed to suffer from the same condition, had mild mental retardation. The mutation in the affected patients, a homozygous substitution in the donor splice site of intron 29, resulted in a shorter transcript due to exclusion of exon 29 and loss of functional protein, as shown by Western blotting (p.S1591HfsX7). In normal human tissue panels, we found CCDC88C ubiquitously expressed, but most prominently in the fetal brain, especially in pons and cerebellum, while expression in the adult brain appeared to be restricted to cortex and medulla oblongata. CCDC88C encodes DAPLE (HkRP2), a Hook-related protein with a binding domain for the central Wnt signalling pathway protein Dishevelled. Targeted quantitative RT-PCR and expression profiling of 84 genes from the Wnt signalling pathway in peripheral blood from the index patient and her healthy mother revealed increased mRNA levels of CCDC88C indicating transcriptional upregulation. Due to loss of CCDC88C function ß-catenin (CTNNB1) and the downstream target LEF1 showed increased mRNA levels in the patient, but many genes from the Wnt pathway and transcriptional target genes showed reduced expression, which might be explained by a complex negative feedback loop. We have thus identified a further essential component of the Wnt signalling pathway in human brain development.

Location and type of mutation in the LIS1 gene do not predict phenotypic severity.

BACKGROUND: Lissencephaly is a neuronal migration disorder leading to absent or reduced gyration and a broadened but poorly organized cortex. The most common form of lissencephaly is isolated, referred as classic or type 1 lissencephaly. Type 1 lissencephaly is mostly associated with a heterozygous deletion of the entire LIS1 gene, whereas intragenic heterozygous LIS1 mutations or hemizygous DCX mutations in males are less common. METHODS: Eighteen unrelated patients with type 1 lissencephaly were clinically and genetically assessed. In addition, patients with subcortical band heterotopia (n = 1) or lissencephaly with cerebellar hypoplasia (n = 2) were included. RESULTS: Fourteen new and seven previously described LIS1 mutations were identified. We observed nine truncating mutations (nonsense, n = 2; frameshift, n = 7), six splice site mutations, five missense mutations, and one in-frame deletion. Somatic mosaicism was assumed in three patients with partial subcortical band heterotopia in the occipital-parietal lobes or mild pachygyria. We report three mutations in exon 11, including a frameshift which extends the LIS1 protein, leading to type 1 lissencephaly and illustrating the functional importance of the WD domains at the C terminus. Furthermore, we present two patients with novel LIS1 mutations in exon 10 associated with lissencephaly with cerebellar hypoplasia type a. CONCLUSION: In contrast to previous reports, our data suggest that neither type nor position of intragenic mutations in the LIS1 gene allows an unambiguous prediction of the phenotypic severity. Furthermore, patients presenting with mild cerebral malformations such as subcortical band heterotopia or cerebellar hypoplasia should be considered for genetic analysis of the LIS1 gene.

Novel truncating and missense mutations of the KCC3 gene associated with Andermann syndrome.

BACKGROUND: Andermann syndrome (OMIM 218000) is an autosomal recessive motor-sensory neuropathy associated with developmental and neurodegenerative defects. The cerebral MRI reveals a variable degree of agenesis of the corpus callosum. Recently, truncating mutations of the KCC3 gene (also known as SLC12A6) have been associated with Andermann syndrome. METHODS: The authors assessed clinically and genetically three isolated cases from Germany and Turkey with symptoms consistent with Andermann syndrome. RESULTS: The authors detected four novel mutations within the KCC3 gene in their patients: two different truncating mutations in the first patient, a homozygous truncating mutation in the second, and a homozygous missense mutation in the third patient. In contrast to the classic phenotype of the Andermann syndrome linked to truncating KCC3 mutations the phenotype and the course of the disease linked to the missense mutation appeared to be different (i.e., showing additional features like diffuse and widespread white matter abnormalities). CONCLUSIONS: Not only truncating but also missense mutations of the KCC3 gene are associated with Andermann syndrome. Different types of KCC3 mutations may determine different clinical phenotypes.

Further clinical and genetic characterization of SPG11: hereditary spastic paraplegia with thin corpus callosum.

Hereditary spastic paraplegias (HSPs) are a heterogeneous group of neurodegenerative disorders leading to progressive spasticity of the lower limbs. Clinically, HSPs are divided into "pure" and "complicated" forms. In pure HSP, the spasticity of the lower limbs is the sole symptom, whereas in complicated forms additional neurological and non-neurological features are observed. Genetically, HSPs are divided into autosomal dominant (AD), autosomal recessive (AR) and X-linked (XL) forms. Up to date, 30 different HSPs are linked to different chromosomal loci and 11 genes could be defined for AR-HSP, AD-HSP and XL-HSP. SPG11, an AR-HSP (synonym: HSP11), is a complicated HSP associated with a slowly progressive spastic paraparesis, mental impairment and the development of a thin corpus callosum (TCC) during the course of the disease. SPG11 has been previously linked to chromosomal region 15q13 - 15. First, we applied rigid diagnostic criteria to systematically examine 20 Turkish families with autosomal recessive HSP for characteristic features of SPG11. We detected four large Turkish families with AR-HSP and TCC consistent with SPG11. Subsequent genetic linkage analysis of those 4 families refines the SPG11 locus further down to a small region of 2.93 cM with a maximum lod score of 11.84 at marker D15S659 and will guide further candidate gene analysis.