A ZPR1 mutation is associated with a novel syndrome of growth restriction, distinct craniofacial features, alopecia, and hypoplastic kidneys.

A novel autosomal recessive disorder characterized by pre- and postnatal growth restriction with microcephaly, distinctive craniofacial features, congenital alopecia, hypoplastic kidneys with renal insufficiency, global developmental delay, severe congenital sensorineural hearing loss, early mortality, hydrocephalus, and genital hypoplasia was observed in 4 children from 3 families of New Mexican Hispanic heritage. Three of the children died before 3 years of age from uremia and/or sepsis. Exome sequencing of the surviving individual identified a homozygous c.587T>C (p.Ile196Thr) mutation in ZPR1 Zinc Finger (ZPR1) that segregated appropriately in her family. In a second family, the identical variant was shown to be heterozygous in the affected individual's parents and not homozygous in any of her unaffected siblings. ZPR1 is a ubiquitously expressed, highly conserved protein postulated to transmit proliferative signals from the cell membrane to the nucleus. Structural modeling reveals that p.Ile196Thr disrupts the hydrophobic core of ZPR1. Patient fibroblast cells showed no detectable levels of ZPR1 and the cells showed a defect in cell cycle progression where a significant number of cells remained arrested in the G1 phase. We provide genetic and molecular evidence that a homozygous missense mutation in ZPR1 is associated with a rare and recognizable multisystem syndrome.

Whole-exome sequencing is a valuable diagnostic tool for inherited peripheral neuropathies: Outcomes from a cohort of 50 families.

The inherited peripheral neuropathies (IPNs) are characterized by marked clinical and genetic heterogeneity and include relatively frequent presentations such as Charcot-Marie-Tooth disease and hereditary motor neuropathy, as well as more rare conditions where peripheral neuropathy is associated with additional features. There are over 250 genes known to cause IPN-related disorders but it is estimated that in approximately 50% of affected individuals a molecular diagnosis is not achieved. In this study, we examine the diagnostic utility of whole-exome sequencing (WES) in a cohort of 50 families with 1 or more affected individuals with a molecularly undiagnosed IPN with or without additional features. Pathogenic or likely pathogenic variants in genes known to cause IPN were identified in 24% (12/50) of the families. A further 22% (11/50) of families carried sequence variants in IPN genes in which the significance remains unclear. An additional 12% (6/50) of families had variants in novel IPN candidate genes, 3 of which have been published thus far as novel discoveries (KIF1A, TBCK, and MCM3AP). This study highlights the use of WES in the molecular diagnostic approach of highly heterogeneous disorders, such as IPNs, places it in context of other published neuropathy cohorts, while further highlighting associated benefits for discovery.

Utility of whole-exome sequencing for those near the end of the diagnostic odyssey: time to address gaps in care.

An accurate diagnosis is an integral component of patient care for children with rare genetic disease. Recent advances in sequencing, in particular whole-exome sequencing (WES), are identifying the genetic basis of disease for 25-40% of patients. The diagnostic rate is probably influenced by when in the diagnostic process WES is used. The Finding Of Rare Disease GEnes (FORGE) Canada project was a nation-wide effort to identify mutations for childhood-onset disorders using WES. Most children enrolled in the FORGE project were toward the end of the diagnostic odyssey. The two primary outcomes of FORGE were novel gene discovery and the identification of mutations in genes known to cause disease. In the latter instance, WES identified mutations in known disease genes for 105 of 362 families studied (29%), thereby informing the impact of WES in the setting of the diagnostic odyssey. Our analysis of this dataset showed that these known disease genes were not identified prior to WES enrollment for two key reasons: genetic heterogeneity associated with a clinical diagnosis and atypical presentation of known, clinically recognized diseases. What is becoming increasingly clear is that WES will be paradigm altering for patients and families with rare genetic diseases.

LIMS2 mutations are associated with a novel muscular dystrophy, severe cardiomyopathy and triangular tongues.

Limb girdle muscular dystrophy (LGMD) is a heterogeneous group of genetic disorders leading to progressive muscle degeneration and often associated with cardiac complications. We present two adult siblings with childhood-onset of weakness progressing to a severe quadriparesis with the additional features of triangular tongues and biventricular cardiac dysfunction. Whole exome sequencing identified compound heterozygous missense mutations that are predicted to be pathogenic in LIMS2. Biopsy of skeletal muscle demonstrated disrupted immunostaining of LIMS2. This is the first report of mutations in LIMS2 and resulting disruption of the integrin linked kinase (ILK)-LIMS-parvin complex associated with LGMD.

Whole-exome sequencing broadens the phenotypic spectrum of rare pediatric epilepsy: a retrospective study.

Whole-exome sequencing (WES) has transformed our ability to detect mutations causing rare diseases. FORGE (Finding Of Rare disease GEnes) and Care4Rare Canada are nation-wide projects focused on identifying disease genes using WES and translating this technology to patient care. Rare forms of epilepsy are well-suited for WES and we retrospectively selected FORGE and Care4Rare families with clinical descriptions that included childhood-onset epilepsy or seizures not part of a recognizable syndrome or an early-onset encephalopathy where standard-of-care investigations were unrevealing. Nine families met these criteria and a diagnosis was made in seven, and potentially eight, of the families. In the eight families we identified mutations in genes associated with known neurological and epilepsy disorders: ASAH1, FOLR1, GRIN2A (two families), SCN8A, SYNGAP1 and SYNJ1. A novel and rare mutation was identified in KCNQ2 and was likely responsible for the benign seizures segregating in the family though additional evidence would be required to be definitive. In retrospect, the clinical presentation of four of the patients was considered atypical, thereby broadening the phenotypic spectrum of these conditions. Given the extensive clinical and genetic heterogeneity associated with epilepsy, our findings suggest that WES may be considered when a specific gene is not immediately suspected as causal.

Evidence for clinical, genetic and biochemical variability in spinal muscular atrophy with progressive myoclonic epilepsy.

Spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME) is a recently delineated, autosomal recessive condition caused by rare mutations in the N-acylsphingosine amidohydrolase 1 (acid ceramidase) ASAH1 gene. It is characterized by motor neuron disease followed by progressive myoclonic seizures and eventual death due to respiratory insufficiency. Here we report an adolescent female who presented with atonic and absence seizures and myoclonic jerks and was later diagnosed as having myoclonic-absence seizures. An extensive genetic and metabolic work-up was unable to arrive at a molecular diagnosis. Whole exome sequencing (WES) identified two rare, deleterious mutations in the ASAH1 gene: c.850G>T;p.Gly284X and c.456A>C;p.Lys152Asn. These mutations were confirmed by Sanger sequencing in the patient and her parents. Functional studies in cultured fibroblasts showed that acid ceramidase was reduced in both overall amount and enzymatic activity. Ceramide level was doubled in the patient's fibroblasts as compared to control cells. The results of the WES and the functional studies prompted an electromyography (EMG) study that showed evidence of motor neuron disease despite only mild proximal muscle weakness. These findings expand the phenotypic spectrum of SMA-PME caused by novel mutations in ASAH1 and highlight the clinical utility of WES for rare, intractable forms of epilepsy.

LRRK2 screening in a Canadian Parkinson's disease cohort.

BACKGROUND: Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) have become the most common known cause for developing Parkinson's disease. The frequency of mutations described in the literature varies widely depending on the population studied with most reports focusing only on screening for the most common G2019S mutation in exon 41. METHODS: In this study seven exons (19, 24, 25, 31, 35, 38, and 41) in LRRK2 where mutations have been reported were screened in 230 unselected Parkinson's disease patients using denaturing high-performance liquid chromatography. RESULTS: The sequencing of samples with heteroduplex profiles revealed five novel and two known intronic sequence variants. In our cohort, we were unable to detect any of the known mutations in these exons or identify novel mutations within the LRRK2 gene. CONCLUSIONS: Therefore, despite the availability of diagnostic LRRK2 genetic testing it is unlikely to yield a positive result in this population.

Neurofilament M gene in a French-Canadian population with Parkinson's disease.

BACKGROUND: Recently, a single base pair substitution (G1747A) mutation of the neurofilament M (NF-M) gene was reported in a French-Canadian patient with early onset Parkinson's disease (PD). Three unaffected siblings were found to be heterozygotes for the NF-M Gly336Ser mutation but, to date, no other affected PD individuals have been found with a similar mutation. No other individuals with Parkinson's disease and of similar ethnic background have been screened for this mutation. METHODS: We screened 102 French-Canadian patients with definite PD and 45 French-Canadian controls for this substitution in the NF-M gene using a PCR-restriction enzyme digestion method. RESULTS: None of the patients or controls carried this mutation. CONCLUSION: Our results would indicate that this mutation is not common even in a PD population of similar ethnic background and suggest this change represents a rare variant. However, these results do not exclude the possibility that other mutations in this gene could be present.

Genetic mapping of a new Lafora progressive myoclonus epilepsy locus (EPM2B) on 6p22.

BACKGROUND: Lafora disease is a progressive myoclonus epilepsy with polyglucosan accumulations and a peculiar neurodegeneration with generalised organellar disintegration. It causes severe seizures, leading to dementia and eventually death in early adulthood. METHODS: One Lafora disease gene, EPM2A, has been identified on chromosome 6q24. Locus heterogeneity led us to search for a second gene using a genome wide linkage scan in French-Canadian families. RESULTS: We mapped a second Lafora disease locus, EPM2B, to a 2.2 Mb region at 6p22, a region known to code for several proteins, including kinesins. Kinesins are microtubule dependent motor proteins that are involved in transporting cellular components. In neurones, they play a major role in axonal and dendritic transport. CONCLUSION: Analysis of the present locus in other non-EPM2A families will reveal whether there is further locus heterogeneity. Identification of the disease gene will be of major importance towards our understanding of the pathogenesis of Lafora disease.

Mutations in the epsilon-sarcoglycan gene found to be uncommon in seven myoclonus-dystonia families.

Myoclonus-dystonia syndrome (MDS) is a disorder for which the major cause appears to be mutations in the epsilon-sarcoglycan gene (SGCE). The authors have now performed mutation screening in 22 affected individuals from seven families with findings of typical MDS. A novel 5-bp deletion in exon 7 of the gene in one family and the previously reported R102X nonsense mutation in exon 3 in two other families were identified. Mutations in the SGCE gene were found in the minority of families screened in this series.

A novel locus for inherited myoclonus-dystonia on 18p11.

OBJECTIVE: Inherited myoclonus-dystonia (IMD) is a new term for an autosomal dominant disorder characterized by myoclonus and dystonia. Recently, IMD was linked to a region on chromosome 11q23 with two different mutations identified in the D2 dopamine receptor gene and linked to chromosome 7q with five different loss-of-function mutations identified in the epsilon-sarcoglycan gene. METHODS: These two regions and genes were excluded in a large Canadian family with IMD in whom 13 individuals are affected. A 25-cM genome scan of this large family with 32 individuals was performed. RESULTS: Two-point linkage analysis revealed a maximum lod score of 3.5 (recombination fraction 0.00; affected only) for the microsatellite marker GATA185C06-18 and a multipoint lod score of 3.9 across the 18p11 region. Haplotype analysis demonstrates that all the affected individuals shared a common haplotype between markers D18S1132 and D18S843 that defines the disease gene within a span of 16.9 cM. CONCLUSIONS: These findings indicate that a novel IMD gene exists on chromosome 18p11.

Parkinson's genetics--creating exciting new insights.

Parkinson's disease is a complex disorder in which the genetic aspects are only just being realized. The underlying cause for the degeneration of dopaminergic substantia nigra neurons and the formation of Lewy bodies in Parkinson's disease is unknown. The identification of clear inherited forms of the disease has provided important clues as to how this complex process may be occurring. Mutations have now been identified in the alpha-synuclein (4q21.3-23), parkin (6q25.2-27), and ubiquitin carboxy terminal hydrolase-L1 (4p16.3) genes in families with Parkinson's disease. Four additional chromosomal locations; 2p13, 4p14-15, 1p35-36, and 12p11.2-q13.1 have been linked to Parkinson's disease families but no pathologic gene mutations have been identified to date. As additional Parkinson's disease loci are mapped and their genes identified we will continue to add to our understating of the critical biochemical pathways involved and be able to develop effective disease altering treatments.

Clinical and radiological assessment of a family with mild brachydactyly type A1: the usefulness of metacarpophalangeal profiles.

The brachydactylies are a group of conditions in which various subtypes have been defined based upon the specific pattern of digital bones involved. Type A1 brachydactyly is principally characterised by maximal involvement of the middle phalanges. We report an extended family with a mild brachydactyly A1 which was, except for some short stature, not associated with any of the additional clinical findings reported in several published families. While all the hand bones tended to be small, the principal features of the affected members were shortened middle and distal phalanges, proximal 1st phalanges, and 5th metacarpals. The feet were similarly involved and tended to have a broad, slightly adducted forefoot. The two affected children showed multiple coned epiphyses. This paper provides a detailed description of the family including the radiographic signs and metacarpophalangeal profiles, which proved to be useful in distinguishing the mildly affected persons.

Evidence favoring genetic heterogeneity for febrile convulsions.

PURPOSE: Two large Canadian kindreds appearing to segregate febrile convulsions as an autosomal dominant trait were evaluated for linkage to three known FC loci, as well as other epilepsy loci. METHODS: Members of the two families were genotyped with microsatellite markers linked to the previously identified febrile convulsion loci, FEB1, FEB2, and GEFS+, and we performed two-point linkage analyses by assuming an autosomal dominant mode of inheritance. RESULTS: We report the exclusion of the FC trait in our families to FEB1 on 8q13-21 and to a second febrile convulsion locus on 19p13. Furthermore, we also excluded the GEFS+ locus on 19q13.1 as the cause of febrile convulsions in both kindreds. Microsatellite markers linked to juvenile myoclonic epilepsy (EJM1), benign neonatal familial convulsions EBN1 and EBN2, autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), idiopathic generalized epilepsy (EGI), progressive myoclonic epilepsy of Unverricht-Lundborg (EPM1), and partial epilepsy with auditory features (EPT), were also excluded as potential loci linked to the FC trait in our families. CONCLUSIONS: These findings favor considerable genetic heterogeneity for febrile convulsions.

The human skeletal muscle Na channel mutation R669H associated with hypokalemic periodic paralysis enhances slow inactivation.

Missense mutations of the human skeletal muscle voltage-gated Na channel (hSkM1) underlie a variety of diseases, including hyperkalemic periodic paralysis (HyperPP), paramyotonia congenita, and potassium-aggravated myotonia. Another disorder of sarcolemmal excitability, hypokalemic periodic paralysis (HypoPP), which is usually caused by missense mutations of the S4 voltage sensors of the L-type Ca channel, was associated recently in one family with a mutation in the outermost arginine of the IIS4 voltage sensor (R669H) of hSkM1 (Bulman et al., 1999). Intriguingly, an arginine-to-histidine mutation at the homologous position in the L-type Ca(2+) channel (R528H) is a common cause of HypoPP. We have studied the gating properties of the hSkM1-R669H mutant Na channel experimentally in human embryonic kidney cells and found that it has no significant effects on activation or fast inactivation but does cause an enhancement of slow inactivation. R669H channels exhibit an approximately 10 mV hyperpolarized shift in the voltage dependence of slow inactivation and a twofold to fivefold prolongation of recovery after prolonged depolarization. In contrast, slow inactivation is often disrupted in HyperPP-associated Na channel mutants. These results demonstrate that, in R669H-associated HypoPP, enhanced slow inactivation does not preclude, and may contribute to, prolonged attacks of weakness and add support to previous evidence implicating the IIS4 voltage sensor in slow-inactivation gating.

A novel sodium channel mutation in a family with hypokalemic periodic paralysis.

OBJECTIVE: To identify the cause of hypokalemic periodic paralysis (HOKPP) in a family whose disease is not caused by a mutation in the dihydropyridine-sensitive (DHP) receptor alpha1-subunit gene (CACNA1S). BACKGROUND: Hypokalemic periodic paralysis is primarily caused by mutations within CACNA1S. Genetic heterogeneity for HOKPP has been reported, but no other locus has been identified. METHODS: Single-stranded conformational polymorphism (SSCP) analysis and PCR direct sequencing were used to screen the skeletal muscle alpha1-sodium channel gene (SCN4A) for a mutation in our family. RESULTS: SSCP analysis showed an abnormally migrating conformer in exon 12. Direct sequencing of the conformer showed a guanine to adenine transition at position 2006 in the cDNA sequence; this results in an amino acid substitution of a highly conserved arginine (Arg) to histidine (His) at position 669. This sequence alteration segregated only with the affected members of the kindred and was not found in a panel of 100 DNA samples from healthy controls. The amino acid substitution alters the outermost positive charge in the membrane spanning segment DII/S4, which is involved in voltage sensing. CONCLUSIONS: The first arginine in DII/S4 and in DIV/S4 within the skeletal muscle sodium channel and the L-type calcium channel genie CACNA1S appear to be critical for normal function. In all four cases, Arg to His mutations result in a disease phenotype. The identification of a mutation within the skeletal muscle sodium channel resulting in hypokalemic periodic paralysis represents a novel finding.