Leigh syndrome is a mitochondrial disease caused by pathogenic variants in over 85 genes. Whole exome sequencing of a patient with Leigh-like syndrome identified homozygous protein-truncating variants in two genes associated with Leigh syndrome; a reported pathogenic variant in PDHX (NP_003468.2:p.(Arg446*)), and an uncharacterized variant in complex I (CI) assembly factor TIMMDC1 (NP_057673.2:p.(Arg225*)). The TIMMDC1 variant was predicted to truncate 61 amino acids at the C-terminus and functional studies demonstrated a hypomorphic impact of the variant on CI assembly. However, the mutant protein could still rescue CI assembly in TIMMDC1 knockout cells and the patient's clinical phenotype was not clearly distinct from that of other patients with the same PDHX defect. Our data suggest that the hypomorphic effect of the TIMMDC1 protein-truncating variant does not constitute a dual diagnosis in this individual. We recommend cautious assessment of variants in the C-terminus of TIMMDC1 and emphasize the need to consider the caveats detailed within the American College of Medical Genetics and Genomics (ACMG) criteria when assessing variants.

Cognitive impairment is a core feature of schizophrenia and impacts negatively the functioning of affected individuals. Cognitive decline correlates with aging, and is the primary cause of loss of independence and reduced quality of life. The klotho gene is a key modulator of aging, with expression deficiency resulting in premature aging, while overexpression extends lifespan and enhances cognition. A haplotype and functional human variant of the gene, KL-VS, increases expression and promotes longevity. KL-VS heterozygosity is associated with enhanced cognition and a larger volume of the right dorsolateral prefrontal cortex, a region involved in planning and decision-making, which is especially susceptible to shrinkage with age. We examined the effect of KL-VS heterozygosity on cognition in 497 schizophrenia patients and 316 healthy controls from the Western Australian Family Study of Schizophrenia (WAFSS) who had been comprehensively characterised by neurocognitive tests and classified into cognitively deficient (CD) and cognitively "spared" (CS) clusters. An older, cognitively normal population sample from the Health in Men Study (HIMS) was included to allow assessment of heterozygosity and memory in aged individuals. We show that heterozygosity is associated with better learning and memory in the younger WAFSS healthy controls but not in the aging HIMS sample. However, in schizophrenia patients, KL-VS has a selective effect on memory, with heterozygotes in CD and CS clusters performing worse than non-carriers. This effect was significant and more severe in the CD cluster, reinforcing the utility of subtyping patients into CD and CS clusters that may differ in their genetic underpinnings.

The importance of genomic copy number variants (CNVs) has long been recognized in the etiology of neurodevelopmental diseases. We report here the results from the CNV analysis of whole-genome sequences from 91 multiplex schizophrenia families. Employing four algorithms (CNVnator, Cn.mops, DELLY and LUMPY) to identify CNVs, we find 1231 rare deletions and 287 rare duplications in 300 individuals (77 with schizophrenia (SZ), 32 with schizoaffective disorder (SAD), 82 with another neuropsychiatric diagnosis and 109 unaffected). The size of the CNVs ranges from a few hundred base-pairs to about 1.3Mb. The total burden of CNVs does not differ significantly between affected (SZ and SAD) and unaffected individuals. Parent-to-child transmission rate for rare CNVs affecting exonic regions is significantly higher for affected (SZ and SAD) probands as compared to their siblings, but rates for all CNVs is not. We observe heterogeneity between families in terms of genes involved in CNVs, and find several CNVs involving genes previously implicated in either schizophrenia or other neuropsychiatric disorders.

Objective To describe a large series of BIN1 patients, in which a novel founder mutation in the Roma population of southern Spain has been identified. Methods Patients diagnosed with centronuclear myopathy (CNM) at 5 major reference centers for neuromuscular disease in Spain (n = 53) were screened for BIN1 mutations. Clinical, histologic, radiologic, and genetic features were analyzed. Results Eighteen patients from 13 families carried the p.Arg234Cys variant; 16 of them were homozygous for it and 2 had compound heterozygous p.Arg234Cys/p.Arg145Cys mutations. Both BlIV1 variants have only been identified in Roma, causing 100% of CNM in this ethnic group in our cohort. The haplotype analysis confirmed all families are related. In addition to clinical features typical of CNM, such as proximal limb weakness and ophthalmoplegia, most patients in our cohort presented with prominent axial weakness, often associated with rigid spine. Severe fat replacement of paravertebral muscles was demonstrated by muscle imaging. This phenotype seems to be specific to the p.Arg234Cys mutation, not reported in other BIN1 mutations. Extreme clinical variability was observed in the 2 compound heterozygous patients for the p.Arg234Cys/p.Arg145Cys mutations, from a congenital onset with catastrophic outcome to a late-onset disease. Screening of European Roma controls (n = 758) for the p.Arg234Cys variant identified a carrier frequency of 3.5% among the Spanish Roma. Conclusion We have identified a BIN1 founder Roma mutation associated with a highly specific phenotype, which is, from the present cohort, the main cause of CNM in Spain.

The exome array assays rare-but-recurrent, likely deleterious, exonic variants and represents an intermediary between single nucleotide polymorphism (SNP) arrays and sequencing for genetic association studies. Multiplex families with multiple affected individuals may be enriched for disease-associated variants of this class compared to unrelated populations. We present an exome array study of schizophrenia in 99 multiplex families (n=341, including 118 cases) from the Western Australian Family Study of Schizophrenia (WAFSS). Compared to 55,726 individuals from the DIAGRAM sample not selected for schizophrenia, overall allele frequency of exome variants was higher in the WAFSS (P<2.2E-16). This was pronounced in variants nominally associated (P<0.05) with schizophrenia. Genes harbouring variants present only in WAFSS cases were enriched (FDR-corrected P=0.05) for membership of the 'extracellular matrix (ECM) - receptor interaction' biological pathway, adding to evidence that processes affecting the composition or turnover of ECM may contribute to neuropsychiatric disease. We did not find individual variants significantly associated with schizophrenia, although like previous studies, power to detect associations of small effect size was low. Cases did not exhibit a higher burden of variants compared to their unaffected relatives and the finding of previous exome chip studies of unrelated samples that 'schizophrenia gene-sets' were enriched for case-only variants was not replicated in the WAFSS. The higher frequency of moderately rare, exonic variants in these multiplex families compared to a population-based sample may account for some of their genetic liability to schizophrenia, and adds to evidence for a role of exome array variants from previous studies of unrelated samples.

BIN1 mutations are a rare cause of dominant and recessive centronuclear myopathies. We have identified a founder BIN1 c.700C > T (p.C234R) mutation in the gypsy Spanish population present in 13 patients with autosomal recessive centronuclear myopathy belonging to 8 families from Andalucía (10p), Valencia (2p) and Cataluña (1p). Eleven patients are homozygous for the BIN1 c.700C > T mutation and two of them have compound heterozygous mutations with a BIN1 c.433C > T. The haplotype analysis confirmed a common origin for the founder mutation in this population group. In addition to proximal weakness ptosis and ophthalmoplegia, the most common clinical signs included axial weakness and rigid spine, which seems to be specific to the mutation, as it has not been described in association with other BIN1 mutations. In addition, compound heterozygous BIN1 c.433C > T and c.700C > T mutations were identified in a severe congenital myotubular myopathy. To establish the prevalence of the mutation, we reviewed all muscle biopsies diagnosed with a centronuclear myopathy excluding myotubular myopathies in the last 10 years in four of the main reference centres of myopathology in Spain. Twenty-one cases were identified. Eight patients were gypsies, eight were of European non-gypsy ethnicity and in five the ethnic origin could not be determined. All gypsy patients carried the BIN1 c.700C > T mutation in homozygosity or as a compound heterozygous mutation with c.433 C > T. These eight patients are included in the previously described 13 patients. Neither the European non-gypsy nor the patients of unknown ethnic origin had the mutation. In normal gypsy controls (N = 718) a 5% of heterozygous carriers was identified among the Spanish gypsies and 0% in European non-Spanish gypsies. In conclusion, we have identified a BIN1 founder mutation in the Gypsy Spanish population which is the most frequent centronuclear myopathy cause identified in the screened populations in Spain.

Introduction: Klotho variants (KL-VS) have been associated with increased longevity and better cognitive function. It is unclear whether they modulate dementia risk.Methods: We recruited 527 men aged 71-87 years who were free of cognitive impairment. We used data linkage to track the onset of dementia over 10 years. KL-VS genotyping (rs9536314 T/G) followed standard procedures.Results: The annual rate of dementia was 17.2%o (95%CI = 14.0-21.1; total = 5053 person-years), and 14.0%o (95%CI = 10.6-18.4; 3582 person-years), 23.5%o (95%CI = 16.6-33.2; 1363 person-years) and 46.4%o (95%CI = 19.3-111.5; 108 person-years) for men with the TT, TG and GG genotypes. Compared with the TT genotype, the sub-hazard ratios of dementia associated with the TG and GG genotypes were 1.6 (95%CI = 1.0, 2.5; p = 0.030) and 3.5 (95%CI = 1.3, 9.1; p = 0.011).Discussion: The Klotho KL-VS variant is associated with an increase in the incidence of dementia in older men, in a dose-dependent fashion (intermediate for heterozygosis and highest for homozygosis).

Objective: To identify the gene defect in patients with hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) who are negative for TUBB4A mutations.Methods: We performed homozygosity mapping and whole exome sequencing (WES) to detect the disease-causing variant. We used a Taqman assay for population screening. We developed a luciferase reporter construct to investigate the effect of the promoter mutation on expression.Results: Sixteen patients from 14 families from different countries fulfilling the MRI criteria for H-ABC exhibited a similar, severe clinical phenotype, including lack of development and a severe epileptic encephalopathy. The majority of patients had a known Roma ethnic background. Single nucleotide polymorphism array analysis in 5 patients identified one large overlapping homozygous region on chromosome 13. WES in 2 patients revealed a homozygous deletion in the promoter region of UFM1. Sanger sequencing confirmed homozygosity for this variant in all 16 patients. All patients shared a common haplotype, indicative of a founder effect. Screening of 1,000 controls from different European Roma panels demonstrated an overall carrier rate of the mutation of 3%-25%. Transfection assays showed that the deletion significantly reduced expression in specific CNS cell lines.Conclusions: UFM1 encodes ubiquitin-fold modifier 1 (UFM1), a member of the ubiquitin-like family involved in posttranslational modification of proteins. Its exact biological role is unclear. This study associates a UFM1 gene defect with a disease and sheds new light on possible UFM1 functional networks.

RNA polymerase III is essential for the transcription of non-coding RNAs, including tRNAs. Mutations in the genes encoding its largest subunits are known to cause hypomyelinating leukodystrophies (HLD7) with pathogenetic mechanisms hypothesised to involve impaired availability of tRNAs. We have identified a founder mutation in the POLR3A gene that leads to aberrant splicing, a premature termination codon and partial deficiency of the canonical full-length transcript. Our clinical and imaging data showed no evidence of the previously reported white matter or cerebellar involvement; instead the affected brain structures included the striatum and red nuclei with the ensuing clinical manifestations. Our transcriptome-wide investigations revealed an overall decrease in the levels of Pol III-transcribed tRNAs and an imbalance in the levels of regulatory ncRNAs such as small nuclear and nucleolar RNAs (snRNAs and snoRNAs). In addition, the Pol III mutation was found to exert complex downstream effects on the Pol II transcriptome, affecting the general regulation of RNA metabolism.

We compared genotype data from the HumanExomeCore Array in peripheral blood mononuclear cells and low passage lymphoblastoid cell lines from the same 24 individuals to test for genotypic errors caused by the Epstein–Barr Virus transformation process. Genotype concordance across the 24 comparisons was 99.57% for unfiltered genotype data, and 99.63% following standard genotype quality control filters. Mendelian error rates and levels of heterozygosity were not significantly different between lymphoblastoid cell lines and their parent peripheral blood mononuclear cells. These results show that at low passage numbers, genotype discrepancies are minimal even before stringent quality control, and extend current evidence qualifying the use of low-passage lymphoblastoid cell lines as a reliable DNA source for genotype analysis.

Renal hypodysplasia (RHD) is a heterogeneous condition encompassing a spectrum of kidney development defects including renal agenesis, hypoplasia, and (cystic) dysplasia. Heterozygous mutations of several genes have been identified as genetic causes of RHD with various severity. However, these genes and mutations are not associated with bilateral renal agenesis, except for RET mutations, which could be involved in a few cases. The pathophysiological mechanisms leading to total absence of kidney development thus remain largely elusive. By using a whole-exome sequencing approach in families with several fetuses with bilateral renal agenesis, we identified recessive mutations in the integrin alpha 8-encoding gene ITGA8 in two families. Itga8 homozygous knockout in mice is known to result in absence of kidney development. We provide evidence of a damaging effect of the human ITGA8 mutations. These results demonstrate that mutations of ITGA8 are a genetic cause of bilateral renal agenesis and that, at least in some cases, bilateral renal agenesis is an autosomal-recessive disease.

Investigation of 31 of Roma patients with congenital lactic acidosis (CLA) from Bulgaria identified homozygosity for the R446* mutation in the PDHX gene as the most common cause of the disorder in this ethnic group. It accounted for around 60% of patients in the study and over 25% of all CLA cases referred to the National Genetic Laboratory in Bulgaria. The detection of a homozygous patient from Hungary and carriers among population controls from Romania and Slovakia suggests a wide spread of the mutation in the European Roma population. The clinical phenotype of the twenty R446* homozygotes was relatively homogeneous, with lactic acidosis crisis in the first days or months of life as the most common initial presentation (15/20 patients) and delayed psychomotor development and/or seizures in infancy as the leading manifestations in a smaller group (5/20 patients). The subsequent clinical picture was dominated by impaired physical growth and a very consistent pattern of static cerebral palsy-like encephalopathy with spasticity and severe to profound mental retardation seen in over 80% of cases. Most patients had a positive family history. We propose testing for the R446* mutation in PDHX as a rapid first screening in Roma infants with metabolic acidosis. It will facilitate and accelerate diagnosis in a large proportion of cases, allow early rehabilitation to alleviate the chronic clinical course, and prevent further affected births in high-risk families.

Abstract The Roma, also known as Gypsies, are a transnational founder population, resembling a mosaic of socially and genetically divergent groups. Common origins from a small group of related founders result in sharing of maternal and paternal lineages and of ancient disease‐causing mutations across endogamous Romani groups in different countries. Genetic differentiation between groups, with an impact on genetic epidemiology, is the product of bottleneck events, random genetic drift and differential admixture, and correlates with the migrational history of the Roma in Europe.

This chapter focuses on human claims to land in Madagascar, and the shifting relationship over time between the island's peoples and the environment. It also discusses how haploid DNA markers have contributed in tracing the genetic trail to the geographic region of origin of the mtDNA and Y chromosome DNA found in living people in Madagascar. In addition, the chapter examines the use of a few autosomal DNA markers in assessing population affinity and gene admixture in the Malagasy. Before 1990, few genetic studies focused on elucidating the origins of the Malagasy. Colleagues in Jenkins' laboratory investigated the distribution of the sickle-cell trait adopting a DNA approach that, through restriction fragment length polymorphisms (RFLPs), examined genetic variation within the beta-globin gene. The Y chromosome contains the largest non-recombining block in the human genome and is therefore extremely important for evolutionary genetic studies. Keywords: beta-globin gene; genetic trail; haploid DNA markers; Jenkins' laboratory; Madagascar; Y chromosome DNA

Recessive ataxias are heterogeneous group of rare monogenic disorders with genetic pathology largely unresolved.

Autosomal-recessive congenital cerebellar ataxia was identified in Roma patients originating from a small subisolate with a known strong founder effect. Patients presented with global developmental delay, moderate to severe stance and gait ataxia, dysarthria, mild dysdiadochokinesia, dysmetria and tremors, intellectual deficit, and mild pyramidal signs. Brain imaging revealed progressive generalized cerebellar atrophy, and inferior vermian hypoplasia and/or a constitutionally small brain were observed in some patients. Exome sequencing, used for linkage analysis on extracted SNP genotypes and for mutation detection, identified two novel (i.e., not found in any database) variants located 7 bp apart within a unique 6q24 linkage region. Both mutations cosegregated with the disease in five affected families, in which all ten patients were homozygous. The mutated gene, GRM1, encodes metabotropic glutamate receptor mGluR1, which is highly expressed in cerebellar Purkinje cells and plays an important role in cerebellar development and synaptic plasticity. The two mutations affect a gene region critical for alternative splicing and the generation of receptor isoforms; they are a 3 bp exon 8 deletion and an intron 8 splicing mutation (c.2652_2654del and c.2660+2T>G, respectively [RefSeq accession number NM_000838.3]). The functional impact of the deletion is unclear and is overshadowed by the splicing defect. Although ataxia lymphoblastoid cell lines expressed GRM1 at levels comparable to those of control cells, the aberrant transcripts skipped exon 8 or ended in intron 8 and encoded various species of nonfunctional receptors either lacking the transmembrane domain and containing abnormal intracellular tails or completely missing the tail. The study implicates mGluR1 in human hereditary ataxia. It also illustrates the potential of the Roma founder populations for mutation identification by exome sequencing.