Categorized Genetic Analysis in Childhood-Onset Cardiomyopathy.

BACKGROUND: Childhood-onset cardiomyopathy is a heterogeneous group of conditions the cause of which is largely unknown. The influence of consanguinity on the genetics of cardiomyopathy has not been addressed at a large scale. METHODS: To unravel the genetic cause of childhood-onset cardiomyopathy in a consanguineous population, a categorized approach was adopted. Cases with childhood-onset cardiomyopathy were consecutively recruited. Based on the likelihood of founder mutation and on the clinical diagnosis, genetic test was categorized to either (1) targeted genetic test with targeted mutation test, single-gene test, or multigene panel for Noonan syndrome, or (2) untargeted genetic test with whole-exome sequencing or whole-genome sequencing. Several bioinformatics tools were used to filter the variants. RESULTS: Two-hundred five unrelated probands with various forms of cardiomyopathy were evaluated. The median age of presentation was 10 months. In 30.2% (n=62), targeted genetic test had a yield of 82.7% compared with 33.6% for whole-exome sequencing/whole-genome sequencing (n=143) giving an overall yield of 53.7%. Strikingly, 96.4% of the variants were homozygous, 9% of which were found in 4 dominant genes. Homozygous variants were also detected in 7 novel candidates (ACACB, AASDH, CASZ1, FLII, RHBDF1, RPL3L, ULK1). CONCLUSIONS: Our work demonstrates the impact of consanguinity on the genetics of childhood-onset cardiomyopathy, the value of adopting a categorized population-sensitive genetic approach, and the opportunity of uncovering novel genes. Our data suggest that if a founder mutation is not suspected, adopting whole-exome sequencing/whole-genome sequencing as a first-line test should be considered.

The morbid genome of ciliopathies: an update.

PURPOSE: Ciliopathies are highly heterogeneous clinical disorders of the primary cilium. We aim to characterize a large cohort of ciliopathies phenotypically and molecularly. METHODS: Detailed phenotypic and genomic analysis of patients with ciliopathies, and functional characterization of novel candidate genes. RESULTS: In this study, we describe 125 families with ciliopathies and show that deleterious variants in previously reported genes, including cryptic splicing variants, account for 87% of cases. Additionally, we further support a number of previously reported candidate genes (BBIP1, MAPKBP1, PDE6D, and WDPCP), and propose nine novel candidate genes (CCDC67, CCDC96, CCDC172, CEP295, FAM166B, LRRC34, TMEM17, TTC6, and TTC23), three of which (LRRC34, TTC6, and TTC23) are supported by functional assays that we performed on available patient-derived fibroblasts. From a phenotypic perspective, we expand the phenomenon of allelism that characterizes ciliopathies by describing novel associations including WDR19-related Stargardt disease and SCLT1- and CEP164-related Bardet-Biedl syndrome. CONCLUSION: In this cohort of phenotypically and molecularly characterized ciliopathies, we draw important lessons that inform the clinical management and the diagnostics of this class of disorders as well as their basic biology.

Whole Exome Sequencing Identifies Three Novel Mutations in the ASPM Gene From Saudi Families Leading to Primary Microcephaly.

Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental defect that is characterized by reduced head circumference at birth along with non-progressive intellectual disability. Till date, 25 genes related to MCPH have been reported so far in humans. The ASPM (abnormal spindle-like, microcephaly-associated) gene is among the most frequently mutated MCPH gene. We studied three different families having primary microcephaly from different regions of Saudi Arabia. Whole exome sequencing (WES) and Sanger sequencing were done to identify the genetic defect. Collectively, three novel variants were identified in the ASPM gene from three different primary microcephaly families. Family 1, showed a deletion mutation leading to a frameshift mutation c.1003del. (p.Val335*) in exon 3 of the ASPM gene and family 2, also showed deletion mutation leading to frameshift mutation c.1047del (p.Gln349Hisfs*18), while in family 3, we identified a missense mutation c.5623A>G leading to a change in protein (p.Lys1875Glu) in exon 18 of the ASPM gene underlying the disorder. The identified respective mutations were ruled out in 100 healthy control samples. In conclusion, we found three novel mutations in the ASPM gene in Saudi families that will help to establish a disease database for specified mutations in Saudi population and will further help to identify strategies to tackle primary microcephaly in the kingdom.

Identification of TMC1 as a relatively common cause for nonsyndromic hearing loss in the Saudi population.

Hearing loss (HL) is the most common sensory disorder worldwide and genetic factors contribute to approximately half of congenital HL cases. HL is subject to extensive genetic heterogeneity, rendering molecular diagnosis difficult. Mutations of the transmembrane channel-like 1 (TMC1) gene cause hearing defects in humans and mice. The precise function of TMC1 protein in the inner ear is unknown, although it is predicted to be involved in functional maturation of cochlear hair cells. TMC1 mutations result in autosomal recessive (DFNB7/11) and sometimes dominant (DFNA36) nonsyndromic HL. Mutations in TMC1 are responsible for a significant portion of HL, particularly in consanguineous populations. To evaluate the importance of TMC1 mutations in the Saudi population, we used a combination of autozygome-guided candidate gene mutation analysis and targeted next generation sequencing in 366 families with HL previously shown to lack mutations in GJB2. We identified 12 families that carried five causative TMC1 mutations; including three novel (c.362+3A > G; c.758C > T [p.Ser253Phe]; c.1396_1398delACC [p.Asn466del]) and two reported mutations (c.100C > T [p.Arg34Ter]; c.1714G > A [p.Asp572Asn]). Each of the identified recessive mutation was classified as severe, by both age of onset and severity of HL. Similarly, consistent with the previously reported dominant variant p.Asp572Asn, the HL phenotype was progressive. Eight families in our cohort were found to share the pathogenic p.Arg34Ter mutation and linkage disequilibrium was observed between p.Arg34Ter and SNPs investigated. Our results indicate that TMC1 mutations account for about 3.3% (12/366) of Saudi HL cases and that the recurrent TMC1 mutation p.Arg34Ter is likely to be a founder mutation.

Chromosomal Micro-aberration in a Saudi Family with Juvenile Myoclonic Epilepsy.

BACKGROUND AND OBJECTIVE: Epilepsy is etiologically and genetically complex neurological disorder affecting millions of people worldwide. Juvenile myoclonic epilepsy (JME) is the most common epilepsy syndrome that starts in the teen age group commonly between ages 12, 18, and lasts till adulthood. One out of fourteen people with epilepsy suffers with JME. Myoclonic seizures and muscle twitching or uncontrolled jerking are the most common type of seizures in the people suffering with JME. METHOD: To observe the novel CNVs involved in JME, we investigated a Saudi family with nine siblings including one male and one female affected members. In this study we used high density whole genome Agilent sure print G3 Hmn CGH 2x 400K array-CGH chips. Our results showed CNVs including the amplifications and deletions in different chromosomal regions in the patients as compared to the normal members of the family. Amplifications were observed in the chromosome 22 cytoband 22q11.23 with LDL receptor related protein 5 like (LRP5L), Immunoglobulin Lambda-Like Polypeptide 3 (IGLL3) and crystallin beta B2 pseudogene (CRYBB2P) genes respectively whereas the deletions were observed in the chromosomal regions 4q22.2 with Glutamate receptor, ionotropic, delta 2 (GRID2) as potential gene cytoband 1p31.1 with potential Neuronal Growth Regulator 1 gene (NEGR1) gene in this region and NME/NM23 family member (NME7) gene cytoband 1q24. Moreover, the array CGH resulting in deletions and duplication were also validated by using primer for simple PCR or also by using quantitative real time PCR analysis. We found deletions and duplication in JME patients in our study for the first time in Saudi population. RESULTS & CONCLUSION: The findings in this study suggest that the array-CGH may be considered as a first line of genetic testing for diagnosis of epilepsy unless strong evidence is presented for a monogenic syndrome. The use of high throughput technique in this study will help to identify novel mechanisms underlying epileptic disorder in order to lower the burden of epilepsy in Saudi Arabia.

The genetic landscape of familial congenital hydrocephalus.

OBJECTIVE: Congenital hydrocephalus is an important birth defect, the genetics of which remains incompletely understood. To date, only 4 genes are known to cause Mendelian diseases in which congenital hydrocephalus is the main or sole clinical feature, 2 X-linked (L1CAM and AP1S2) and 2 autosomal recessive (CCDC88C and MPDZ). In this study, we aimed to determine the genetic etiology of familial congenital hydrocephalus with the assumption that these cases represent Mendelian forms of the disease. METHODS: Exome sequencing combined, where applicable, with positional mapping. RESULTS: We identified a likely causal mutation in the majority of these families (21 of 27, 78%), spanning 16 genes, none of which is X-linked. Ciliopathies and dystroglycanopathies were the most common etiologies of congenital hydrocephalus in our cohort (19% and 26%, respectively). In 1 family with 4 affected members, we identified a homozygous truncating variant in EML1, which we propose as a novel cause of congenital hydrocephalus in addition to its suggested role in cortical malformation. Similarly, we show that recessive mutations in WDR81, previously linked to cerebellar ataxia, mental retardation, and disequilibrium syndrome 2, cause severe congenital hydrocephalus. Furthermore, we confirm the previously reported candidacy of MPDZ by presenting a phenotypic spectrum of congenital hydrocephalus associated with 5 recessive alleles. INTERPRETATION: Our study highlights the importance of recessive mutations in familial congenital hydrocephalus and expands the locus heterogeneity of this condition. Ann Neurol 2017;81:890-897.

A novel WDR62 mutation causes primary microcephaly in a large consanguineous Saudi family.

BACKGROUND: Primary microcephaly (MCPH) is a rare developmental defect characterized by impaired cognitive functions, retarded neurodevelopment and reduced brain size. It is genetically heterogeneous and more than 17 genes so far have been identified that are associated with this disease. OBJECTIVE: To study the genetic defect in a consanguineous Saudi family with primary microcephaly. DESIGN: Cross-sectional clinical genetics study of a Saudi family. SETTING: Medical genomics research center. PATIENTS AND METHODS: Blood samples collected from six members of a family of healthy consanguineous parents were analyzed by whole exome sequencing to identify the underlying pathogenic mutations in two members of the family (23-year-old female and 7-year-old male) who presented with primary microcephaly, intellectual disability, delayed psychomotor development and walking difficulty, speech impedi-ments and seizures. MAIN OUTCOME MEASURE(S): Detection of mutation in the WD repeat domain 62 (WDR62) gene in a family segregating autosomal recessive primary microcephaly. RESULTS: The exome variant analysis identified a novel missense mutation (c.3878C > A) in WDR62 gene in exon 30 resulting in amino acid change from alanine to aspartate (p.Ala1293Asp). Further validation in the affected patients and healthy members of family and 100 unrelated healthy persons as controls confirmed it to be pathogenic. CONCLUSIONS: Functional impairment of the WDR62 gene can lead to severe neurodevelopmental de-fects, brain malformations and reduced head size. A missense mutation of exon 30 changed alanine to aspartate in the WDR62 protein leading to the typical MCPH phenotype. LIMITATIONS: Mutation was identified in a single family.

Microcephaly-capillary malformation syndrome: Brothers with a homozygous STAMBP mutation, uncovered by exome sequencing.

We describe two brothers from a consanguineous family of Egyptian ancestry, presenting with microcephaly, apparent global developmental delay, seizures, spasticity, congenital blindness, and multiple cutaneous capillary malformations. Through exome sequencing, we uncovered a homozygous missense variant in STAMBP (p.K303R) in the two siblings, inherited from heterozygous carrier parents. Mutations in STAMBP are known to cause microcephaly-capillary malformation syndrome (MIC-CAP) and the phenotype in this family is consistent with this diagnosis. We compared the findings in the present brothers with those of earlier reported patients. © 2016 Wiley Periodicals, Inc.

Accelerating novel candidate gene discovery in neurogenetic disorders via whole-exome sequencing of prescreened multiplex consanguineous families.

Our knowledge of disease genes in neurological disorders is incomplete. With the aim of closing this gap, we performed whole-exome sequencing on 143 multiplex consanguineous families in whom known disease genes had been excluded by autozygosity mapping and candidate gene analysis. This prescreening step led to the identification of 69 recessive genes not previously associated with disease, of which 33 are here described (SPDL1, TUBA3E, INO80, NID1, TSEN15, DMBX1, CLHC1, C12orf4, WDR93, ST7, MATN4, SEC24D, PCDHB4, PTPN23, TAF6, TBCK, FAM177A1, KIAA1109, MTSS1L, XIRP1, KCTD3, CHAF1B, ARV1, ISCA2, PTRH2, GEMIN4, MYOCD, PDPR, DPH1, NUP107, TMEM92, EPB41L4A, and FAM120AOS). We also encountered instances in which the phenotype departed significantly from the established clinical presentation of a known disease gene. Overall, a likely causal mutation was identified in >73% of our cases. This study contributes to the global effort toward a full compendium of disease genes affecting brain function.

IFT27, encoding a small GTPase component of IFT particles, is mutated in a consanguineous family with Bardet-Biedl syndrome.

Bardet-Biedl syndrome (BBS) is an autosomal recessive ciliopathy with multisystem involvement. So far, 18 BBS genes have been identified and the majority of them are essential for the function of BBSome, a protein complex involved in transporting membrane proteins into and from cilia. Yet defects in the identified genes cannot account for all the BBS cases. The genetic heterogeneity of this disease poses significant challenge to the identification of additional BBS genes. In this study, we coupled human genetics with functional validation in zebrafish and identified IFT27 as a novel BBS gene (BBS19). This is the first time an intraflagellar transport (IFT) gene is implicated in the pathogenesis of BBS, highlighting the genetic complexity of this disease.

Genomic analysis of primordial dwarfism reveals novel disease genes.

Primordial dwarfism (PD) is a disease in which severely impaired fetal growth persists throughout postnatal development and results in stunted adult size. The condition is highly heterogeneous clinically, but the use of certain phenotypic aspects such as head circumference and facial appearance has proven helpful in defining clinical subgroups. In this study, we present the results of clinical and genomic characterization of 16 new patients in whom a broad definition of PD was used (e.g., 3M syndrome was included). We report a novel PD syndrome with distinct facies in two unrelated patients, each with a different homozygous truncating mutation in CRIPT. Our analysis also reveals, in addition to mutations in known PD disease genes, the first instance of biallelic truncating BRCA2 mutation causing PD with normal bone marrow analysis. In addition, we have identified a novel locus for Seckel syndrome based on a consanguineous multiplex family and identified a homozygous truncating mutation in DNA2 as the likely cause. An additional novel PD disease candidate gene XRCC4 was identified by autozygome/exome analysis, and the knockout mouse phenotype is highly compatible with PD. Thus, we add a number of novel genes to the growing list of PD-linked genes, including one which we show to be linked to a novel PD syndrome with a distinct facial appearance. PD is extremely heterogeneous genetically and clinically, and genomic tools are often required to reach a molecular diagnosis.

Genomic analysis of Meckel-Gruber syndrome in Arabs reveals marked genetic heterogeneity and novel candidate genes.

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.

A comprehensive introduction to the genetic basis of non-syndromic hearing loss in the Saudi Arabian population.

BACKGROUND: Hearing loss is a clinically and genetically heterogeneous disorder. Mutations in the DFNB1 locus have been reported to be the most common cause of autosomal recessive non-syndromic hearing loss worldwide. Apart from DFNB1, many other loci and their underlying genes have also been identified and the basis of our study was to provide a comprehensive introduction to the delineation of the molecular basis of non-syndromic hearing loss in the Saudi Arabian population. This was performed by screening DFNB1 and to initiate prioritized linkage analysis or homozygosity mapping for a pilot number of families in which DFNB1 has been excluded. METHODS: Individuals from 130 families of Saudi Arabian tribal origin diagnosed with an autosomal recessive non-syndromic sensorineural hearing loss were screened for mutations at the DFNB1 locus by direct sequencing. If negative, genome wide linkage analysis or homozygosity mapping were performed using Affymetrix GeneChip® Human Mapping 250K/6.0 Arrays to identify regions containing any known-deafness causing genes that were subsequently sequenced. RESULTS: Our results strongly indicate that DFNB1 only accounts for 3% of non-syndromic hearing loss in the Saudi Arabian population of ethnic ancestry. Prioritized linkage analysis or homozygosity mapping in five separate families established that their hearing loss was caused by five different known-deafness causing genes thus confirming the genetic heterogeneity of this disorder in the kingdom. CONCLUSION: The overall results of this study are highly suggestive that underlying molecular basis of autosomal recessive non-syndromic deafness in Saudi Arabia is very genetically heterogeneous. In addition, we report that the preliminary results indicate that there does not seem to be any common or more prevalent loci, genes or mutations in patients with autosomal recessive non-syndromic hearing loss in patients of Saudi Arabian tribal origin.