Lessons Learned from Large-Scale, First-Tier Clinical Exome Sequencing in a Highly Consanguineous Population.

We report the results of clinical exome sequencing (CES) on >2,200 previously unpublished Saudi families as a first-tier test. The predominance of autosomal-recessive causes allowed us to make several key observations. We highlight 155 genes that we propose to be recessive, disease-related candidates. We report additional mutational events in 64 previously reported candidates (40 recessive), and these events support their candidacy. We report recessive forms of genes that were previously associated only with dominant disorders and that have phenotypes ranging from consistent with to conspicuously distinct from the known dominant phenotypes. We also report homozygous loss-of-function events that can inform the genetics of complex diseases. We were also able to deduce the likely causal variant in most couples who presented after the loss of one or more children, but we lack samples from those children. Although a similar pattern of mostly recessive causes was observed in the prenatal setting, the higher proportion of loss-of-function events in these cases was notable. The allelic series presented by the wealth of recessive variants greatly expanded the phenotypic expression of the respective genes. We also make important observations about dominant disorders; these observations include the pattern of de novo variants, the identification of 74 candidate dominant, disease-related genes, and the potential confirmation of 21 previously reported candidates. Finally, we describe the influence of a predominantly autosomal-recessive landscape on the clinical utility of rapid sequencing (Flash Exome). Our cohort's genotypic and phenotypic data represent a unique resource that can contribute to improved variant interpretation through data sharing.

Autozygome and high throughput confirmation of disease genes candidacy.

PURPOSE: Establishing links between Mendelian phenotypes and genes enables the proper interpretation of variants therein. Autozygome, a rich source of homozygous variants, has been successfully utilized for the high throughput identification of novel autosomal recessive disease genes. Here, we highlight the utility of the autozygome for the high throughput confirmation of previously published tentative links to diseases. METHODS: Autozygome and exome analysis of patients with suspected Mendelian phenotypes. All variants were classified according to the American College of Medical Genetics and Genomics guidelines. RESULTS: We highlight 30 published candidate genes (ACTL6B, ADAM22, AGTPBP1, APC, C12orf4, C3orf17 (NEPRO), CENPF, CNPY3, COL27A1, DMBX1, FUT8, GOLGA2, KIAA0556, LENG8, MCIDAS, MTMR9, MYH11, QRSL1, RUBCN, SLC25A42, SLC9A1, TBXT, TFG, THUMPD1, TRAF3IP2, UFC1, UFM1, WDR81, XRCC2, ZAK) in which we identified homozygous likely deleterious variants in patients with compatible phenotypes. We also identified homozygous likely deleterious variants in 18 published candidate genes (ABCA2, ARL6IP1, ATP8A2, CDK9, CNKSR1, DGAT1, DMXL2, GEMIN4, HCN2, HCRT, MYO9A, PARS2, PLOD3, PREPL, SCLT1, STX3, TXNRD2, WIPI2) although the associated phenotypes are sufficiently different from the original reports that they represent phenotypic expansion or potentially distinct allelic disorders. CONCLUSIONS: Our results should facilitate the timely relabeling of these candidate disease genes in relevant databases to improve the yield of clinical genomic sequencing.

Identification of a Recognizable Progressive Skeletal Dysplasia Caused by RSPRY1 Mutations.

Skeletal dysplasias are highly variable Mendelian phenotypes. Molecular diagnosis of skeletal dysplasias is complicated by their extreme clinical and genetic heterogeneity. We describe a clinically recognizable autosomal-recessive disorder in four affected siblings from a consanguineous Saudi family, comprising progressive spondyloepimetaphyseal dysplasia, short stature, facial dysmorphism, short fourth metatarsals, and intellectual disability. Combined autozygome/exome analysis identified a homozygous frameshift mutation in RSPRY1 with resulting nonsense-mediated decay. Using a gene-centric "matchmaking" system, we were able to identify a Peruvian simplex case subject whose phenotype is strikingly similar to the original Saudi family and whose exome sequencing had revealed a likely pathogenic homozygous missense variant in the same gene. RSPRY1 encodes a hypothetical RING and SPRY domain-containing protein of unknown physiological function. However, we detect strong RSPRY1 protein localization in murine embryonic osteoblasts and periosteal cells during primary endochondral ossification, consistent with a role in bone development. This study highlights the role of gene-centric matchmaking tools to establish causal links to genes, especially for rare or previously undescribed clinical entities.

The landscape of genetic diseases in Saudi Arabia based on the first 1000 diagnostic panels and exomes.

In this study, we report the experience of the only reference clinical next-generation sequencing lab in Saudi Arabia with the first 1000 families who span a wide-range of suspected Mendelian phenotypes. A total of 1019 tests were performed in the period of March 2016-December 2016 comprising 972 solo (index only), 14 duo (parents or affected siblings only), and 33 trio (index and parents). Multigene panels accounted for 672 tests, while whole exome sequencing (WES) represented the remaining 347 tests. Pathogenic or likely pathogenic variants that explain the clinical indications were identified in 34% (27% in panels and 43% in exomes), spanning 279 genes and including 165 novel variants. While recessive mutations dominated the landscape of solved cases (71% of mutations, and 97% of which are homozygous), a substantial minority (27%) were solved on the basis of dominant mutations. The highly consanguineous nature of the study population also facilitated homozygosity for many private mutations (only 32.5% of the recessive mutations are founder), as well as the first instances of recessive inheritance of previously assumed strictly dominant disorders (involving ITPR1, VAMP1, MCTP2, and TBP). Surprisingly, however, dual molecular diagnosis was only observed in 1.5% of cases. Finally, we have encountered candidate variants in 75 genes (ABHD6, ACY3, ADGRB2, ADGRG7, AGTPBP1, AHNAK2, AKAP6, ASB3, ATXN1L, C17orf62, CABP1, CCDC186, CCP110, CLSTN2, CNTN3, CNTN5, CTNNA2, CWC22, DMAP1, DMKN, DMXL1, DSCAM, DVL2, ECI1, EP400, EPB41L5, FBXL22, GAP43, GEMIN7, GIT1, GRIK4, GRSF1, GTRP1, HID1, IFNL1, KCNC4, LRRC52, MAP7D3, MCTP2, MED26, MPP7, MRPS35, MTDH, MTMR9, NECAP2, NPAT, NRAP, PAX7, PCNX, PLCH2, PLEKHF1, PTPN12, QKI, RILPL2, RIMKLA, RIMS2, RNF213, ROBO1, SEC16A, SIAH1, SIRT2, SLAIN2, SLC22A20, SMDT1, SRRT, SSTR1, ST20, SYT9, TSPAN6, UBR4, VAMP4, VPS36, WDR59, WDYHV1, and WHSC1) not previously linked to human phenotypes and these are presented to accelerate post-publication matchmaking. Two of these genes were independently mutated in more than one family with similar phenotypes, which substantiates their link to human disease (AKAP6 in intellectual disability and UBR4 in early dementia). If the novel candidate disease genes in this cohort are independently confirmed, the yield of WES will have increased to 83%, which suggests that most "negative" clinical exome tests are unsolved due to interpretation rather than technical limitations.

Vosoritide Therapy in Children with Achondroplasia: Early Experience and Practical Considerations for Clinical Practice.

INTRODUCTION: Vosoritide is the first precision medical therapy approved to increase growth velocity in children with achondroplasia. Sharing early prescribing experiences across different regions could provide a framework for developing practical guidance for the real-world use of vosoritide. METHODS: Two meetings were held to gather insight and early experience from experts in Europe, the Middle East, and the USA. The group comprised geneticists, pediatric endocrinologists, pediatricians, and orthopedic surgeons. Current practices and considerations for vosoritide were discussed, including administration practicalities, assessments, and how to manage expectations. RESULTS: A crucial step in the management of achondroplasia is to determine if adequate multidisciplinary support is in place. Training for families is essential, including practical information on administration of vosoritide, and how to recognize and manage injection-site reactions. Advocated techniques include establishing a routine, empowering patients by allowing them to choose injection sites, and managing pain. Patients may discontinue vosoritide if they cannot tolerate daily injections or are invited to participate in a clinical trial. Clinicians in Europe and the Middle East emphasized the importance of assessing adherence to daily injections, as non-adherence may impact response and reimbursement. Protocols for monitoring patients receiving vosoritide may be influenced by regional differences in reimbursement and healthcare systems. Core assessments may include pubertal staging, anthropometry, radiography to confirm open physes, the review of adverse events, and discussion of concomitant or new medications-but timing of these assessments may also differ regionally and vary across institutions. Patients and families should be informed that response to vosoritide can vary in both magnitude and timing. Keeping families informed regarding vosoritide clinical trial data is encouraged. CONCLUSION: The early real-world experience with vosoritide is generally positive. Sharing these insights is important to increase understanding of the practicalities of treatment with vosoritide in the clinical setting.

Identification of a novel DLX5 mutation in a family with autosomal recessive split hand and foot malformation.

BACKGROUND: Split hand and foot malformation (SHFM) refers to a genetically heterogeneous developmental disorder of the hands and feet that presents as median ray deficiency of varying severity. 7q21.3 (SHFM1) is one of six loci described to date, and although DLX5 and DLX6 are compelling candidates in that locus, no intragenic mutations have been described in either of these genes. METHODS: The authors combined autozygome analysis and exome sequencing to study a consanguineous family with a highly unusual SHFM phenotype, where there is associated dorsalisation of the hands. RESULTS: A novel missense mutation in a highly conserved residue of the homeobox domain of DLX5 was identified. Unlike previously reported position effect mutations in SHFM1, this first documented intragenic DLX5 mutation is also accompanied by abnormal dorsal-ventral patterning. CONCLUSION: This study identified the first intragenic DLX5 mutation in SHFM and raises interesting possibilities about a dual role for DLX5 in limb development.

Mutation of CANT1 causes Desbuquois dysplasia.

Desbuquois dysplasia is an autosomal recessive dysplasia characterized by severe growth restriction and distinct hand and proximal femur appearance in addition to cognitive impairment. The critical interval for this disease has been mapped to 17q25.3 using homozygosity mapping. We have identified a newborn with classical features of the disease whose parents are first cousins. Assuming genetic homogeneity of this disorder, we were able to narrow the critical interval to a region that only contained 10 annotated genes by combining the results of our homozygosity mapping with those of others. Serial sequencing of the genes contained within the interval revealed a 5 bp duplication in Calcium-Activated Nucleotidase 1 gene (CANT1), consistent with the very recent report by Huber et al. [Huber et al. (2009); Am J Hum Genet 85:706-710]. This report cements the role of CANT1 in the causation of this dysplasia and demonstrates the high value of even single cases in the setting of genetically homogeneous disorders when homozygosity mapping is used.

KDM5A mutations identified in autism spectrum disorder using forward genetics.

Autism spectrum disorder (ASD) is a constellation of neurodevelopmental disorders with high phenotypic and genetic heterogeneity, complicating the discovery of causative genes. Through a forward genetics approach selecting for defective vocalization in mice, we identified Kdm5a as a candidate ASD gene. To validate our discovery, we generated a Kdm5a knockout mouse model (Kdm5a-/-) and confirmed that inactivating Kdm5a disrupts vocalization. In addition, Kdm5a-/- mice displayed repetitive behaviors, sociability deficits, cognitive dysfunction, and abnormal dendritic morphogenesis. Loss of KDM5A also resulted in dysregulation of the hippocampal transcriptome. To determine if KDM5A mutations cause ASD in humans, we screened whole exome sequencing and microarray data from a clinical cohort. We identified pathogenic KDM5A variants in nine patients with ASD and lack of speech. Our findings illustrate the power and efficacy of forward genetics in identifying ASD genes and highlight the importance of KDM5A in normal brain development and function.

The Erlenmeyer flask bone deformity in the skeletal dysplasias.

Erlenmeyer flask bone deformity (EFD) is a long-standing term used to describe a specific abnormality of the distal femora. The deformity consists of lack of modeling of the di-metaphysis with abnormal cortical thinning and lack of the concave di-metaphyseal curve resulting in an Erlenmeyer flask-like appearance. Utilizing a literature review and cohort study of 12 disorders we found 20 distinct disorders were associated with EFD. We interrogated the International Skeletal Dysplasia Registry (ISDR) radiographic database (1988-2007) to determine which skeletal dysplasias or syndromes were highly associated with EFD, whether it was a uniform finding in these disorders, and if forms of EFD could be differentiated. EFD was classified into three groups. The first catogory was the typical EFD shaped bone (EFD-T) resultant from absent normal di-metaphyseal modeling with relatively normal appearing radiographic trabecular bone. EFD-T was identified in: frontometaphyseal dysplasia, craniometaphyseal dysplasia, craniodiaphyseal dysplasia, diaphyseal dysplasia-Engelmann type, metaphyseal dysplasia-Pyle type, Melnick-Needles osteodysplasty, and otopalatodigital syndrome type I. The second group was the atypical type (EFD-A) due to absence of normal di-metaphyseal modeling with abnormal radiographic appearance of trabecular bone and was seen in dysosteosclerosis and osteopetrosis. The third group was EFD-marrow expansion type (EFD-ME) in which bone marrow hyperplasia or infiltration leads to abnormal modeling (e.g., Gaucher disease). Further, radiographic review determined that it was not always a consistent finding and that there was variability in both appearance and location within the skeleton. This analysis and classification aided in differentiating disorders with the finding of EFD.