Beyond the exome: utility of long-read whole genome sequencing in exome-negative autosomal recessive diseases.

BACKGROUND: Long-read whole genome sequencing (lrWGS) has the potential to address the technical limitations of exome sequencing in ways not possible by short-read WGS. However, its utility in autosomal recessive Mendelian diseases is largely unknown. METHODS: In a cohort of 34 families in which the suspected autosomal recessive diseases remained undiagnosed by exome sequencing, lrWGS was performed on the Pacific Bioscience Sequel IIe platform. RESULTS: Likely causal variants were identified in 13 (38%) of the cohort. These include (1) a homozygous splicing SV in TYMS as a novel candidate gene for lethal neonatal lactic acidosis, (2) a homozygous non-coding SV that we propose impacts STK25 expression and causes a novel neurodevelopmental disorder, (3) a compound heterozygous SV in RP1L1 with complex inheritance pattern in a family with inherited retinal disease, (4) homozygous deep intronic variants in LEMD2 and SNAP91 as novel candidate genes for neurodevelopmental disorders in two families, and (5) a promoter SNV in SLC4A4 causing non-syndromic band keratopathy. Surprisingly, we also encountered causal variants that could have been identified by short-read exome sequencing in 7 families. The latter highlight scenarios that are especially challenging at the interpretation level. CONCLUSIONS: Our data highlight the continued need to address the interpretation challenges in parallel with efforts to improve the sequencing technology itself. We propose a path forward for the implementation of lrWGS sequencing in the setting of autosomal recessive diseases in a way that maximizes its utility.

Human 'knockouts' of CSF3 display severe congenital neutropenia.

Colony-stimulating factor 3 (CSF3) is a key factor in neutrophil production and function, and recombinant forms have been used clinically for decades to treat congenital and acquired neutropenia. Although biallelic inactivation of its receptor CSF3R is a well-established cause of severe congenital neutropenia (SCN), no corresponding Mendelian disease has been ascribed to date to CSF3. Here, we describe three patients from two families each segregating a different biallelic inactivating variant in CSF3 with SCN. Complete deficiency of CSF3 as a result of nonsense-mediated decay (NMD) could be demonstrated on RT-PCR using skin fibroblasts-derived RNA. The phenotype observed in this cohort mirrors that documented in mouse and zebrafish models of CSF3 deficiency. Our results suggest that CSF3 deficiency in humans causes a novel autosomal recessive form of SCN.

Diagnostic implications of pitfalls in causal variant identification based on 4577 molecularly characterized families.

Despite large sequencing and data sharing efforts, previously characterized pathogenic variants only account for a fraction of Mendelian disease patients, which highlights the need for accurate identification and interpretation of novel variants. In a large Mendelian cohort of 4577 molecularly characterized families, numerous scenarios in which variant identification and interpretation can be challenging are encountered. We describe categories of challenges that cover the phenotype (e.g. novel allelic disorders), pedigree structure (e.g. imprinting disorders masquerading as autosomal recessive phenotypes), positional mapping (e.g. double recombination events abrogating candidate autozygous intervals), gene (e.g. novel gene-disease assertion) and variant (e.g. complex compound inheritance). Overall, we estimate a probability of 34.3% for encountering at least one of these challenges. Importantly, our data show that by only addressing non-sequencing-based challenges, around 71% increase in the diagnostic yield can be expected. Indeed, by applying these lessons to a cohort of 314 cases with negative clinical exome or genome reports, we could identify the likely causal variant in 54.5%. Our work highlights the need to have a thorough approach to undiagnosed diseases by considering a wide range of challenges rather than a narrow focus on sequencing technologies. It is hoped that by sharing this experience, the yield of undiagnosed disease programs globally can be improved.

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.

Confirming the recessive inheritance of PERP-related erythrokeratoderma.

Erythrokeratoderma (EK) is heterogeneous clinical entity characterized by excessive scaling with resulting erythrokeratotic plaques. Several genes have been linked to EK and they encode a number of proteins that are important for the integrity of the keratinocyte layer of the epidermis. PERP is a transcription factor that is activated by both p53 and p63. However, its deficiency in a mouse model appears to only recapitulate p63-mediated role in skin development and organization. We report an extended multiplex consanguineous family in which an EK phenotype with a striking similarity to that observed in Perp-/- mice, is mapped to an autozygous region on chromosome 6 that spans PERP. Whole-exome sequencing revealed a novel variant in PERP that fully segregated with the phenotype. Functional analysis of patient- and control-derived keratinocytes revealed a deleterious effect of the identified variant on the intracellular localization of PERP. A previous report showed that PERP mutation causes a dominant form of keratoderma but a single patient in that report with a homozygous variant in PERP suggests that recessive inheritance is also possible. Our results, therefore, support the establishment of an autosomal recessive PERP-related EK phenotype in humans.

Molecular autopsy in maternal-fetal medicine.

PurposeThe application of genomic sequencing to investigate unexplained death during early human development, a form of lethality likely enriched for severe Mendelian disorders, has been limited.MethodsIn this study, we employed exome sequencing as a molecular autopsy tool in a cohort of 44 families with at least one death or lethal fetal malformation at any stage of in utero development. Where no DNA was available from the fetus, we performed molecular autopsy by proxy, i.e., through parental testing.ResultsPathogenic or likely pathogenic variants were identified in 22 families (50%), and variants of unknown significance were identified in further 15 families (34%). These variants were in genes known to cause embryonic or perinatal lethality (ALPL, GUSB, SLC17A5, MRPS16, THSD1, PIEZO1, and CTSA), genes known to cause Mendelian phenotypes that do not typically include embryonic lethality (INVS, FKTN, MYBPC3, COL11A2, KRIT1, ASCC1, NEB, LZTR1, TTC21B, AGT, KLHL41, GFPT1, and WDR81) and genes with no established links to human disease that we propose as novel candidates supported by embryonic lethality of their orthologs or other lines of evidence (MS4A7, SERPINA11, FCRL4, MYBPHL, PRPF19, VPS13D, KIAA1109, MOCS3, SVOPL, FEN1, HSPB11, KIF19, and EXOC3L2).ConclusionOur results suggest that molecular autopsy in pregnancy losses is a practical and high-yield alternative to traditional autopsy, and an opportunity for bringing precision medicine to the clinical practice of perinatology.

A null mutation in MICU2 causes abnormal mitochondrial calcium homeostasis and a severe neurodevelopmental disorder.

Mitochondrial calcium homeostasis is a tightly controlled process that is required for a variety of cellular functions. The mitochondrial calcium uniporter complex plays a critical role in this process. MICU2 is a major component of the mitochondrial calcium uniporter complex and its deficiency has been shown to impair mitochondrial calcium [Ca2+]m homeostasis although the exact mechanism remains unclear. We used exome sequencing, positional mapping, and functional characterization of MICU2 deficiency to investigate the role of MICU2 in calcium homeostasis. Using combined autozygome/exome analysis, a homozygous truncating mutation in MICU2 was found to fully segregate with a neurodevelopmental disorder in the form of severe cognitive impairment, spasticity, and white matter involvement in a multiplex consanguineous family. Patient-derived MICU2-deficient cells displayed impaired [Ca2+]m homeostasis, with associated increase in mitochondrial sensitivity to oxidative stress, and abnormal regulation of inner mitochondrial membrane potential. This is the first demonstration of MICU2 deficiency in humans, which we suggest causes a distinct neurodevelopmental phenotype secondary to impaired mitochondrial calcium uniporter-mediated regulation of intracellular calcium homeostasis.

A novel mechanism for variable phenotypic expressivity in Mendelian diseases uncovered by an AU-rich element (ARE)-creating mutation.

BACKGROUND: Variable expressivity is a well-known phenomenon in which patients with mutations in one gene display varying degrees of clinical severity, potentially displaying only subsets of the clinical manifestations associated with the multisystem disorder linked to the gene. This remains an incompletely understood phenomenon with proposed mechanisms ranging from allele-specific to stochastic. RESULTS: We report three consanguineous families in which an isolated ocular phenotype is linked to a novel 3' UTR mutation in SLC4A4, a gene known to be mutated in a syndromic form of intellectual disability with renal and ocular involvement. Although SLC4A4 is normally devoid of AU-rich elements (AREs), a 3' UTR motif that mediates post-transcriptional control of a subset of genes, the mutation we describe creates a functional ARE. We observe a marked reduction in the transcript level of SLC4A4 in patient cells. Experimental confirmation of the ARE-creating mutation is shown using a post-transcriptional reporter system that reveals consistent reduction in the mRNA-half life and reporter activity. Moreover, the neo-ARE binds and responds to the zinc finger protein ZFP36/TTP, an ARE-mRNA decay-promoting protein. CONCLUSIONS: This novel mutational mechanism for a Mendelian disease expands the potential mechanisms that underlie variable phenotypic expressivity in humans to also include 3' UTR mutations with tissue-specific pathology.

GZF1 Mutations Expand the Genetic Heterogeneity of Larsen Syndrome.

Larsen syndrome is characterized by the dislocation of large joints and other less consistent clinical findings. Heterozygous FLNB mutations account for the majority of Larsen syndrome cases, but biallelic mutations in CHST3 and B4GALT7 have been more recently described, thus confirming the existence of recessive forms of the disease. In a multiplex consanguineous Saudi family affected by severe and recurrent large joint dislocation and severe myopia, we identified a homozygous truncating variant in GZF1 through a combined autozygome and exome approach. Independently, the same approach identified a second homozygous truncating GZF1 variant in another multiplex consanguineous family affected by severe myopia, retinal detachment, and milder skeletal involvement. GZF1 encodes GDNF-inducible zinc finger protein 1, a transcription factor of unknown developmental function, which we found to be expressed in the eyes and limbs of developing mice. Global transcriptional profiling of cells from affected individuals revealed a shared pattern of gene dysregulation and significant enrichment of genes encoding matrix proteins, including P3H2, which hints at a potential disease mechanism. Our results suggest that GZF1 mutations cause a phenotype of severe myopia and significant articular involvement not previously described in Larsen syndrome.

Mutations in SMG9, Encoding an Essential Component of Nonsense-Mediated Decay Machinery, Cause a Multiple Congenital Anomaly Syndrome in Humans and Mice.

Nonsense-mediated decay (NMD) is an important process that is best known for degrading transcripts that contain premature stop codons (PTCs) to mitigate their potentially harmful consequences, although its regulatory role encompasses other classes of transcripts as well. Despite the critical role of NMD at the cellular level, our knowledge about the consequences of deficiency of its components at the organismal level is largely limited to model organisms. In this study, we report two consanguineous families in which a similar pattern of congenital anomalies was found to be most likely caused by homozygous loss-of-function mutations in SMG9, encoding an essential component of the SURF complex that generates phospho-UPF1, the single most important step in NMD. By knocking out Smg9 in mice via CRISPR/Cas9, we were able to recapitulate the major features of the SMG9-related multiple congenital anomaly syndrome we observed in humans. Surprisingly, human cells devoid of SMG9 do not appear to have reduction of PTC-containing transcripts but do display global transcriptional dysregulation. We conclude that SMG9 is required for normal human and murine development, most likely through a transcriptional regulatory role, the precise nature of which remains to be determined.

Chromosome 12q24.31-q24.33 deletion causes multiple dysmorphic features and developmental delay: First mosaic patient and overview of the phenotype related to 12q24qter defects.

BACKGROUND: Genomic imbalances of the 12q telomere are rare; only a few patients having 12q24.31-q24.33 deletions were reported. Interestingly none of these were mosaic. Although some attempts have been made to establish phenotype/genotype interaction for the deletions in this region, no clear relationship has been established to date. RESULTS: We have clinically screened more than 100 patients with dysmorphic features, mental retardation and normal karyotype using high density oligo array-CGH (aCGH) and identified a ~9.2 Mb hemizygous interstitial deletion at the 12q telomere (Chromosome 12: 46,XY,del(12)(q24.31q24.33) in a severely developmentally retarded patient having dysmorphic features such as low set ears, microcephaly, undescended testicles, bent elbow, kyphoscoliosis, and micropenis. Parents were found to be not carriers. MLPA experiments confirmed the aCGH result. Interphase FISH revealed mosaicism in cultured peripheral blood lymphocytes. CONCLUSIONS: Since conventional G-Banding technique missed the abnormality; this work re-confirms that any child with unexplained developmental delay and systemic involvement should be studied by aCGH techniques. The FISH technique, however, would still be useful to further delineate the research work and identify such rare mosaicism. Among the 52 deleted genes, P2RX2, ULK1, FZD10, RAN, NCOR2 STX2, TESC, FBXW8, and TBX3 are noteworthy since they may have a role in observed phenotype.