Genomic and transcriptomic correlates of Richter transformation in chronic lymphocytic leukemia.

The transformation of chronic lymphocytic leukemia (CLL) to high-grade B-cell lymphoma is known as Richter syndrome (RS), a rare event with dismal prognosis. In this study, we conducted whole-genome sequencing (WGS) of paired circulating CLL (PB-CLL) and RS biopsies (tissue-RS) from 17 patients recruited into a clinical trial (CHOP-O). We found that tissue-RS was enriched for mutations in poor-risk CLL drivers and genes in the DNA damage response (DDR) pathway. In addition, we identified genomic aberrations not previously implicated in RS, including the protein tyrosine phosphatase receptor (PTPRD) and tumor necrosis factor receptor-associated factor 3 (TRAF3). In the noncoding genome, we discovered activation-induced cytidine deaminase-related and unrelated kataegis in tissue-RS affecting regulatory regions of key immune-regulatory genes. These include BTG2, CXCR4, NFATC1, PAX5, NOTCH-1, SLC44A5, FCRL3, SELL, TNIP2, and TRIM13. Furthermore, differences between the global mutation signatures of pairs of PB-CLL and tissue-RS samples implicate DDR as the dominant mechanism driving transformation. Pathway-based clonal deconvolution analysis showed that genes in the MAPK and DDR pathways demonstrate high clonal-expansion probability. Direct comparison of nodal-CLL and tissue-RS pairs from an independent cohort confirmed differential expression of the same pathways by RNA expression profiling. Our integrated analysis of WGS and RNA expression data significantly extends previous targeted approaches, which were limited by the lack of germline samples, and it facilitates the identification of novel genomic correlates implicated in RS transformation, which could be targeted therapeutically. Our results inform the future selection of investigative agents for a UK clinical platform study. This trial was registered at www.clinicaltrials.gov as #NCT03899337.

The complete costs of genome sequencing: a microcosting study in cancer and rare diseases from a single center in the United Kingdom.

PURPOSE: The translation of genome sequencing into routine health care has been slow, partly because of concerns about affordability. The aspirational cost of sequencing a genome is $1000, but there is little evidence to support this estimate. We estimate the cost of using genome sequencing in routine clinical care in patients with cancer or rare diseases. METHODS: We performed a microcosting study of Illumina-based genome sequencing in a UK National Health Service laboratory processing 399 samples/year. Cost data were collected for all steps in the sequencing pathway, including bioinformatics analysis and reporting of results. Sensitivity analysis identified key cost drivers. RESULTS: Genome sequencing costs £6841 per cancer case (comprising matched tumor and germline samples) and £7050 per rare disease case (three samples). The consumables used during sequencing are the most expensive component of testing (68-72% of the total cost). Equipment costs are higher for rare disease cases, whereas consumable and staff costs are slightly higher for cancer cases. CONCLUSION: The cost of genome sequencing is underestimated if only sequencing costs are considered, and likely surpasses $1000/genome in a single laboratory. This aspirational sequencing cost will likely only be achieved if consumable costs are considerably reduced and sequencing is performed at scale.

Identification of a new VHL exon and complex splicing alterations in familial erythrocytosis or von Hippel-Lindau disease.

Chuvash polycythemia is an autosomal recessive form of erythrocytosis associated with a homozygous p.Arg200Trp mutation in the von Hippel-Lindau (VHL) gene. Since this discovery, additional VHL mutations have been identified in patients with congenital erythrocytosis, in a homozygous or compound-heterozygous state. VHL is a major tumor suppressor gene, mutations in which were first described in patients presenting with VHL disease, which is characterized by the development of highly vascularized tumors. Here, we identify a new VHL cryptic exon (termed E1') deep in intron 1 that is naturally expressed in many tissues. More importantly, we identify mutations in E1' in 7 families with erythrocytosis (1 homozygous case and 6 compound-heterozygous cases with a mutation in E1' in addition to a mutation in VHL coding sequences) and in 1 large family with typical VHL disease but without any alteration in the other VHL exons. In this study, we show that the mutations induced a dysregulation of VHL splicing with excessive retention of E1' and were associated with a downregulation of VHL protein expression. In addition, we demonstrate a pathogenic role for synonymous mutations in VHL exon 2 that altered splicing through E2-skipping in 5 families with erythrocytosis or VHL disease. In all the studied cases, the mutations differentially affected splicing, correlating with phenotype severity. This study demonstrates that cryptic exon retention and exon skipping are new VHL alterations and reveals a novel complex splicing regulation of the VHL gene. These findings open new avenues for diagnosis and research regarding the VHL-related hypoxia-signaling pathway.

Clinical-grade validation of whole genome sequencing reveals robust detection of low-frequency variants and copy number alterations in CLL.

The 100 000 Genome Project aims to develop a diagnostics platform by introducing whole genome sequencing (WGS) into clinical practice. Samples from patients with chronic lymphocytic leukaemia were subjected to WGS. WGS detection of single nucleotide variants and insertion/deletions were validated by targeted next generation sequencing showing high concordance (96·3%), also for detection of sub-clonal variants and low-frequency TP53 variants. Copy number alteration detection was verified by fluorescent in situ hybridisation and genome-wide single nucleotide polymorphism array (concordances of 86·7% and 92·9%, respectively), confirming adequate sensitivity by WGS. Our results confirm that WGS can provide comprehensive genomic characterisation for clinical trials, drug discovery and, ultimately, precision medicine.

Clinical whole-genome sequencing from routine formalin-fixed, paraffin-embedded specimens: pilot study for the 100,000 Genomes Project.

PURPOSE: Fresh-frozen (FF) tissue is the optimal source of DNA for whole-genome sequencing (WGS) of cancer patients. However, it is not always available, limiting the widespread application of WGS in clinical practice. We explored the viability of using formalin-fixed, paraffin-embedded (FFPE) tissues, available routinely for cancer patients, as a source of DNA for clinical WGS. METHODS: We conducted a prospective study using DNAs from matched FF, FFPE, and peripheral blood germ-line specimens collected from 52 cancer patients (156 samples) following routine diagnostic protocols. We compared somatic variants detected in FFPE and matching FF samples. RESULTS: We found the single-nucleotide variant agreement reached 71% across the genome and somatic copy-number alterations (CNAs) detection from FFPE samples was suboptimal (0.44 median correlation with FF) due to nonuniform coverage. CNA detection was improved significantly with lower reverse crosslinking temperature in FFPE DNA extraction (80 °C or 65 °C depending on the methods). Our final data showed somatic variant detection from FFPE for clinical decision making is possible. We detected 98% of clinically actionable variants (including 30/31 CNAs). CONCLUSION: We present the first prospective WGS study of cancer patients using FFPE specimens collected in a routine clinical environment proving WGS can be applied in the clinic.

Germline recessive mutations in PI4KA are associated with perisylvian polymicrogyria, cerebellar hypoplasia and arthrogryposis.

Polymicrogyria (PMG) is a structural brain abnormality involving the cerebral cortex that results from impaired neuronal migration and although several genes have been implicated, many cases remain unsolved. In this study, exome sequencing in a family where three fetuses had all been diagnosed with PMG and cerebellar hypoplasia allowed us to identify regions of the genome for which both chromosomes were shared identical-by-descent, reducing the search space for causative variants to 8.6% of the genome. In these regions, the only plausibly pathogenic mutations were compound heterozygous variants in PI4KA, which Sanger sequencing confirmed segregated consistent with autosomal recessive inheritance. The paternally transmitted variant predicted a premature stop mutation (c.2386C>T; p.R796X), whereas the maternally transmitted variant predicted a missense substitution (c.5560G>A; p.D1854N) at a conserved residue within the catalytic domain. Functional studies using expressed wild-type or mutant PI4KA enzyme confirmed the importance of p.D1854 for kinase activity. Our results emphasize the importance of phosphoinositide signalling in early brain development.

Mutations in PIGY: expanding the phenotype of inherited glycosylphosphatidylinositol deficiencies.

Glycosylphosphatidylinositol (GPI)-anchored proteins are ubiquitously expressed in the human body and are important for various functions at the cell surface. Mutations in many GPI biosynthesis genes have been described to date in patients with multi-system disease and together these constitute a subtype of congenital disorders of glycosylation. We used whole exome sequencing in two families to investigate the genetic basis of disease and used RNA and cellular studies to investigate the functional consequences of sequence variants in the PIGY gene. Two families with different phenotypes had homozygous recessive sequence variants in the GPI biosynthesis gene PIGY. Two sisters with c.137T>C (p.Leu46Pro) PIGY variants had multi-system disease including dysmorphism, seizures, severe developmental delay, cataracts and early death. There were significantly reduced levels of GPI-anchored proteins (CD55 and CD59) on the surface of patient-derived skin fibroblasts (∼20-50% compared with controls). In a second, consanguineous family, two siblings had moderate development delay and microcephaly. A homozygous PIGY promoter variant (c.-540G>A) was detected within a 7.7 Mb region of autozygosity. This variant was predicted to disrupt a SP1 consensus binding site and was shown to be associated with reduced gene expression. Mutations in PIGY can occur in coding and non-coding regions of the gene and cause variable phenotypes. This article contributes to understanding of the range of disease phenotypes and disease genes associated with deficiencies of the GPI-anchor biosynthesis pathway and also serves to highlight the potential importance of analysing variants detected in 5'-UTR regions despite their typically low coverage in exome data.

Mutations in PGAP3 impair GPI-anchor maturation, causing a subtype of hyperphosphatasia with mental retardation.

Glycosylphophatidylinositol (GPI)-anchored proteins play important roles in many biological processes, and mutations affecting proteins involved in the synthesis of the GPI anchor are reported to cause a wide spectrum of intellectual disabilities (IDs) with characteristic additional phenotypic features. Here, we describe a total of five individuals (from three unrelated families) in whom we identified mutations in PGAP3, encoding a protein that is involved in GPI-anchor maturation. Three siblings in a consanguineous Pakistani family presented with profound developmental delay, severe ID, no speech, psychomotor delay, and postnatal microcephaly. A combination of autozygosity mapping and exome sequencing identified a 13.8 Mb region harboring a homozygous c.275G>A (p.Gly92Asp) variant in PGAP3 region 17q11.2-q21.32. Subsequent testing showed elevated serum alkaline phosphatase (ALP), a GPI-anchored enzyme, in all three affected children. In two unrelated individuals in a cohort with developmental delay, ID, and elevated ALP, we identified compound-heterozygous variants c.439dupC (p.Leu147Profs(∗)16) and c.914A>G (p.Asp305Gly) and homozygous variant c.314C>G (p.Pro105Arg). The 1 bp duplication causes a frameshift and nonsense-mediated decay. Further evidence supporting pathogenicity of the missense mutations c.275G>A, c.314C>G, and c.914A>G was provided by the absence of the variants from ethnically matched controls, phylogenetic conservation, and functional studies on Chinese hamster ovary cell lines. Taken together with recent data on PGAP2, these results confirm the importance of the later GPI-anchor remodelling steps for normal neuronal development. Impairment of PGAP3 causes a subtype of hyperphosphatasia with ID, a congenital disorder of glycosylation that is also referred to as Mabry syndrome.

Presence of multiple recurrent mutations confers poor trial outcome of relapsed/refractory CLL.

Although TP53, NOTCH1, and SF3B1 mutations may impair prognosis of patients with chronic lymphocytic leukemia (CLL) receiving frontline therapy, the impact of these mutations or any other, alone or in combination, remains unclear at relapse. The genome of 114 relapsed/refractory patients included in prospective trials was screened using targeted next-generation sequencing of the TP53, SF3B1, ATM, NOTCH1, XPO1, SAMHD1, MED12, BIRC3, and MYD88 genes. We performed clustering according to both number and combinations of recurrent gene mutations. The number of genes affected by mutation was ≥ 2, 1, and 0 in 43 (38%), 49 (43%), and 22 (19%) respectively. Recurrent combinations of ≥ 2 mutations of TP53, SF3B1, and ATM were found in 22 (19%) patients. This multiple-hit profile was associated with a median progression-free survival of 12 months compared with 22.5 months in the remaining patients (P = .003). Concurrent gene mutations are frequent in patients with relapsed/refractory CLL and are associated with worse outcome.

Identification of Intragenic Exon Deletions and Duplication of TCF12 by Whole Genome or Targeted Sequencing as a Cause of TCF12-Related Craniosynostosis.

TCF12-related craniosynostosis can be caused by small heterozygous loss-of-function mutations in TCF12. Large intragenic rearrangements, however, have not been described yet. Here, we present the identification of four large rearrangements in TCF12 causing TCF12-related craniosynostosis. Whole-genome sequencing was applied on the DNA of 18 index cases with coronal synostosis and their family members (43 samples in total). The data were analyzed using an autosomal-dominant disease model. Structural variant analysis reported intragenic exon deletions (of sizes 84.9, 8.6, and 5.4 kb) in TCF12 in three different families. The results were confirmed by deletion-specific PCR and dideoxy-sequence analysis. Separately, targeted sequencing of the TCF12 genomic region in a patient with coronal synostosis identified a tandem duplication of 11.3 kb. The pathogenic effect of this duplication was confirmed by cDNA analysis. These findings indicate the importance of screening for larger rearrangements in patients suspected to have TCF12-related craniosynostosis.

SAMHD1 is mutated recurrently in chronic lymphocytic leukemia and is involved in response to DNA damage.

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase and a nuclease that restricts HIV-1 in noncycling cells. Germ-line mutations in SAMHD1 have been described in patients with Aicardi-Goutières syndrome (AGS), a congenital autoimmune disease. In a previous longitudinal whole genome sequencing study of chronic lymphocytic leukemia (CLL), we revealed a SAMHD1 mutation as a potential founding event. Here, we describe an AGS patient carrying a pathogenic germ-line SAMHD1 mutation who developed CLL at 24 years of age. Using clinical trial samples, we show that acquired SAMHD1 mutations are associated with high variant allele frequency and reduced SAMHD1 expression and occur in 11% of relapsed/refractory CLL patients. We provide evidence that SAMHD1 regulates cell proliferation and survival and engages in specific protein interactions in response to DNA damage. We propose that SAMHD1 may have a function in DNA repair and that the presence of SAMHD1 mutations in CLL promotes leukemia development.

Cellular interference in craniofrontonasal syndrome: males mosaic for mutations in the X-linked EFNB1 gene are more severely affected than true hemizygotes.

Craniofrontonasal syndrome (CFNS), an X-linked disorder caused by loss-of-function mutations of EFNB1, exhibits a paradoxical sex reversal in phenotypic severity: females characteristically have frontonasal dysplasia, craniosynostosis and additional minor malformations, but males are usually more mildly affected with hypertelorism as the only feature. X-inactivation is proposed to explain the more severe outcome in heterozygous females, as this leads to functional mosaicism for cells with differing expression of EPHRIN-B1, generating abnormal tissue boundaries-a process that cannot occur in hemizygous males. Apparently challenging this model, males occasionally present with a more severe female-like CFNS phenotype. We hypothesized that such individuals might be mosaic for EFNB1 mutations and investigated this possibility in multiple tissue samples from six sporadically presenting males. Using denaturing high performance liquid chromatography, massively parallel sequencing and multiplex-ligation-dependent probe amplification (MLPA) to increase sensitivity above standard dideoxy sequencing, we identified mosaic mutations of EFNB1 in all cases, comprising three missense changes, two gene deletions and a novel point mutation within the 5' untranslated region (UTR). Quantification by Pyrosequencing and MLPA demonstrated levels of mutant cells between 15 and 69%. The 5' UTR variant mutates the stop codon of a small upstream open reading frame that, using a dual-luciferase reporter construct, was demonstrated to exacerbate interference with translation of the wild-type protein. These results demonstrate a more severe outcome in mosaic than in constitutionally deficient males in an X-linked dominant disorder and provide further support for the cellular interference mechanism, normally related to X-inactivation in females.

Mutations in multidomain protein MEGF8 identify a Carpenter syndrome subtype associated with defective lateralization.

Carpenter syndrome is an autosomal-recessive multiple-congenital-malformation disorder characterized by multisuture craniosynostosis and polysyndactyly of the hands and feet; many other clinical features occur, and the most frequent include obesity, umbilical hernia, cryptorchidism, and congenital heart disease. Mutations of RAB23, encoding a small GTPase that regulates vesicular transport, are present in the majority of cases. Here, we describe a disorder caused by mutations in multiple epidermal-growth-factor-like-domains 8 (MEGF8), which exhibits substantial clinical overlap with Carpenter syndrome but is frequently associated with abnormal left-right patterning. We describe five affected individuals with similar dysmorphic facies, and three of them had either complete situs inversus, dextrocardia, or transposition of the great arteries; similar cardiac abnormalities were previously identified in a mouse mutant for the orthologous Megf8. The mutant alleles comprise one nonsense, three missense, and two splice-site mutations; we demonstrate in zebrafish that, in contrast to the wild-type protein, the proteins containing all three missense alterations provide only weak rescue of an early gastrulation phenotype induced by Megf8 knockdown. We conclude that mutations in MEGF8 cause a Carpenter syndrome subtype frequently associated with defective left-right patterning, probably through perturbation of signaling by hedgehog and nodal family members. We did not observe any subject with biallelic loss-of function mutations, suggesting that some residual MEGF8 function might be necessary for survival and might influence the phenotypes observed.

De novo and rare inherited mutations implicate the transcriptional coregulator TCF20/SPBP in autism spectrum disorder.

BACKGROUND: Autism spectrum disorders (ASDs) are common and have a strong genetic basis, yet the cause of ∼70-80% ASDs remains unknown. By clinical cytogenetic testing, we identified a family in which two brothers had ASD, mild intellectual disability and a chromosome 22 pericentric inversion, not detected in either parent, indicating de novo mutation with parental germinal mosaicism. We hypothesised that the rearrangement was causative of their ASD and localised the chromosome 22 breakpoints. METHODS: The rearrangement was characterised using fluorescence in situ hybridisation, Southern blotting, inverse PCR and dideoxy-sequencing. Open reading frames and intron/exon boundaries of the two physically disrupted genes identified, TCF20 and TNRC6B, were sequenced in 342 families (260 multiplex and 82 simplex) ascertained by the International Molecular Genetic Study of Autism Consortium (IMGSAC). RESULTS: IMGSAC family screening identified a de novo missense mutation of TCF20 in a single case and significant association of a different missense mutation of TCF20 with ASD in three further families. Through exome sequencing in another project, we independently identified a de novo frameshifting mutation of TCF20 in a woman with ASD and moderate intellectual disability. We did not identify a significant association of TNRC6B mutations with ASD. CONCLUSIONS: TCF20 encodes a transcriptional coregulator (also termed SPBP) that is structurally and functionally related to RAI1, the critical dosage-sensitive protein implicated in the behavioural phenotypes of the Smith-Magenis and Potocki-Lupski 17p11.2 deletion/duplication syndromes, in which ASD is frequently diagnosed. This study provides the first evidence that mutations in TCF20 are also associated with ASD.

Recessive mutations in SPTBN2 implicate ß-III spectrin in both cognitive and motor development.

ß-III spectrin is present in the brain and is known to be important in the function of the cerebellum. Heterozygous mutations in SPTBN2, the gene encoding ß-III spectrin, cause Spinocerebellar Ataxia Type 5 (SCA5), an adult-onset, slowly progressive, autosomal-dominant pure cerebellar ataxia. SCA5 is sometimes known as "Lincoln ataxia," because the largest known family is descended from relatives of the United States President Abraham Lincoln. Using targeted capture and next-generation sequencing, we identified a homozygous stop codon in SPTBN2 in a consanguineous family in which childhood developmental ataxia co-segregates with cognitive impairment. The cognitive impairment could result from mutations in a second gene, but further analysis using whole-genome sequencing combined with SNP array analysis did not reveal any evidence of other mutations. We also examined a mouse knockout of ß-III spectrin in which ataxia and progressive degeneration of cerebellar Purkinje cells has been previously reported and found morphological abnormalities in neurons from prefrontal cortex and deficits in object recognition tasks, consistent with the human cognitive phenotype. These data provide the first evidence that ß-III spectrin plays an important role in cortical brain development and cognition, in addition to its function in the cerebellum; and we conclude that cognitive impairment is an integral part of this novel recessive ataxic syndrome, Spectrin-associated Autosomal Recessive Cerebellar Ataxia type 1 (SPARCA1). In addition, the identification of SPARCA1 and normal heterozygous carriers of the stop codon in SPTBN2 provides insights into the mechanism of molecular dominance in SCA5 and demonstrates that the cell-specific repertoire of spectrin subunits underlies a novel group of disorders, the neuronal spectrinopathies, which includes SCA5, SPARCA1, and a form of West syndrome.

Discovery of previously unidentified genomic disorders from the duplication architecture of the human genome.

Genomic disorders are characterized by the presence of flanking segmental duplications that predispose these regions to recurrent rearrangement. Based on the duplication architecture of the genome, we investigated 130 regions that we hypothesized as candidates for previously undescribed genomic disorders. We tested 290 individuals with mental retardation by BAC array comparative genomic hybridization and identified 16 pathogenic rearrangements, including de novo microdeletions of 17q21.31 found in four individuals. Using oligonucleotide arrays, we refined the breakpoints of this microdeletion, defining a 478-kb critical region containing six genes that were deleted in all four individuals. We mapped the breakpoints of this deletion and of four other pathogenic rearrangements in 1q21.1, 15q13, 15q24 and 17q12 to flanking segmental duplications, suggesting that these are also sites of recurrent rearrangement. In common with the 17q21.31 deletion, these breakpoint regions are sites of copy number polymorphism in controls, indicating that these may be inherently unstable genomic regions.

A new chromosome 17q21.31 microdeletion syndrome associated with a common inversion polymorphism.

Submicroscopic genomic copy number changes have been identified only recently as an important cause of mental retardation. We describe the detection of three interstitial, overlapping 17q21.31 microdeletions in a cohort of 1,200 mentally retarded individuals associated with a clearly recognizable clinical phenotype of mental retardation, hypotonia and a characteristic face. The deletions encompass the MAPT and CRHR1 genes and are associated with a common inversion polymorphism.

Structural and non-coding variants increase the diagnostic yield of clinical whole genome sequencing for rare diseases.

BACKGROUND: Whole genome sequencing is increasingly being used for the diagnosis of patients with rare diseases. However, the diagnostic yields of many studies, particularly those conducted in a healthcare setting, are often disappointingly low, at 25-30%. This is in part because although entire genomes are sequenced, analysis is often confined to in silico gene panels or coding regions of the genome. METHODS: We undertook WGS on a cohort of 122 unrelated rare disease patients and their relatives (300 genomes) who had been pre-screened by gene panels or arrays. Patients were recruited from a broad spectrum of clinical specialties. We applied a bioinformatics pipeline that would allow comprehensive analysis of all variant types. We combined established bioinformatics tools for phenotypic and genomic analysis with our novel algorithms (SVRare, ALTSPLICE and GREEN-DB) to detect and annotate structural, splice site and non-coding variants. RESULTS: Our diagnostic yield was 43/122 cases (35%), although 47/122 cases (39%) were considered solved when considering novel candidate genes with supporting functional data into account. Structural, splice site and deep intronic variants contributed to 20/47 (43%) of our solved cases. Five genes that are novel, or were novel at the time of discovery, were identified, whilst a further three genes are putative novel disease genes with evidence of causality. We identified variants of uncertain significance in a further fourteen candidate genes. The phenotypic spectrum associated with RMND1 was expanded to include polymicrogyria. Two patients with secondary findings in FBN1 and KCNQ1 were confirmed to have previously unidentified Marfan and long QT syndromes, respectively, and were referred for further clinical interventions. Clinical diagnoses were changed in six patients and treatment adjustments made for eight individuals, which for five patients was considered life-saving. CONCLUSIONS: Genome sequencing is increasingly being considered as a first-line genetic test in routine clinical settings and can make a substantial contribution to rapidly identifying a causal aetiology for many patients, shortening their diagnostic odyssey. We have demonstrated that structural, splice site and intronic variants make a significant contribution to diagnostic yield and that comprehensive analysis of the entire genome is essential to maximise the value of clinical genome sequencing.

Exome sequencing can detect pathogenic mosaic mutations present at low allele frequencies.

The development of next generation sequencing (NGS) has radically transformed the scientific landscape, making it possible to sequence the exome of any given individual in a cost-effective way. The power of this approach has been demonstrated by a number of groups who have identified pathogenic mutations in small pedigrees that have been resistant to traditional genetic mapping. Recently it has become clear that exome sequencing has great potential with respect to sporadic disease and the identification of de novo mutations. This is highlighted by studies reporting whole-exome sequencing of patient-parental trios affected by learning disability, autism and schizophrenia. It is widely anticipated that the introduction of this technique into a clinical setting will revolutionise genetic diagnosis. However, the sensitivity of NGS exome sequencing is currently unclear. Here, we describe the exome sequencing of DNA samples from a patient with double cortex syndrome and her parents, resulting in the detection of a mosaic splicing mutation in LIS1. This variant was found at an allele frequency of just 18%, demonstrating that NGS methods have the capacity to identify pathogenic mosaic mutations present at a low level.

A novel nonsense CDK5RAP2 mutation in a Somali child with primary microcephaly and sensorineural hearing loss.

Primary microcephaly is a genetically heterogeneous condition characterized by reduced head circumference (-3 SDS or more) and mild-to-moderate learning disability. Here, we describe clinical and molecular investigations of a microcephalic child with sensorineural hearing loss. Although consanguinity was unreported initially, detection of 13.7 Mb of copy neutral loss of heterozygosity (cnLOH) on chromosome 9 implicated the CDK5RAP2 gene. Targeted sequencing identified a homozygous E234X mutation, only the third mutation to be described in CDK5RAP2, the first in an individual of non-Pakistani descent. Sensorineural hearing loss is not generally considered to be consistent with autosomal recessive microcephaly and therefore it seems likely that the deafness in this individual is caused by the co-occurrence of a further gene mutation, independent of CDK5RAP2. Nevertheless, further detailed clinical descriptions of rare CDK5RAP2 patients, including hearing assessments will be needed to resolve fully the phenotypic range associated with mutations in this gene. This study also highlights the utility of SNP-array testing to guide disease gene identification where an autosomal recessive condition is plausible.