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Primary microcephaly (PM) is defined as a significant reduction in occipito-frontal circumference (OFC) of prenatal onset. Clinical and genetic heterogeneity of PM represents a diagnostic challenge. We performed detailed phenotypic and genomic analyses in a large cohort (n=169) of patients referred for PM, and could establish a molecular diagnosis in 38 patients. Pathogenic variants in ASPM and WDR62 were the most frequent causes in non-consanguineous patients in our cohort. In consanguineous patients, microarray and targeted gene panel analyses reached a diagnostic yield of 67%, which contrasts with a much lower rate in outbred patients (9%). Our series includes 15 previously unreported families and 11 novel pathogenic variants, and we identify novel candidate genes including IGF2BP3, DNAH2, and TSR1. We confirm progression of microcephaly over time in affected children. Epilepsy was an important associated feature in our PM cohort, affecting 34% of patients, with various degrees of severity and seizure types. Our findings will help to prioritize genomic investigations, accelerate molecular diagnoses and improve management of PM patients.

Figure 2 Clinical features of Patient 2 demonstrating microcephaly, flat forehead, dysplastic ears, short nose, retrognathia and irregular insertion of the toes.

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Study question Can we reliably detect aneuploidy in trophectoderm samples using SNP array data, independent of haplotyping? Summary answer We identified all chromosomes with meiotic copy number gains and non-mosaic copy number losses larger than 5Mb that had been detected with Karyomapping. What is known already Currently, generic methods such as Karyomapping (Vitrolife) and OnePGT (Agilent), are commonly used for preimplantation genetic testing of monogenic disorders (PGT-M). The genome-wide approach has the advantage that also aneuploidy can be detected by visualizing the haplotypes, the raw B-allele frequency (BAF) and the copynumber (CN) or Log2 ratio (Log2R) values. However, these haplotyping methods have considerable shortcomings for PGT-A including the need of reference DNA samples from both sides of the family and the long time required to access all haplotype information. We aimed to circumvent these shortcomings with our own proprietary method. Study design, size, duration We retrospectively re-analyzed the available raw SNP array data from 359 embryos with trophectoderm biopsy between September 1 2015 and December 31 2017 that were diagnosed with Karyomapping (Vitrolife) as being not genetically transferable. The local ethical committee approved this study under number B.U.N. 143201731745. Written informed consent was available. We intended to identify anomalies >5Mb that are detectable by haplotyping. Participants/materials, setting, methods SNPs were categorized based on the parental SNP genotypes. Next, the BAF values for each category of SNPs (cBAF) were extracted from the raw SNP array data and visualized on genomewide plots. The obtained cBAF plots were analyzed for the presence of aneuploidy together with raw BAF and Log2R SNP data. The results from the interpretation of these profiles were compared with the findings previously obtained with Karyomapping (Log2R, raw BAF and haplotype profiles). Main results and the role of chance A low level maternal contamination was observed in 5 embryos by analysis of cBAF profiles that had not been detected using Karyomapping. In five other embryos, an abnormal ploidy was observed with both methods. In the remaining 8027 chromosomes from 349 embryos, all 70 chromosomes with both parental homolog (BPH) anomalies larger than 5Mb detected with Karyomapping were identified by analysis of cBAF profiles. In 68 chromosomes the type (segmental or whole chromosome) and the copy number (+1 or + 2 copies) of the detected BPH anomaly was identical with both methods. All 93 chromosomes with a copy number loss larger than 5Mb detected with Karyomapping were identified by analysis of cBAF profiles. For 92 out of 93 chromosomes the type of non-mosaic copy number loss was identical (segmental or whole chromosome). Out of 17 single parental homolog (SPH) copy number gains interpreted as non-mosaic by Karyomapping, 16 were interpreted as non-mosaic based on cBAF profiles. In one instance, the SPH copy number detected with Karyomapping was interpreted as a mosaic SPH trisomy using cBAF. Two chromosomes with mosaic anomalies and one chromosome without diagnosis with Karyomapping were interpreted as non-mosaic anomalies using cBAF. Limitations, reasons for caution In case of consanguinity aneuploidy may be more difficult to detect. The limit of detection for the size of segmental anomalies remains to be determined. Wider implications of the findings By analyzing the cBAF profiles we were able to detect all types of chromosome anomaly that are detectable by haplotyping. We were able to identify meiotic BPH copy number gains and determine the parent of origin for all anomalies without haplotyping and hence without the need for DNA from familymembers. Trial registration number not applicable