Treacher Collins syndrome (TCS) is a rare congenital craniofacial disorder, typically inherited as an autosomal dominant condition. Here, we report on a family in which germline mosaicism for TCS was likely present. The proband was diagnosed with TCS based on the typical clinical features and a pathogenic variant TCOF1 (c.4369_4373delAAGAA, p.K1457Efs*12). The mutation was not detected in his parents\' peripheral blood DNA samples, suggesting a de novo mutation had occurred in the proband. However, a year later, the proband\'s mother became pregnant, and the amniotic fluid puncture revealed that the fetus carried the same mutation as the proband. Prenatal ultrasound also indicated a maxillofacial dysplasia with unilateral microtia. The mother then disclosed a previous birth history in which a baby had died of respiratory distress shortly after birth, displaying a TCS-like phenotype. Around the same time, the proband\'s father was diagnosed with mild bilateral conductive hearing loss. Based on array data, we concluded that the father may have had germline mosaicism for TCOF1 mutation. Our findings highlight the importance of considering germline mosaicism in sporadic de novo TCOF1 mutations when providing genetic consulting, and prenatal diagnosis is important when the proband\'s parents become pregnant again.

UNASSIGNED: Facioscapulohumeral muscular dystrophy type 1 (FSHD1) is one of the most common forms of autosomal-dominant muscular dystrophies characterized by variable disease penetrance due to shortened D4Z4 repeat units on 4q35. The molecular diagnosis of FSHD1 is usually made by Southern blotting, which is complex, time-consuming, and lacks clinical practicality. Therefore, in this study, optical genome mapping (OGM) is employed for the genetic diagnosis of FSHD1. Furthermore, epigenetic heterogeneity is determined from methylation analysis.
UNASSIGNED: Genomic DNA samples from four members of the same family were subjected to whole-exome sequencing. OGM was used to identify structural variations in D4Z4, while sodium bisulfite sequencing helped identify the methylation levels of CpG sites in a region located distally to the D4Z4 array. A multidisciplinary team collected the clinical data, and comprehensive family analyses aided in the assessment of phenotypes and genotypes.
UNASSIGNED: Whole-exome sequencing did not reveal variants related to clinical phenotypes in the patients. OGM showed that the proband was a compound heterozygote for the 4qA allele with four and eight D4Z4 repeat units, whereas the affected younger brother had only one 4qA allele with four D4Z4 repeat units. Both the proband and her younger brother were found to display asymmetric weakness predominantly involving the facial, shoulder girdle, and upper arm muscles, whereas the younger brother had more severe clinical symptoms. The proband\'s father, who was found to be normal after a neurological examination, also carried the 4qA allele with eight D4Z4 repeat units. The unaffected mother exhibited 49 D4Z4 repeat units of the 4qA allele and a minor mosaic pattern with four D4Z4 repeat units of the 4qA allele. Consequently, the presence of the 4qA allele in the four D4Z4 repeat units strongly pointed to the occurrence of maternal germline mosaicism. The CpG6 methylation levels were lower in symptomatic patients compared to those in the asymptomatic parents. The older sister had lower clinical scores and ACSS and higher CpG6 methylation levels than that of her younger brother.
UNASSIGNED: In this study, two siblings with FSHD1 with phenotypically normal parents were identified by OGM. Our findings suggest that the 4qA allele of four D4Z4 repeats was inherited through maternal germline mosaicism. The clinical phenotype heterogeneity is influenced by the CpG6 methylation levels. The results of this study greatly aid in the molecular diagnosis of FSHD1 and in also understanding the clinical phenotypic variability underlying the disease.

BACKGROUND: KIF2A-related tubulinopathy (MIM: #615411) is a very rare disorder that was clinically characterized as microcephaly, epilepsy, motor developmental disorder (MDD), and various malformations of cortical development, but intellectual disability (ID) or global developmental delay (GDD) was rarely reported in the patients.
METHODS: Quad whole-exome sequencing (WES) was performed on the proband, the older brother, and their parents. Sanger sequencing was used to verify the candidate gene variant.
RESULTS: The proband, a 23-month-old boy, was previously diagnosed with GDD, and his brother, aged nine years, had ID; both were born to a healthy couple. Quad-WES detected a novel heterozygous KIF2A variant, c.1318G>A (p.G440R), in both the brothers but not in the parents. In-silico analysis revealed that the variants G440R and G318R (which were previously reported in the only reported patient with GDD) lead to markedly enlarged side chains and hinder ATP\'s emplacement in the NBD pocket.
CONCLUSIONS: The type of KIF2A variants that sterically hinder ATP emplacing in KIF2A NBD pocket may be associated with the intellectual disability phenotype; however, further studies are needed. Findings in this case also suggest a rare parental germline mosaicism of KIF2A G440R.

Preimplantation genetic testing (PGT) for monogenic disorders (PGT-M) for germline mosaicism was previously highly dependent on polymerase chain reaction (PCR)-based directed mutation detection combined with linkage analysis of short tandem repeats (STRs). However, the number of STRs is usually limited. In addition, designing suitable probes and optimizing the reaction conditions for multiplex PCR are time-consuming and laborious. Here, we evaluated the effectiveness of next generation sequencing (NGS)-based haplotype linkage analysis in PGT of germline mosaicism.
PGT-M with NGS-based haplotype linkage analysis was performed for two families with maternal germline mosaicism for an X-linked Duchenne muscular dystrophy (DMD) mutation (del exon 45-50) or an autosomal TSC1 mutation (c.2074C > T). Trophectoderm biopsy and multiple displacement amplification (MDA) were performed for a total of nine blastocysts. NGS and Sanger sequencing were performed in genomic DNA of family members and embryonic MDA products to detect DMD deletion and TSC1 mutation, respectively. Single nucleotide polymorphism (SNP) sites closely linked to pathogenic mutations were detected with NGS and served in haplotype linkage analysis. NGS-based aneuploidy screening was performed for all embryos to reduce the risk of pregnancy loss.
All nine blastocytes showed conclusive PGT results. Each family underwent one or two frozen-thawed embryo transfer cycles to obtain a clinical pregnancy, and the prenatal diagnosis showed that the fetus was genotypically normal and euploid for both families.
NGS-SNP could effectively realize PGT for germline mosaicism. Compared with PCR-based methods, the NGS-SNP method with increased polymorphic informative markers can achieve a greater diagnostic accuracy. Further studies are warranted to verify the effectiveness of NGS-based PGT of germline mosaicism cases in the absence of surviving offsprings.

UNASSIGNED: Alport syndrome is a hereditary glomerulopathy featured by haematuria, proteinuria, and progressive renal failure. X-linked Alport syndrome (XLAS) due to COL4A5 disease-causing variants is the most common form. In the case of XLAS resulting from 10-18% presumed de novo COL4A5 disease-causing variants, there are only a few studies for mosaicism in the probands or parents. Very low-level (<1.0%) somatic mosaicism for COL4A5 disease-causing variants has not been published.
UNASSIGNED: Chinese XLAS families with suspected parental mosaicism were enrolled in the present study to evaluate the forms of mosaicism, to offer more appropriate genetic counseling. PCR and direct sequencing were used to detect COL4A5 disease-causing variants harbored by the affected probands in parental multi-tissue DNAs (peripheral blood, urine sediments, saliva, hair), and droplet digital PCR (ddPCR) was used to quantify the mutant COL4A5 allelic fractions in parental different samples such as peripheral blood, saliva, and urine sediments.
UNASSIGNED: A Chinese asymptomatic female with suspected somatic and germline mosaicism was enrolled in the present study. She gave birth to two boys with XLAS caused by a hemizygous disease-causing variant c. 2245-1G>A in COL4A5 (NM_033380) intron 28, whereas this disease-causing variant was not detected in genomic DNA extracted from peripheral blood leukocytes in the woman using Sanger sequencing. She had multiple normal urine test results, and continuous linear immunofluorescence staining of α2 (IV) and α5 (IV) chains of skin tissue. Sanger sequencing demonstrated that COL4A5 disease-causing variant c. 2245-1G>A was not detected in her genomic DNAs isolated from urine sediments, saliva, and hair roots. Using ddPCR, the wild-type and mutant-type (c.2245-1G>A) COL4A5 was identified in the female\'s genomic DNAs isolated from peripheral blood, saliva, and urine sediments. The mutant allelic fractions in these tissues were 0.26% (peripheral blood), 0.73% (saliva), and 1.39% (urine), respectively.
UNASSIGNED: Germline and very low-level somatic mosaicism for a COL4A5 splicing variant was detected in an asymptomatic female, which highlights that parental mosaicism should be excluded when a COL4A5 presumed de novo disease-causing variant is detected.

BACKGROUND: Nicolaides-Baraitser syndrome (NCBRS) is a severe neurodevelopmental disorder with multiple abnormalities. To date, all pathogenic variants in SMARCA2 causing NCBRS are de novo and most are missense variants located in the ATPase domain of SMARCA2 protein.
METHODS: In this study, a familial trio whole-exome sequencing was performed on the proband presenting with intellectual disability, early-onset epilepsy, and autistic features. A novel missense variant c.553C>G (p.Gln185Glu) in SMARCA2 was identified, which is located in the QLQ domain. The same variant was subsequently also found in the mother\'s ongoing pregnancy. Samples from accessible tissues such as saliva and sperm other than blood were collected from the parents, and the detection of the target variant was performed by amplicon-based deep sequencing.
RESULTS: Low-level mosaicism of the target variant c.553C>G (p.Gln185Glu) was detected in the father\'s sperm with allele fraction of 2.8% by amplicon-based deep sequencing, which was not detected in either parents\' blood or saliva specimens. Heterozygosity of this variant was confirmed in the proband.
CONCLUSIONS: This is the first report of paternal germline mosaicism for a SMARCA2 disease-causing variant. In addition, the missense variant c.553C>G (p.Gln185Glu) in the QLQ domain causes mainly neurological and developmental phenotypes with unremarkable characteristic facial features and limb abnormalities. Our findings expand the phenotypic spectrum and mode of genetic transmission associated with the SMARCA2 variants.

Germline mosaicism should be suspected when the same de novo mutations are identified in a second pregnancy with asymptomatic parents. Our study aims to find a feasible approach to reveal the existence of germline mosaicism. Multiplex Ligation-dependent Probe Amplification was performed on a Duchenne muscular dystrophy affected pedigree to detect deletion mutations. Then gap-polymerase chain reaction was performed to amplify the breakpoints junction sequence. Droplet digital polymerase chain reaction was utilized to identify the mutation frequencies in healthy parents. The same deletion in the exon 51 of the dystrophin gene, which was 50,035 bp in size, was detected in the proband and the fetus but not in their parents. Droplet digital polymerase chain reaction analysis of peripheral blood samples revealed mutant alleles of 3.53% in maternal blood cells. We here report a case of maternal low-level mosaicism confirmed by droplet digital polymerase chain reaction in peripheral blood samples, which reveals the existence of germline mosaicism. Gap-polymerase chain reaction combined with droplet digital polymerase chain reaction provide insights into the detection of germline mosaicism.

Familial Rubinstein-Taybi syndrome (RSTS) with recurrent RSTS siblings and apparently unaffected parents is rare; such cases might result from parental somatic and/or germline mosaicism. Parental low-level (<10%) germline mosaicism in the CREBBP-associated RSTS family has not been reported. Here, we present our studies of a Chinese family with two RSTS siblings and apparently unaffected parents. We detected the apparent de novo variant (DNV) c.3235C>T (p.Gln1079*) in CREBBP in the siblings via trio whole-exome sequencing. High-depth next-generation sequencing (NGS) for the parents revealed a low-level (<10%) mosaic variant in both the peripheral blood (3.64%) and buccal mucosa (1.94%) of the unaffected mother, indicating maternal somatic and germline mosaicism. Peripheral blood RNA-sequencing analysis for the patients and normal individuals indicated that the c.3235C>T (p.Gln1079*) non-sense variant did not trigger nonsense-mediated mRNA decay to reduce CREBBP mRNA levels. Transcriptome analysis revealed 151 downregulated mRNAs and 132 upregulated mRNAs between the patients and normal individuals. This study emphasizes that high-depth NGS using multiple specimens might be applied for a family with an affected sibling caused by an apparent CREBBP DNV to identify potential low-level parental mosaicism and provide an assessment of recurrence risk.

Prezygotic de novo mutations may be inherited from parents with germline mosaicism and are often overlooked when the resulting phenotype affects only one child. We aimed to identify paternal germline mosaicism in an index family and provide a strategy to determine germline mosaicism.\'
Whole-exome sequencing was performed on an Alport syndrome-affected child. Variants were validated using Sanger sequencing in the pedigree analysis. An apparent de novo mutation was tested by next-generation sequencing (NGS) following chromosome microdissection of the mutant region (MicroSeq) to clarify its homologous chromosome source. Mosaic mutation in sperm samples was detected using targeted next-generation sequencing (TNGS). Self-prepared mosaic DNA samples of the 3% and 0.1% mutant fractions were used to evaluate the TNGS detection sensitivity.
Two novel heterozygous variants, maternally inherited c.1322delT (p.Ile441Thrfs*17) and the de novo mutation c.2939T>A (p.Leu980Ter), in the COL4A3 gene were discovered in the propositus. MicroSeq identified c.2939T>A in the paternal chromosome, which was in trans with c.1322delT. The frequency of c.2937A was 2.65% in the father\'s sperm sample. We also showed that a 500X depth coverage may detect a mosaic mutation with an allele frequency as low as 2%-3% using TNGS.
MicroSeq is a valuable tool to identify the allele source of de novo mutations in a single patient. TNGS can be used to assess the mosaic ratios of known sites. We provided a systematic algorithm to detect germinal mosaicism in a single patient. This algorithm may have implications for genetic and reproductive counseling on germline mosaicism.