Mosaicism of a TCOF1 mutation in an individual clinically unaffected with Treacher Collins syndrome

Treacher Collins syndrome (TCS) or mandibulofacial dysostosis is an autosomal dominant disorder of craniofacial development with 60% of its cases arising de novo. Other modes of inheritance such as autosomal recessive, gonadal mosaicism, and chromosomal rearrangement have also been proposed. This syndrome can result from TCOF1 gene mutations. In this study we identified a TCOF1 1408delAG heterozygous mutation in a patient with the clinical diagnosis of TCS. This same mutation was found in the clinically unaffected mother's leukocytes, hair root bulbs, buccal mucosa, urine, and stool. The mother has a clinically unaffected child and the maternal grandparents do not have the mutation. Because the mother has the mutation in cells derived from all three germ layers, we suspected the mutation was nonpenetrant. However, we could not detect the mutation in her skin fibroblasts, suggesting she is mosaic secondary to cell type specific selection.     

A familial case with interstitial 2q36 deletion: Variable phenotypic expression in full and mosaic state

Submicroscopic chromosomal anomalies play an important role in the etiology of craniofacial malformations, including midline facial defects with hypertelorism (MFDH). MFDH is a common feature combination in several conditions, of which Frontonasal Dysplasia is the most frequently encountered manifestation; in most cases the etiology remains unknown. We identified a parent to child transmission of a 6.2 Mb interstitial deletion of chromosome region 2q36.1q36.3 by array-CGH and confirmed by FISH and microsatellite analysis. The patient and her mother both presented an MFDH phenotype although the phenotype in the mother was much milder than her daughter. Inspection of haplotype segregation within the family of 2q36.1 region suggests that the deletion arose on a chromosome derived from the maternal grandfather. Evidences based on FISH, microsatellite and array-CGH analysis point to a high frequency mosaicism for presence of a deleted region 2q36 occurring in blood of the mother. The frequency of mosaicism in other tissues could not be determined. We here suggest that the milder phenotype observed in the proband's mother can be explained by the mosaic state of the deletion. This most likely arose by an early embryonic deletion in the maternal embryo resulting in both gonadal and somatic mosaicism of two cell lines, with and without the deleted chromosome. The occurrence of gonadal mosaicism increases the recurrence risk significantly and is often either underestimated or not even taken into account in genetic counseling where new mutation is suspected.     

Bohring‐Opitz syndrome caused by an ASXL1 mutation inherited from a germline mosaic mother

Bohring‐Opitz syndrome (BOS) is characterized clinically by severe developmental delays, microcephaly, failure to thrive, and characteristic facial features (prominent eyes, facial nevus simplex [flammeus], and others). Most patients meeting the clinical criteria for BOS (MIM: 605039) have a de novo nonsense or frameshift variant in ASXL1. We report a case of BOS caused by a pathogenic ASXL1 variant inherited from a germline mosaic mother. The ASXL1 mutation was detected via trio exome sequencing. The sequencing data demonstrated that the variant was inherited maternally but that the maternal variant was underrepresented in comparison to the normal allele. These results suggested maternal mosaicism for the variant. Additional testing on the mother was performed on buccal cell DNA, which was also consistent with mosaicism. The mother had been reported to be healthy and the family history is unremarkable. This is the first report of BOS caused by a mutation inherited from an unaffected, presumed germline mosaic parent. This phenomenon has been reported for other traditionally de novo dominant disorders like CHARGE syndrome and Cornelia de Lange syndrome. This case emphasizes the need for accurate low but non‐negative recurrence risk counseling for families with children with BOS and it impacts exome interpretation strategy.     

Germline and gonosomal mosaicism in the ATR-X syndrome

We have identified two females who are mosaic for an ATRX mutation. One case, in whom the mutation was undetectable in peripheral blood and buccal cells, has two affected sons and is therefore presumed to be a germline mosaic. In another case, the ATRX mutation is weakly detectable in the peripheral blood but only one of her three children who share the disease-associated haplotype carries the mutation and therefore it is concluded that she is a gonosomal mosaic. These cases provide the first molecular evidence for the occurrence of post-zygotic mutation in X-linked alpha thalassaemia mental retardation syndrome. The possibility of germline mosaicism must therefore be considered in the genetic counselling of ATR-X families.     

Evaluation of pharmacological induction of fatty acid beta-oxidation in X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is an inherited neurometabolic disorder associated with elevated levels of saturated unbranched very-long-chain fatty acids (VLCFA; C > 22:0) in plasma and tissues, and reduced VLCFA beta-oxidation in fibroblasts, white blood cells, and amniocytes from X-ALD patients. The X-ALD gene (ABCD1) at Xq28 encodes the adrenoleukodystrophy protein (ALDP) that is related to the peroxisomal ATP-binding cassette (ABCD) transmembrane half-transporter proteins. The function of ALDP is unknown and its role in VLCFA accumulation unresolved. Previously, our laboratory has shown that sodium 4-phenylbutyrate (4PBA) treatment of X-ALD fibroblasts results in increased peroxisomal VLCFA beta-oxidation activity and increased expression of the X-ALD-related protein, ALDRP, encoded by the ABCD2 gene. In this study, the effect of various pharmacological agents on VLCFA beta-oxidation in ALD mouse fibroblasts is tested. 4PBA, styrylacetate and benzyloxyacetate (structurally related to 4PBA), and trichostatin A (functionally related to 4PBA) increase both VLCFA (peroxisomal) and long-chain fatty acid [LCFA (peroxisomal and mitochondrial)] beta-oxidation. Isobutyrate, zaprinast, hydroxyurea, and 5-azacytidine had no effect on VLCFA or LCFA beta-oxidation. Lovastatin had no effect on fatty acid beta-oxidation under normal tissue culture conditions but did result in an increase in both VLCFA and LCFA beta-oxidation when ALD mouse fibroblasts were cultured in the absence of cholesterol. The effect of trichostatin A on peroxisomal VLCFA beta-oxidation is shown to be independent of an increase in ALDRP expression, suggesting that correction of the biochemical abnormality in X-ALD is not dependent on pharmacological induction of a redundant gene (ABCD2). These studies contribute to a better understanding of the role of ALDP in VLCFA accumulation and may lead to the development of more effective pharmacological therapies.     

A very long-chain acyl-CoA synthetase-deficient mouse and its relevance to X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative and endocrine disorder resulting from mutations in ABCD1 which encodes a peroxisomal membrane protein in the ATP binding cassette superfamily. The biochemical signature of X-ALD is increased levels of saturated very long-chain fatty acids (VLCFA; carbon chains of 22 or more) in tissues and plasma that has been associated with decreased peroxisomal very long-chain acyl-CoA synthetase (VLCS) activity and decreased peroxisomal VLCFA beta-oxidation. It has been hypothesized that ABCD1, which has no demonstrable VLCS activity itself, has an indirect effect on peroxisomal VLCS activity and VLCFA beta-oxidation by transporting fatty acid substrates, VLCS protein or some required co-factor into peroxisomes. Here we report the characterization of a Vlcs knockout mouse that exhibits decreased peroxisomal VLCS activity and VLCFA beta-oxidation but does not accumulate VLCFA. The XALD/Vlcs double knockout mouse has the biochemical abnormalities observed in the individual knockout mice but does not display a more severe X-ALD phenotype. These data lead us to conclude that (1) VLCFA levels are independent of peroxisomal fatty acid beta-oxidation, (2) there is no ABCD1/VLCS interaction and (3) the common severe forms of X-ALD cannot be modeled by decreasing peroxisomal VLCS activity in the XALD mouse.     

Parental gonadal but not somatic mosaicism leading to de novo NFIX variants shared by two brothers with Malan syndrome

The importance of gonadal mosaicism in families with apparently de novo mutations is being increasingly recognized. We report on two affected brothers initially suggestive of X‐linked or autosomal recessive inheritance. Malan syndrome due to shared NFIX variants was diagnosed in the brothers using exome sequencing. The boys shared the same paternal but not maternal haplotype around NFIX, and deep amplicon sequencing showed ~7% of the variant in paternal sperm but not in paternal blood and saliva. We performed review of previous cases of gonadal mosaicism, which suggests that the phenomenon is not uncommon. Gonadal mosaicism is often not accompanied by somatic mosaicism in tissues routinely used for testing, and if both types of mosaicism are present, the frequency of the variant in sperm is often higher than in somatic cells. In families with shared apparently de novo variants without evidence of parental somatic mosaicism, the transmitting parent may be determined through haplotyping of exome variants. Gonadal mosaicism has important consequences for recurrence risks and should be considered in genetic counseling in families with de novo variants.     

A de novo MLH1 germ line mutation in a 31-year-old colorectal cancer patient

Hereditary nonpolyposis colorectal cancer is an autosomal dominant cancer predisposition syndrome caused by inherited germ line mutations in DNA mismatch repair genes, predominantly MSH2 and MLH1. Here we report the first proven de novo germ line mutation in MLH1 (c.666dupA) identified in a 31-year-old colorectal cancer patient with the alteration being present in a heterozygous state in all three germ layers and homozygously in his colon cancer. The mutation was absent in both biological parents and all sibs available. Despite extensive polymorphic marker analysis, the parental origin of c.666dupA could not be conclusively determined, representing either a single mutational event in a parental germ cell or (maternal) gonadal mosaicism. Although rare, consequential application of the Bethesda guidelines for genetic testing should allow the clinician to readily identify colorectal cancer patients below age 50 years who carry de novo mismatch repair gene mutations.     

Identification and quantification of somatic mosaicism for a point mutation in a Duchenne muscular dystrophy family

Relatively few point mutations have been found in the dystrophin gene and of these only two have been associated with mosaicism. A single base insertion has been identified and quantified in a mother of two sons affected with Duchenne muscular dystrophy. It has been determined that she is a somatic mosaic with the mutation present in 25% of lymphocyte DNA. Further tissue lineages have been tested and the time at which the mutation arose was determined to be before the cellular differentiation into the bilaminar disc at approximately eight days after fertilisation. We suggest that the incidence of mosaicism for dystrophin point mutations may be higher than current data suggest.     

A novel mutation of MSX1 inherited from maternal mosaicism causes a severely affected child with nonsyndromic oligodontia

Mutations of MSX1 have been associated with nonsyndromic hypodontia. To seek the causal gene mutation sites in a family with nonsyndromic oligodontia, whole-exome sequencing (WES) was performed to seek the causative locus of the family. The candidate mutation was further identified by Sanger sequencing afterward. Two mutations of MSX1 were found both in the proband and her mother. One novel heterozygous missense mutation (c.C667G, p.R223G) of MSX1 inherited from the asymptomatic mother with mosaic mutation was located in the highly conserved fragment of exon 2. The other was a synonymous mutation (c.C348T, p.G116G) in exon 1, which had been reported. The novel maternal heterozygous missense mutation (c.C667G, p.R223G) was likely to be the major reason for nonsyndromic oligodontia in the family. This is the first mosaic variant that has been recorded of the MSX1 gene. Our study expands the phenotype–genotype correlation associated with MSX1 variants. Our study also suggests that the determination of the mosaicism is essential for precise genetic counseling if a disease appears to arise de novo.     

Germline mosaicism in Rett syndrome identified by prenatal diagnosis

Rett syndrome is an X-linked neurodevelopmental dominant disorder that affects almost exclusively girls. The vast majority of cases are sporadic and are caused by de novo mutations in the MECP2 gene, located in Xq28. Only few familial cases have been reported: in four cases, the mother was an asymptomatic carrier and in other four cases, the germline mosaicism in the mother was postulated. Owing to the above reported cases of germline mosaicism, we decided to offer prenatal diagnosis to all expectant mothers with a Rett daughter despite the absence of the causative mutation in parents' blood. We describe here the outcome of the first nine cases of prenatal diagnosis followed by our center. In eight cases, the fetus did not carry the mutation. In one case, the female fetus did carry the same mutation of the affected sister. The couple decided to interrupt the pregnancy and to devolve fetal tissues for research purposes. Our results indicate that prenatal diagnosis should be proposed to all couples with a Rett daughter, even when the mutation is apparently de novo. Moreover, one positive prenatal test among the first nine cases indicates that germline mosaicism may be seriously considered for the assessment of recurrence risk during genetic counseling.     

Parental SCN1A mutation mosaicism in familial Dravet syndrome

Different SCN1A mutations are known to cause a variety of phenotypes, such as generalized epilepsy with febrile seizures plus (GEFS+), Dravet syndrome and familial hemiplegic migraine (FHM). In Dravet syndrome, most mutations are de novo and familial cases are rare. In this study, Dravet syndrome is observed in two maternal half sisters. They have healthy fathers and their common mother has never experienced seizures, but has a lifelong history of migraine. Direct sequencing of DNA extracted from blood revealed a heterozygous SCN1A nonsense mutation c.3985C>T in the sisters, but not in the mother. The mutation induces a premature stop codon and probably leads to a non-functional protein. Further examination of the mother's DNA showed that she has a mosaicism of the mutation. This report of parental SCN1A nonsense mutation mosaicism in familial Dravet syndrome suggests that mosaicism might be more common than previously suspected and emphasizes the importance of taking mosaicism into account in genetic counselling of Dravet syndrome and SCN1A mutations. Furthermore, whether the migraine of the mother could be influenced by her SCN1A mutation mosaicism is not known, but increased awareness of migraine in future studies of SCN1A related epilepsies could clarify this intriguing link between migraine and epilepsy.     

Carrier detection of haemophilia A using DNA markers in families with an isolated affected male

The carrier status of women in five families with an isolated haemophilia male was assessed by pedigree analysis, coagulation factor assays and DNA markers. In three families, ten women could be given very low risks of being carriers based on DNA analysis. In two of the families the DNA markers identified the mutation as originating in either the maternal or maternal grandfather's germ cell. Combined DNA and coagulation data suggested that the affected male in a third family was a de novo mutation. DNA analysis of the affected male in another family identified a large deletion of the F8 gene which was present in his mother and three sisters, suggesting that the grandmother was a carrier. A combination of coagulation factor data and DNA marker assessment can determine the carrier status of the majority of females in families with isolated affected haemophilia A males.     

Incidence of mosaicism in 1055 de novo NF2 cases: much higher than previous estimates with high utility of next-generation sequencing

PurposeTo evaluate the incidence of mosaicism in de novo neurofibromatosis 2 (NF2).MethodsPatients fulfilling NF2 criteria, but with no known affected family member from a previous generation (n = 1055), were tested for NF2 variants in lymphocyte DNA and where available tumor DNA. The proportion of individuals with a proven or presumed mosaic NF2 variant was assessed and allele frequencies of identified variants evaluated using next-generation sequencing.ResultsThe rate of proven/presumed mosaicism was 232/1055 (22.0%). However, nonmosaic heterozygous pathogenic variants were only identified in 387/1055 (36.7%). When variant detection rates in second generation nonmosaics were applied to de novo cases, we assessed the overall probable mosaicism rate to be 59.7%. This rate differed by age from 21.7% in those presenting with bilateral vestibular schwannoma <20 years to 80.7% in those aged ≥60 years. A mosaic variant was detected in all parents of affected children with a single-nucleotide pathogenic NF2 variant.ConclusionThis study has identified a very high probable mosaicism rate in de novo NF2, probably making NF2 the condition with the highest expressed rate of mosaicism in de novo dominant disease that is nonlethal in heterozygote form. Risks to offspring are small and probably correlate with variant allele frequency detected in blood.     

X-linked adrenoleukodystrophy: very long-chain fatty acid metabolism, ABC half-transporters and the complicated route to treatment

X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene that encodes a peroxisomal membrane located ABC half-transporter named ALDP. Mutations in ALDP result in elevated levels of very long-chain fatty acids (VLCFA) and reduced VLCFA beta-oxidation in peroxisomes. The peroxisomal membrane harbors three additional closely related ABC half-transporters, ALDRP, PMP70 and PMP69 (PMP70R). ABC half-transporters must dimerize to form a functional full-transporter. Whether ALDP forms a homodimer or a heterodimer has not yet been resolved, but most indirect evidence favors homodimerization. The peroxisomal ABC half-transporters are functionally related. Over-expression of ALDRP can correct the biochemical defect both in X-ALD patients cells and the Abcd1 knockout mouse, providing an exciting new possibility for treatment of X-ALD patients. This paper provides an overview of current knowledge and the problems that have been encountered.     

Identification of somatic and germline mosaicism for a keratin 5 mutation in epidermolysis bullosa simplex in a family of which the proband was previously regarded as a sporadic case

Epidermolysis bullosa simplex (EBS) is an autosomal-dominant inherited blistering skin disease characterized by intraepidermal blistering due to mechanical stress-induced degeneration of basal keratinocytes. EBS is caused by mutations in either keratin 5 or keratin 14, the major keratins expressed in the basal layer of the epidermis. We experienced a unique EBS-affected family. The proband had a heterozygous 1649delG mutation in the keratin 5 gene and had been reported as a case of de novo mutation, because the mutations were not detected in the parents' DNA from blood samples. However, the proband's younger sister was revealed to have the same disease at birth and we found the same mutation in her. We reinvestigated the familial segregation of the 1649delG mutation and it was shown that the mother's DNA from hair bulb and buccal cell samples had the 1649delG mutation heterozygously, but her DNA from blood samples did not. A careful check on the mother's history disclosed that she had migratory circinate pigmentation in her skin in childhood, which means maternal somatic and germline mosaicism. The demonstration of somatic and gonadal mosaicism in the keratin 5 gene is important for accurate genetic counselling of families with sporadic cases of EBS.     

Fraternal twins with Aarskog-Scott syndrome due to maternal germline mosaicism

Aarskog-Scott syndrome is a rare X-linked recessive disorder with characteristic facial, skeletal, and genital abnormalities. We report on Aarskog-Scott syndrome in male dizygotic twins with an identical de novo mutation in FGD1 that resulted from germline mosaicism in the phenotypically normal mother. This is the first report of inheritance by germline mosaicism for the FGD1 gene.