H.J.M. Smeets

Rett syndrome in females with CTS hot spot deletions: a disorder profile.

From a series of 107 females with Rett syndrome (RTT), we describe the long‐term history of ten females with a deletion in the C‐terminus of the MECP2 gene. We observed that their disorder profile is clinically recognizable with time and different from other atypical and milder RTT phenotypes. In females with hot spot deletions in the C‐terminus, dystonia is present from childhood and results in a serious spine deformation in spite of preventive measures. Their adaptive behavior is surprisingly better preserved and in contrast with the typical decline in motor functioning. The delineaton of disorder profiles by long‐term clinical observation can teach us about genotype/phenotype relationships and eventually about the effect of epigenetic phenomena on the final phenotype.

Defective complex I assembly due to C20orf7 mutations as a new cause of Leigh syndrome

Background Leigh syndrome is an early onset, progressive, neurodegenerative disorder with developmental and motor skills regression. Characteristic magnetic resonance imaging abnormalities consist of focal bilateral lesions in the basal ganglia and/or the brainstem. The main cause is a deficiency in oxidative phosphorylation due to mutations in an mtDNA or nuclear oxidative phosphorylation gene. Methods and results A consanguineous Moroccan family with Leigh syndrome comprise 11 children, three of which are affected. Marker analysis revealed a homozygous region of 11.5 Mb on chromosome 20, containing 111 genes. Eight possible mitochondrial candidate genes were sequenced. Patients were homozygous for an unclassified variant (p.P193L) in the cardiolipin synthase gene (CRLS1). As this variant was present in 20% of a Moroccan control population and enzyme activity was only reduced to 50%, this could not explain the rare clinical phenotype in our family. Patients were also homozygous for an amino acid substitution (p.L159F) in C20orf7, a new complex I assembly factor. Parents were heterozygous and unaffected sibs heterozygous or homozygous wild type. The mutation affects the predicted S-adenosylmethionine (SAM) dependent methyltransferase domain of C20orf7, possibly involved in methylation of NDUFB3 during the assembly process. Blue native gel electrophoresis showed an altered complex I assembly with only 30–40% of mature complex I present in patients and 70–90% in carriers. Conclusions A new cause of Leigh syndrome can be a defect in early complex I assembly due to C20orf7 mutations.

The unfolding clinical spectrum of holoprosencephaly due to mutations in SHH, ZIC2, SIX3 and TGIF genes

Holoprosencephaly is a severe malformation of the brain characterized by abnormal formation and separation of the developing central nervous system. The prevalence is 1:250 during early embryogenesis, the live-born prevalence is 1:16 000. The etiology of HPE is extremely heterogeneous and can be teratogenic or genetic. We screened four known HPE genes in a Dutch cohort of 86 non-syndromic HPE index cases, including 53 family members. We detected 21 mutations (24.4%), 3 in SHH, 9 in ZIC2 and 9 in SIX3. Eight mutations involved amino-acid substitutions, 7 ins/del mutations, 1 frame-shift, 3 identical poly-alanine tract expansions and 2 gene deletions. Pathogenicity of mutations was presumed based on de novo character, predicted non-functionality of mutated proteins, segregation of mutations with affected family-members or combinations of these features. Two mutations were reported previously. SNP array confirmed detected deletions; one spanning the ZIC2/ZIC5 genes (approx. 100 kb) the other a 1.45 Mb deletion including SIX2/SIX3 genes. The mutation percentage (24%) is comparable with previous reports, but we detected significantly less mutations in SHH: 3.5 vs 10.7% (P=0.043) and significantly more in SIX3: 10.5 vs 4.3% (P=0.018). For TGIF1 and ZIC2 mutation the rate was in conformity with earlier reports. About half of the mutations were de novo, one was a germ line mosaic. The familial mutations displayed extensive heterogeneity in clinical manifestation. Of seven familial index patients only two parental carriers showed minor HPE signs, five were completely asymptomatic. Therefore, each novel mutation should be considered as a risk factor for clinically manifest HPE, with the caveat of reduced clinical penetrance.

Validation of preimplantation genetic diagnosis by PCR analysis: genotype comparison of the blastomere and corresponding embryo, implications for clinical practice

The aim of this study was to validate the overall preimplantation genetic diagnosis (PGD)-PCR procedure and to determine the diagnostic value. Genotyped embryos not selected for embryo transfer (ET) and unsuitable for cryopreservation after PGD were used for confirmatory analysis. The PGD genotyped blastomeres and corresponding embryos were compared, and morphology was scored on Day 4 post fertilization. To establish the validity of the PGD-PCR procedure and the diagnostic value, misdiagnosis rate, false-negative rate and negative predictive value were calculated. Moreover, comparison on the validity was made for the biopsy of one or two blastomeres. For the total embryo group (n = 422), a misdiagnosis rate of 7.1% and a false-negative rate of 3.1% were found. The negative predictive value was 96.1%. Poor morphology Day 4 embryos (Class 1) were over-represented in the embryo group in which the blastomere genotype was not confirmed by the whole embryo genotype. The misdiagnosis rate of Class 1 embryos was 12.5% and the false-negative rate 17.1%. Exclusion of these embryos resulted in a misdiagnosis rate of 6.1%, a false-negative rate of 0.5% and a negative predictive value of 99.3%. The two blastomere biopsies revealed a significant higher positive predictive value, lowering the misdiagnosis rate, whereas the negative predictive value remained the same. In conclusion, the PGD-PCR procedure is a valid diagnostic method to select unaffected embryos for ET. The misdiagnosis and false-negative rates decrease by rejecting Class 1 embryos for ET. The biopsy of a second blastomere improves the positive predictive value, lowering the misdiagnosis rate.

A patient with a mild holoprosencephaly spectrum phenotype and heterotaxy and a 1.3 Mb deletion encompassing GLI2.

Loss‐of‐function mutations of GLI2 are associated with features at the mild end of the holoprosencephaly spectrum, including abnormal pituitary gland formation and/or function, and craniofacial abnormalities. In addition patients may have branchial arch anomalies and polydactyly. Large, microscopically visible, interstitial deletions spanning 2q14.2 have been reported in patients with multiple congenital anomalies and intellectual disability. We report here on a patient with a mild holoprosencephaly spectrum phenotype (bilateral cleft lip and palate and abnormal pituitary gland formation with panhypopituitarism) and normal psychomotor development, who was found to carry a 1.3 Mb submicroscopic heterozygous deletion in 2q14.2, encompassing the GLI2 gene. We review the genotype and phenotype of previously published probands with GLI2 aberrations. Our findings confirm the association of haploinsufficiency of GLI2 and mild HPE spectrum features. Consistent with prior reports, we observed incomplete penetrance of the deletion in the family, illustrating the multifactorial etiology of holoprosencephaly spectrum features. In addition to the holoprosencephaly spectrum features, the proband had heterotaxy of the abdominal organs. Mutations in the known heterotaxy genes (NODAL, ZIC3 and CFC1) were excluded. The deletion contains five genes, in addition to GLI2, including the EPB4.1l5 gene. Based on findings in Epb4.1l5 mutant mice we hypothesize that Epb4.1l5 is a candidate gene for the heterotaxy observed in the proband.

Termination of damaged protein repair defines the occurrence of symptoms in carriers of the m.3243A>G tRNA Leu mutation.

Background: The m.3243A>G mutation in the mitochondrial tRNALeu(UUR) gene is an example of a mutation causing a very heterogeneous phenotype. It is the most frequent cause (80%) of the MELAS syndrome (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes), but it can also lead in addition or separately to type 2 diabetes, deafness, renal tubulopathy and/or cardiomyopathy. Methods: To identify pathogenic processes induced by this mutation, we compared global gene expression levels of muscle biopsies from affected and unaffected mutation carriers with controls. Results and conclusions: Gene expression changes were relatively subtle. In the asymptomatic group 200 transcripts were upregulated and 12 were downregulated, whereas in the symptomatic group 15 transcripts were upregulated and 52 were downregulated. In the asymptomatic group, oxidative phosphorylation (OXPHOS) complex I and IV genes were induced. Protein turnover and apoptosis were elevated, most likely due to the formation of dysfunctional and reactive oxygen species (ROS) damaged proteins. These processes returned to normal in symptomatic patients. Components of the complement system were upregulated in both groups, but the strongest in the symptomatic group, which might indicate muscle regeneration—most likely, protein damage and OXPHOS dysfunction stimulate repair (protein regeneration) and metabolic adaptation (OXPHOS). In asymptomatic individuals these processes suffice to prevent the occurrence of symptoms. However, in affected individuals the repair process terminates, presumably because of excessive damage, and switches to muscle regeneration, as indicated by a stronger complement activation. This switch leaves increasingly damaged tissue in place and muscle pathology becomes manifest. Therefore, the expression of complement components might be a marker for the severity and progression of MELAS clinical course.

Anticipation resulting in elimination of the myotonic dystrophy gene: a follow up study of one extended family.

We have re-examined an extended myotonic dystrophy (DM) family, previously described in 1955, in order to study the long term effects of anticipation in DM and in particular the implications for families affected by this disease. This follow up study provides data on 35 gene carriers and 46 asymptomatic at risk family members in five generations. Clinical anticipation, defined as the cascade of mild, adult, childhood, or congenital disease in subsequent generations, appeared to be a relentless process, occurring in all affected branches of the family. The cascade was found to proceed asynchronously in the different branches, mainly because of an unequal number of generations with mild disease. The transition from the mild to the adult type was associated with transmission through a male parent. Stable transmission of the asymptomatic/mild phenotype showed a female transmission bias. We further examined the extent and causes of gene loss in this pedigree. Gene loss in the patient group was complete, owing to infertility of the male patients with adult onset disease and the fact that mentally retarded patients did not procreate. Out of the 46 at risk subjects in the two youngest generations, only one was found to have a full mutation. This is the only subject who may transmit the gene to the sixth generation. No protomutation carriers were found in the fourth and fifth generations. Therefore it is highly probable that the DM gene will be eliminated from this pedigree within one generation. The high population frequency of DM can at present not be explained by the contribution of asymptomatic cases in the younger generations of known families, but is probably caused by the events in the ancestral generations.

Large scale mtDNA sequencing reveals sequence and functional conservation as major determinants of homoplasmic mtDNA variant distribution

The mitochondrial DNA (mtDNA) is highly variable, containing large numbers of pathogenic mutations and neutral polymorphisms. The spectrum of homoplasmic mtDNA variation was characterized in 730 subjects and compared with known pathogenic sites. The frequency and distribution of variants in protein coding genes were inversely correlated with conservation at the amino acid level. Analysis of tRNA secondary structures indicated a preference of variants for the loops and some acceptor stem positions. This comprehensive overview of mtDNA variants distinguishes between regions and positions which are likely not critical, mainly conserved regions with pathogenic mutations and essential regions containing no mutations at all.

Encephalopathic attacks in a family co-segregating myotonic dystrophy type 1, an intermediate Charcot-Marie-Tooth neuropathy and early hearing loss

Objective: To report new disease components in a unique myotonic dystrophy type 1 (DM1) family previously described by us in which all affected members also had a sensorimotor neuropathy that co-segregated with markers flanking the DM1 locus. Methods: Clinical observations, electrophysiology, audiometry, DNA studies. Results: During a follow-up period of over 25 years, the following were observed: (i) co-segregation of a striking new encephalopathic phenotype. In middle age, five patients were admitted on multiple occasions with attacks of impaired consciousness, psychomotor agitation, fever and, in about half of the cases, focal neurological signs, including unilateral weakness, sensory deficits and dysphasia. Reported onset phenomena consisted of confusion, headache, focal neurological symptoms and nausea; (ii) many patients show an early and severe sensorineural hearing loss; (iii) although they have mothers with the adult onset type, the four affected subjects from the youngest generation do not show any signs or symptoms of childhood or congenital myotonic dystrophy; (iv) the neuropathy meets the criteria of an intermediate type Charcot–Marie–Tooth (CMT), and is more severe in males; and (v) patients presented with an expanded fragment at the DM1 CTG repeat but this allele was refractory to PCR amplification and triplet repeat primed PCR at the 3′ end of the array, indicating the existence of an additional lesion at the 3′ end. Conclusions: The phenotype in this unique family extends beyond myotonic dystrophy and CMT to include encephalopathic attacks and early hearing loss, and is associated with an atypical mutation at the DM1 locus.

Selecting the Right Embryo in Mitochondrial Disorders

Mitochondrial disorders are among the most common inborn errors of metabolism. Based on genetic etiology, they can be divided into several subgroups, which require different approaches for reproductive counseling, addressing differences in recurrence risks and reproductive options. The majority are caused by mutations in nuclear genes, which are currently being rapidly resolved by whole-exome sequencing (WES) and which segregate in a Mendelian way. Prenatal diagnosis (PND) or preimplantation genetic diagnosis (PGD) is available for these families to prevent the birth of another severely affected child. In at least 15 % of cases, mitochondrial diseases are caused by mitochondrial DNA (mtDNA) mutations. Such mtDNA mutations can (1) be the result of a nuclear gene defect (multiple mtDNA deletions), (2) occur de novo (point mutations or large single mtDNA rearrangements), or (3) be maternally inherited (generally point mutations). For the maternally inherited heteroplasmic mtDNA mutations, the mitochondrial bottleneck is an important phenomenon defining the mtDNA mutation load in the offspring, with an often high (or unpredictable) recurrence risk and consequently complex counseling. PND to enable testing for mtDNA mutations is technically possible, but for many carriers of mtDNA point mutations, this approach is not applicable given the limitations in predicting phenotype. A total of 44 cases of PND performed in 35 mtDNA mutation carriers (m.3243A>G, m.9176T>C, m.8993T>G/C, m.8344A>G, m.13513G>A, m.11777C>A, m.10191T>C, m.10158T>C, m.3688G>A) have been described in literature. One additional unpublished case is presented here (m.3303C>T). For mtDNA point mutations which are most likely de novo in the affected child, the recurrence risk has been shown to be very low, and PND can be offered for reassurance. We have performed PND in four such cases, and six additional prenatal diagnoses were reported in literature. PND is also the most suitable option for female carriers with a low mutation load, demonstrating extreme skew such as mutations at nucleotide 8993. A fairly new option for preventing the transmission of mtDNA diseases is preimplantation genetic diagnosis (PGD). Embryos with mutant load below a mutation-specific or, if not possible, general expression threshold are transferred. A systematic review showed ≥95 % probability of being unaffected at (muscle) mutant level of 18 % or less, irrespective of the mutation. A total of 14 PGD cycles in six female carriers of heteroplasmic mtDNA mutations (m.3243A>G, m.8993T>G, m.8344A>G) have been completed at our center to date. All carriers produced oocytes below the threshold, and blastomere mutation load was representative for the whole embryo when two blastomeres were analyzed. A total of 12 PGD cycles in nine mtDNA mutation carriers (m.3243A>G, m.8993T>G) have been reported elsewhere. A total of six children were born after PGD and one pregnancy is ongoing. So, PGD is currently the best reproductive option for most maternally transmitted heteroplasmic mtDNA point mutations. Oocyte donation is a safe option to prevent the transmission of mtDNA disease to a future child for couples who reject PGD. Nuclear genome transfer techniques are currently investigated in research settings and might offer additional reproductive options in specific cases of mtDNA disease in the future.