Genetic landscape of congenital insensitivity to pain and hereditary sensory and autonomic neuropathies.

Congenital insensitivity to pain (CIP) and hereditary sensory and autonomic neuropathies (HSAN) are clinically and genetically heterogeneous disorders exclusively or predominantly affecting the sensory and autonomic neurons. Due to the rarity of the diseases and findings based mainly on single case reports or small case series, knowledge about these disorders is limited. Here, we describe the molecular workup of a large international cohort of CIP/HSAN patients including patients from normally under-represented countries. We identify 80 previously unreported pathogenic or likely pathogenic variants in a total of 73 families in the >20 known CIP/HSAN-associated genes. The data expand the spectrum of disease-relevant alterations in CIP/HSAN, including novel variants in previously rarely recognized entities such as ATL3-, FLVCR1- and NGF-associated neuropathies and previously under-recognized mutation types such as larger deletions. In silico predictions, heterologous expression studies, segregation analyses and metabolic tests helped to overcome limitations of current variant classification schemes that often fail to categorize a variant as disease-related or benign. The study sheds light on the genetic causes and disease-relevant changes within individual genes in CIP/HSAN. This is becoming increasingly important with emerging clinical trials investigating subtype or gene-specific treatment strategies.

Molecular pathophysiology of human MICU1 deficiency.

AIMS: MICU1 encodes the gatekeeper of the mitochondrial Ca2+ uniporter, MICU1 and biallelic loss-of-function mutations cause a complex, neuromuscular disorder in children. Although the role of the protein is well understood, the precise molecular pathophysiology leading to this neuropaediatric phenotype has not been fully elucidated. Here we aimed to obtain novel insights into MICU1 pathophysiology. METHODS: Molecular genetic studies along with proteomic profiling, electron-, light- and Coherent anti-Stokes Raman scattering microscopy and immuno-based studies of protein abundances and Ca2+ transport studies were employed to examine the pathophysiology of MICU1 deficiency in humans. RESULTS: We describe two patients carrying MICU1 mutations, two nonsense (c.52C>T; p.(Arg18*) and c.553C>T; p.(Arg185*)) and an intragenic exon 2-deletion presenting with ataxia, developmental delay and early onset myopathy, clinodactyly, attention deficits, insomnia and impaired cognitive pain perception. Muscle biopsies revealed signs of dystrophy and neurogenic atrophy, severe mitochondrial perturbations, altered Golgi structure, vacuoles and altered lipid homeostasis. Comparative mitochondrial Ca2+ transport and proteomic studies on lymphoblastoid cells revealed that the [Ca2+ ] threshold and the cooperative activation of mitochondrial Ca2+ uptake were lost in MICU1-deficient cells and that 39 proteins were altered in abundance. Several of those proteins are linked to mitochondrial dysfunction and/or perturbed Ca2+ homeostasis, also impacting on regular cytoskeleton (affecting Spectrin) and Golgi architecture, as well as cellular survival mechanisms. CONCLUSIONS: Our findings (i) link dysregulation of mitochondrial Ca2+ uptake with muscle pathology (including perturbed lipid homeostasis and ER-Golgi morphology), (ii) support the concept of a functional interplay of ER-Golgi and mitochondria in lipid homeostasis and (iii) reveal the vulnerability of the cellular proteome as part of the MICU1-related pathophysiology.

Autosomal dominant spinal muscular atrophy with lower extremity predominance: A recognizable phenotype of BICD2 mutations.

INTRODUCTION: Heterozygous BICD2 gene mutations cause a form of autosomal dominant spinal muscular atrophy with lower extremity predominance (SMALED). METHODS: We analyzed the BICD2 gene in a selected group of 25 index patients with neurogenic muscle atrophy. RESULTS: We identified 2 new BICD2 missense mutations, c.2515G>A, p.Gly839Arg, in a family with autosomal dominant inheritance, and c.2202G>T, p.Lys734Asn, as a de novo mutation in an isolated patient with similar phenotype. The patients had congenital foot contractures, muscle atrophy of the legs, and slowly progressive weakness of the shoulder girdle. There was no apparent sensory or brain dysfunction. One patient died of unrelated reasons at age 52 years. Autopsy revealed no upper motor neuron and only moderate lower motor neuron loss, but there was distal corticospinal tract degeneration and marked neurogenic muscular atrophy. CONCLUSION: These findings give further insight into the clinical and pathoanatomical consequences of BICD2 mutations. Muscle Nerve 54: 496-500, 2016.

Patient with three euchromatic supernumerary marker chromosomes derived from chromosomes 1, 12, and 18: characterization and evaluation of the aberrations.

The genetic relevance of small supernumerary marker chromosomes (sSMCs) depends on their content of euchromatin. In case of mosaicism, the phenotype of the carrier furthermore is influenced by the distribution of the marker in the body. In the majority of reported cases no correlation of the degree of mosaicism in the tissue(s) analyzed and the phenotype could be detected. In particular, non-acrocentric derived sSMCs show a strong tendency to appear in mosaic state irrespective of the clinical picture. We present a patient with cognitive disability and mild craniofacial dysmorphisms with mosaicism of three different autosomal marker chromosomes. The extra chromosomes were analyzed by a combination of SNP array and a variety of fluorescence in situ hybridization (FISH) probes. All three markers were identified as ring chromosomes containing different amounts of euchromatic material derived from chromosome 1 (1p12 → q21), 12 (12p13.1 → q13.11) and 18 (18p11.21 → q11.2). The size and the frequency of the sSMCs were strikingly different, besides, we observed an unequal combination of the three derivates.

Closing the Gap - Detection of 5q-Spinal Muscular Atrophy by Short-Read Next-Generation Sequencing and Unexpected Results in a Diagnostic Patient Cohort.

BACKGROUND: The importance of early diagnosis of 5q-Spinal muscular atrophy (5q-SMA) has heightened as early intervention can significantly improve clinical outcomes. In 96% of cases, 5q-SMA is caused by a homozygous deletion of SMN1. Around 4 % of patients carry a SMN1 deletion and a single-nucleotide variant (SNV) on the other allele. Traditionally, diagnosis is based on multiplex ligation probe amplification (MLPA) to detect homozygous or heterozygous exon 7 deletions in SMN1. Due to high homologies within the SMN1/SMN2 locus, sequence analysis to identify SNVs of the SMN1 gene is unreliable by standard Sanger or short-read next-generation sequencing (srNGS) methods. OBJECTIVE: The objective was to overcome the limitations in high-throughput srNGS with the aim of providing SMA patients with a fast and reliable diagnosis to enable their timely therapy. METHODS: A bioinformatics workflow to detect homozygous SMN1 deletions and SMN1 SNVs on srNGS analysis was applied to diagnostic whole exome and panel testing for suggested neuromuscular disorders (1684 patients) and to fetal samples in prenatal diagnostics (260 patients). SNVs were detected by aligning sequencing reads from SMN1 and SMN2 to an SMN1 reference sequence. Homozygous SMN1 deletions were identified by filtering sequence reads for the ,, gene-determining variant" (GDV). RESULTS: 10 patients were diagnosed with 5q-SMA based on (i) SMN1 deletion and hemizygous SNV (2 patients), (ii) homozygous SMN1 deletion (6 patients), and (iii) compound heterozygous SNVs in SMN1 (2 patients). CONCLUSIONS: Applying our workflow in srNGS-based panel and whole exome sequencing (WES) is crucial in a clinical laboratory, as otherwise patients with an atypical clinical presentation initially not suspected to suffer from SMA remain undiagnosed.

Newbornscreening SMA - From Pilot Project to Nationwide Screening in Germany.

Now that targeted therapies for spinal muscular atrophy are available, attempts are being made worldwide to include screening for spinal muscular atrophy in general newborn screening. In Germany, after pilot projects from 2018-2021, it was included in the general newborn screening from October 2021. To ensure a smooth transition, criteria for follow-up were developed together with key stakeholders. At the beginning of the transition to nationwide screening, false positive findings were reported in 3 patients. After optimization of the screening method in the laboratories concerned, all findings have been subsequently confirmed. On average, the first presentation to a neuromuscular center occurred on day 12 of life, and in patients with 2 or 3 SMN2 copies, therapy started on day 26 of life. Compared with the pilot project, there was no significant delay in timing.

Novel phenotype with prominent cerebellar oculomotor dysfunction in spastic paraplegia type 39.

OBJECTIVES: The term hereditary spastic paraplegia comprises an ever-expanding array of neurological disorders with distinct aetiologies. Spastic paraplegia gene 39 is one of the many genetically defined types with features of other organs and neurological systems in addition to paraspasticity. We describe a large kindred with a novel clinical phenotype as, in addition to spastic paraplegia, affected subjects suffered from a prominent cerebellar oculomotor dysfunction with two hitherto undescribed mutations of PNPLA6. METHODS: Three of five genetically tested family members of a large kindred were affected by spastic gait and a unique and prominent cerebellar oculomotor dysfunction. Further clinical, imaging, laboratory and videonystagmographic data were analyzed. Genetic analysis was done using next-generation sequencing. RESULTS: The most salient clinical feature, in addition to paraspasticity, in three of five subjects was cerebellar oculomotor dysfunction with an upbeating nystagmus provoked by downward gaze. Genetic analysis revealed two hitherto unknown sequence variants in the PNPLA6 gene, a splice-site variant c.1635 + 3G > T and a missense variant c.3401A > T, p.(Asp1134Val). In addition to cerebellar oculomotor dysfunction, compound-heterozygous siblings presented with paraspasticity and a moderate hypogonadotropic hypogonadism in the female. A paternal uncle being homozygous for the splice-site variant of PNPLA6 presented with increased lower limb reflexes and an unstable gait. Treatment with 4-aminopyridine, a potassium channel blocker, lead to meaningful improvement of clinical symptoms. CONCLUSIONS: The unique and prominent cerebellar ocular motor disorder in our family broadens the spectrum of clinical phenotypes associated with variations in the PNLA6 gene. The finding of paraspasticity with cerebellar oculomotor dysfunction alongside inconspicuous brainstem imaging may raise suspicion of complex HSP with PNPLA6 mutations.

Newborn Screening for SMA - Can a Wait-and-See Strategy be Responsibly Justified in Patients With Four SMN2 Copies?

BACKGROUND: Early treatment after genetic newborn screening (NBS) for SMA significantly improves outcomes in infantile SMA. However, there is no consensus in the SMA treatment community about early treatment initiation in patients with four copies of SMN2. OBJECTIVE: Approach to a responsible treatment strategy for SMA patients with four SMN2 copies detected in newborn screening. METHODS: Inclusion criteria were a history of SMA diagnosed by NBS, age > 12 months at last examination, and diagnosis of four SMN2 copies at confirmatory diagnosis. RESULTS: 21 patients with SMA and four SMN2 copies were identified in German screening projects over a three-year period. In three of them, the SMN2 copy number had to be corrected later, and three patients were lost to follow-up. Eight of the fifteen patients who were subject to long-term follow-up underwent presymptomatic therapy between 3 and 36 months of age and had no definite disease symptoms to date. Five of the other seven patients who underwent a strict follow-up strategy, showed clinical or electrophysiological disease onset between 1.5 and 4 years of age. In two of them, complete recovery was not achieved despite immediate initiation of treatment after the onset of the first symptoms. CONCLUSION: A remarkable proportion of patients with four copies of SMN2 develop irreversible symptoms within the first four years of life, if a wait-and-see strategy is followed. These data argue for a proactive approach, i.e., early initiation of treatment in this subgroup of SMA patients.

Hereditary Sensory and Autonomic Neuropathy: A Case Series of Six Children.

Objectives: Hereditary sensory and autonomic neuropathy (HSAN) is a group of rare disorders affecting the sensory and autonomic neurons. Herein, we describe the clinical and genetic profile of six children with HSAN. Methods: Hospital records of six children diagnosed with HSAN over 7 years (2011-2018) were retrieved. Clinical features, electrophysiological studies, and genetic reports were collected from the case files. Results: The presenting clinical features in these six cases were developmental delay, recurrent febrile episodes, rhinitis, recurrent nonhealing ulcers, burns, self-mutilations, chronic osteomyelitis, and corneal ulcers. Electrophysiology studies showed predominant sensory axonal neuropathy. Autonomic features noted were recurrent fever, constipation, abdominal distension, hypertension, and vasomotor rhinitis. Genetic testing was done with next-generation sequencing in all six children. Causative genetic variants were identified in the NTRK1, PRDM12, DST gene, and a novel compound heterozygous variant in the FLVCR1 gene. The diagnosis of HSAN was delayed in most of our children due to variable presentation and lack of awareness among the treating paediatricians. Conclusions: Although the clinical presentation of HASN is highly variable, it is dominated by pain and temperature insensitivity and self-mutilation. Our report of six children with HSAN expands the existing knowledge on phenotype and genotype spectrum of HSAN.

Genetic pain loss disorders.

Genetic pain loss includes congenital insensitivity to pain (CIP), hereditary sensory neuropathies and, if autonomic nerves are involved, hereditary sensory and autonomic neuropathy (HSAN). This heterogeneous group of disorders highlights the essential role of nociception in protecting against tissue damage. Patients with genetic pain loss have recurrent injuries, burns and poorly healing wounds as disease hallmarks. CIP and HSAN are caused by pathogenic genetic variants in >20 genes that lead to developmental defects, neurodegeneration or altered neuronal excitability of peripheral damage-sensing neurons. These genetic variants lead to hyperactivity of sodium channels, disturbed haem metabolism, altered clathrin-mediated transport and impaired gene regulatory mechanisms affecting epigenetic marks, long non-coding RNAs and repetitive elements. Therapies for pain loss disorders are mainly symptomatic but the first targeted therapies are being tested. Conversely, chronic pain remains one of the greatest unresolved medical challenges, and the genes and mechanisms associated with pain loss offer new targets for analgesics. Given the progress that has been made, the coming years are promising both in terms of targeted treatments for pain loss disorders and the development of innovative pain medicines based on knowledge of these genetic diseases.

Growth retardation versus overgrowth: Silver-Russell syndrome is genetically opposite to Beckwith-Wiedemann syndrome.

Human growth is a complex process that requires the appropriate interaction of many players. Central members in the growth pathways are regulated epigenetically and thereby reflect the profound significance of imprinting for correct mammalian ontogenesis. In this review, we show that the growth retardation disorder Silver-Russell syndrome (SRS) is a suitable model to decipher the role of imprinting in growth. As we will show, SRS should not only be regarded as the genetically (and clinically) opposite disease to Beckwith-Wiedemann syndrome, but it also represents the first human disorder with imprinting disturbances that affect two different chromosomes (i.e. chromosomes 7 and 11). Thus, a functional interaction between factors encoded by chromosomes 7 and 11 is likely.

Diagnosing small fiber neuropathy in clinical practice: a deep phenotyping study.

BACKGROUND AND AIMS: Small fiber neuropathy (SFN) is increasingly suspected in patients with pain of uncertain origin, and making the diagnosis remains a challenge lacking a diagnostic gold standard. METHODS: In this case-control study, we prospectively recruited 86 patients with a medical history and clinical phenotype suggestive of SFN. Patients underwent neurological examination, quantitative sensory testing (QST), and distal and proximal skin punch biopsy, and were tested for pain-associated gene loci. Fifty-five of these patients additionally underwent pain-related evoked potentials (PREP), corneal confocal microscopy (CCM), and a quantitative sudomotor axon reflex test (QSART). RESULTS: Abnormal distal intraepidermal nerve fiber density (IENFD) (60/86, 70%) and neurological examination (53/86, 62%) most frequently reflected small fiber disease. Adding CCM and/or PREP further increased the number of patients with small fiber impairment to 47/55 (85%). Genetic testing revealed potentially pathogenic gene variants in 14/86 (16%) index patients. QST, QSART, and proximal IENFD were of lower impact. CONCLUSION: We propose to diagnose SFN primarily based on the results of neurological examination and distal IENFD, with more detailed phenotyping in specialized centers.

Newborn screening for spinal muscular atrophy in Germany: clinical results after 2 years.

BACKGROUND: Spinal muscular atrophy (SMA) is the most common neurodegenerative disease in childhood. Since motor neuron injury is usually not reversible, early diagnosis and treatment are essential to prevent major disability. Our objective was to assess the impact of genetic newborn screening for SMA on outcome. METHODS: We provided clinical data from 43 SMA patients, identified via polymerase chain reaction of the SMN1 gene from dried blood spots between January 2018 and January 2020 in Germany. Follow-up included neurophysiological examinations and standardized physiotherapeutic testing. RESULTS: Detection of SMA with newborn screening was consistent with known incidence in Germany. Birth prevalence was 1:6910; 39.5% had 2 SMN2 copies, 23% had 3 SMN2 copies, 32.5% had 4 copies, and 4.5% had 5 copies of the SMN2 gene. Treatment with SMA-specific medication could be started at the age of 14-39 days in 21 patients. Pre-symptomatically treated patients remained throughout asymptomatic within the observation period. 47% of patients with 2 SMN2 copies showed early, presumably intrauterine onset of disease. These patients reached motor milestones with delay; none of them developed respiratory symptoms. Untreated children with 2 SMN2 copies died. Untreated children with 3 SMN2 copies developed proximal weakness in their first year. In patients with ≥ 4 SMN2 copies, a follow-up strategy of "watchful waiting" was applied despite the fact that one of them was treated from the age of 6 months. Two infant siblings with 4 SMN2 copies were identified with a missed diagnosis of SMA type 3. CONCLUSION: Identification of newborns with infantile SMA and prompt SMA-specific treatment substantially improves neurodevelopmental outcome, and we recommend implementation in the public newborn screening in countries where therapy is available. Electrophysiology is a relevant parameter to support the urgency of therapy. There has to be a short time interval between a positive screening result and referral to a therapy-ready specialized treatment center.

Paroxysmal tonic upgaze: A heterogeneous clinical condition responsive to carbonic anhydrase inhibition.

BACKGROUND: Paroxysmal tonic upgaze (PTU), defined as an involuntary upward movement of the eyes, has been considered as a benign phenomenon but may also be associated with ataxia and developmental delay. METHODS: We report eight children with PTU; six of them also exhibiting symptoms of ataxia and/or developmental delay. Treatment with carbonic anhydrase inhibition was offered to children with persisting and/or severe forms. RESULTS: Whole-exome sequencing and genome-wide array analysis (n = 7) did not reveal mutations in the three known genes associated with PTU (CACNA1A, GRID2, SEPSECS), whereas by MLPA a heterozygous deletion of exon 31 of the CACNA1A gene could be detected in one patient, her mother and two further family members. Further exome and array analysis showed no recurrent variants in potentially novel PTU-related genes in more than one patient. A de novo variant at a highly conserved position in the SIM1 gene was detected in one patient, for which a pathogenic effect could be speculated. Carbonic anhydrase inhibition was started in five children and proved at least partially effective in all of them. CONCLUSION: Irrespective of the clinical background and the molecular basic mechanism of PTU, therapeutic carbonic anhydrase inhibition was effective in all five children (acetazolamide, n = 3; sultiame, n = 2) who received this treatment.

Need for a precise molecular diagnosis in Beckwith-Wiedemann and Silver-Russell syndrome: what has to be considered and why it is important.

Molecular diagnostic testing of the 11p15.5-associated imprinting disorders Silver-Russell and Beckwith-Wiedemann syndrome (SRS, BWS) is challenging due to the broad spectrum of molecular defects and their mosaic occurrence. Additionally, the decision on the molecular testing algorithm is hindered by their clinical heterogeneity. However, the precise identification of the type of defect is often a prerequisite for the clinical management and genetic counselling. Four major molecular alterations (epimutations, uniparental disomies, copy number variants, single nucleotide variants) have been identified, but their frequencies vary between SRS and BWS. Due to their molecular aetiology, epimutations in both disorders as well as upd(11)pat in BWS are particular prone to mosaicism which might additionally complicate the interpretation of testing results. We report on our experience of molecular analysis in a total cohort of 1448 patients referred for diagnostic testing of BWS and SRS, comprising a dataset from 737 new patients and from 711 cases from a recent study. Though the majority of positively tested patients showed the expected molecular results, we identified a considerable number of clinically unexpected molecular alterations as well as not yet reported changes and discrepant mosaic distributions. Additionally, the rate of multilocus imprinting disturbances among the patients with epimutations and uniparental diploidies could be further specified. Altogether, these cases show that comprehensive testing strategies have to be applied in diagnostic testing of SRS and BWS. The precise molecular diagnosis is required as the basis for a targeted management (e.g. ECG (electrocardiogram) and tumour surveillance in BWS, growth treatment in SRS). The molecular diagnosis furthermore provides the basis for genetic counselling. However, it has to be considered that recurrence risk calculation is determined by the phenotypic consequences of each molecular alteration and mechanism by which the alteration arose. KEY MESSAGES: The detection rates for the typical molecular defects of Beckwith-Wiedemann syndrome or Silver-Russell syndrome (BWS, SRS) are lower in routine cohorts than in clinically well-characterised ones. A broad spectrum of (unexpected) molecular alterations in both disorders can be identified. Multilocus imprinting disturbances (MLID) are less frequent in SRS than expected. The frequency of MLID and uniparental diploidy in BWS is confirmed. Mosaicism is a diagnostic challenge in BWS and SRS. The precise determination of the molecular defects affecting is the basis for a targeted clinical management and genetic counselling.

Differential diagnosis of vacuolar myopathies in the NGS era.

Altered autophagy accompanied by abnormal autophagic (rimmed) vacuoles detectable by light and electron microscopy is a common denominator of many familial and sporadic non-inflammatory muscle diseases. Even in the era of next generation sequencing (NGS), late-onset vacuolar myopathies remain a diagnostic challenge. We identified 32 adult vacuolar myopathy patients from 30 unrelated families, studied their clinical, histopathological and ultrastructural characteristics and performed genetic testing in index patients and relatives using Sanger sequencing and NGS including whole exome sequencing (WES). We established a molecular genetic diagnosis in 17 patients. Pathogenic mutations were found in genes typically linked to vacuolar myopathy (GNE, LDB3/ZASP, MYOT, DES and GAA), but also in genes not regularly associated with severely altered autophagy (FKRP, DYSF, CAV3, COL6A2, GYG1 and TRIM32) and in the digenic facioscapulohumeral muscular dystrophy 2. Characteristic histopathological features including distinct patterns of myofibrillar disarray and evidence of exocytosis proved to be helpful to distinguish causes of vacuolar myopathies. Biopsy validated the pathogenicity of the novel mutations p.(Phe55*) and p.(Arg216*) in GYG1 and of the p.(Leu156Pro) TRIM32 mutation combined with compound heterozygous deletion of exon 2 of TRIM32 and expanded the phenotype of Ala93Thr-caveolinopathy and of limb-girdle muscular dystrophy 2i caused by FKRP mutation. In 15 patients no causal variants were detected by Sanger sequencing and NGS panel analysis. In 12 of these cases, WES was performed, but did not yield any definite mutation or likely candidate gene. In one of these patients with a family history of muscle weakness, the vacuolar myopathy was eventually linked to chloroquine therapy. Our study illustrates the wide phenotypic and genotypic heterogeneity of vacuolar myopathies and validates the role of histopathology in assessing the pathogenicity of novel mutations detected by NGS. In a sizable portion of vacuolar myopathy cases, it remains to be shown whether the cause is hereditary or degenerative.

Infants Diagnosed with Spinal Muscular Atrophy and 4 SMN2 Copies through Newborn Screening - Opportunity or Burden?

Although the value of newborn screening (NBS) for early detection and treatment opportunity in SMA patients is generally accepted, there is still an ongoing discussion about the best strategy in children with 4 and more copies of the SMN2 gene. This gene is known to be the most important but not the only disease modifier.In our SMA-NBS pilot project in Germany comprising 278,970 infants screened between January 2018 and November 2019 were 38 positive cases with a homozygous SMN1 deletion. 40% of them had 4 or more SMN2 copies. The incidence for homozygous SMN1 deletion was 1 : 7350, which is within the known range of SMA incidence in Germany.Of the 15 SMA children with 4 SMN2 copies, one child developed physical signs of SMA by the age of 8 months. Reanalysis of the SMN2 copy number by a different test method revealed 3 copies. Two children had affected siblings with SMA Type III, who were diagnosed only after detection of the index patient in the NBS. One had a positive family history with an affected aunt (onset of disease at the age of 3 years). Three families were lost to medical follow up; two because of socioeconomic reasons and one to avoid the psychological stress associated with the appointments.Decisions on how to handle patients with 4 SMN2 copies are discussed in the light of the experience gathered from our NBS pilot SMA program.

The genetic landscape of axonal neuropathies in the middle-aged and elderly: Focus on MME.

OBJECTIVE: To test the hypothesis that monogenic neuropathies such as Charcot-Marie-Tooth disease (CMT) contribute to frequent but often unexplained neuropathies in the elderly, we performed genetic analysis of 230 patients with unexplained axonal neuropathies and disease onset ≥35 years. METHODS: We recruited patients, collected clinical data, and conducted whole-exome sequencing (WES; n = 126) and MME single-gene sequencing (n = 104). We further queried WES repositories for MME variants and measured blood levels of the MME-encoded protein neprilysin. RESULTS: In the WES cohort, the overall detection rate for assumed disease-causing variants in genes for CMT or other conditions associated with neuropathies was 18.3% (familial cases 26.4%, apparently sporadic cases 12.3%). MME was most frequently involved and accounted for 34.8% of genetically solved cases. The relevance of MME for late-onset neuropathies was further supported by detection of a comparable proportion of cases in an independent patient sample, preponderance of MME variants among patients compared to population frequencies, retrieval of additional late-onset neuropathy patients with MME variants from WES repositories, and low neprilysin levels in patients' blood samples. Transmission of MME variants was often consistent with an incompletely penetrant autosomal-dominant trait and less frequently with autosomal-recessive inheritance. CONCLUSIONS: A detectable fraction of unexplained late-onset axonal neuropathies is genetically determined, by variants in either CMT genes or genes involved in other conditions that affect the peripheral nerves and can mimic a CMT phenotype. MME variants can act as completely penetrant recessive alleles but also confer dominantly inherited susceptibility to axonal neuropathies in an aging population.

A new splice site mutation in the SMN1 gene causes discrepant results in SMN1 deletion screening approaches.

In most patients with infantile spinal muscular atrophy (SMA) both exons 7 and 8 of the SMN1 gene are deleted, but the deletion may also be restricted to exon 7. We report on an SMA type I patient who was initially diagnosed to be homozygous for an exon 7 deletion only. However, multiplex ligation-dependent probe amplification (MLPA) analyses revealed a heterozygous deletion of exons 7 and 8 of the SMN1 gene. By sequencing a new subtle splice site mutation (IVS6-2A>G) was identified. This variant affects the target sequence of oligonucleotides of all applied tests in a way that it has contrary effects on the efficiencies of the different assays. The results have major impacts on genetic counselling and carrier detection of the patient's paternal relatives.

Search for subtelomeric imbalances by multiplex ligation-dependent probe amplification in Silver-Russell syndrome.

Chromosomal aberrations are typically associated with primordial growth retardation, psychomotoric constrictions, and dysmorphisms. Since these features may be present in patients with Silver-Russell syndrome (SRS) and chromosomal disturbances are also detected in a subgroup of SRS patients, we screened a cohort of 45 SRS patients for cryptic subtelomeric imbalances. Submicroscopic deletions/duplications in the telomere regions are meanwhile well known to cause a broad spectrum of conspicuous phenotypes, characterized by mental retardation and multiple further congenital anomalies. We hypothesize that SRS might represent at the mild end of the broad phenotypic range of subtelomeric imbalances. Screening of the patients was performed by multiplex ligation-dependent probe amplification (MLPA), a technique that has already been shown to be effective and reliable for measuring copy numbers. We excluded pathogenetically relevant copy number variations in the subtelomeres in our SRS patient cohort, but one patient carried an apathogenic polymorphic Yq deletion. It can therefore be concluded that this type of chromosomal aberration does not belong to the genetic causes of SRS and it is not necessary to include this test in the diagnostic algorithm of the disease.