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The storage of cultured fibroblasts, like the retention of DNA samples, is one of the prerequisites for later genetic diagnosis of unexplained disorders. Some types of fibroblast study are particularly important

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Golgi and endoplasmic reticulum disorders (congenital defects of glycosylation)

Attaching sugar molecules to proteins or lipids (glycosylation) is mediated by the Golgi apparatus or complex and by the endoplastic reticulum and is necessary for numerous biochemical functions.

Defects may be divided into soluble and structural abnormalities.

The 'soluble' congenital defects of glycosylation (CDG) - predominantly defects of N-glycosylation - are many and increasing in number but by far the most important is CDG1a. 'structural' defects of O-glycosylation comprise congenital and later onset muscular dystrophies and are investigated by completely different methods.

Clinical clues to CDG

The symptomatology may be highly variable even within the same family but some of the following features may be seen:

• facial and hand dysmorphism often mild
• Fat pads, particularly on the buttocks
• Flat or inverted nipples (inverted nipples are more common in those without CDG!)
• Slow cognitive development
• Slow motor development
• Strabismus
• Feeding difficulties
• Stroke-like episodes (hemipareses)
• Epileptic seizures (not as an isolated phenomenon)
• Retinopathy
• Hepatomegaly or disorder of liver function
• Cardiomyopathy
• Palpable kidneys
• Endocrine defects, e.g. hypothyroidism, hyperinsulinism, hypergonadotrophic hypogonadism (in females)
• Miscellaneous defects, skeleton, etc.

Neorological phenotypes similar to those well recognized in mitochondrial disorders may be seen and it is wise to ensure that congenital defects of glycosylation have been excluded before one proceeds to invasive investigation for mitochondrial disorders.

Investigation Result in CDG

Isoelectric focusing (IEF) for transferrins (sialotransferrins) is the major primary diagnostic investigation. The clinical spectrum of CDG is so wide that transferrin IEF must always be done when the diagnosis is a possibility.

Other investigations that may be abnormal in CDG and may give clues towards the diagnosis are as follows:

• EEG during stroke-like episodes showing repetitive spike complexes contralateral to the hemiparesis
• Slow nerve conduction
• Cerebellar atrophy or pontocerebellar atrophy and particularly progressive cerebellar atrophy
• CSF protein increase
• Coagulation abnormality, in particular decreased factor IX and antithrombin III, protein C and protein S
• Abnormal liver function tests, especially increased transaminases.

The precise biochemical defect causing the CDG can be elucidated by performing leukocyte or fibroblast enzyme assays and then by DNA mutation studies in cases where the clinical phenotype points towards a specific enzymatic deficiency. Where this is not the case, powerful tandem mass spectrometry techniques can be utilized to provide information on the glycoprotein in question, hence providing a clue towards the precise enzymatic block. 

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Peroxisomes are spherical 1pm diameter organelles with a multitude of oxidative and other enzymes packed within a single-layered membrane (http://www.peroxisomedb. org)

• global' peroxisomal disorders with impaired peroxisomal biogenesis (fewer or even no peroxisomes), together with those single peroxisomal enzyme defects that induce a similar phenotype (in particular, D-bifunctional protein deficiency)
• adrenoleukodystrophy, due to an X-linked defect of the peroxisomal ABCD1 gene that codes for the peroxisomal membrane protein ABCD1, a member of the ATP-hmding cassette transporters.

While estimation of very long chain fatty acids (VLCFA) may detect many peroxisomal isomers there is no 'metabolic screen' that will detect them all. 

CLINICAL CLUES TO PROXISOMAL DISORDERS

• Neonatal hypotonia (especially extreme hypotonia of the neck) Neonatal seizures (refractory or phenytoin-responsive in a hypotonic baby)
• Neonatal unresponsiveness (except reflex crying)
• Non-development
• Retinal blindness ('Leber amaurosis')
• Sensorineural deafness
• Gross dysmorphic features of Zellweger syndrome type (high forehead, flat face, simian creases, simple genitalia)
• Mild dysmorphism (large fontanelle with open metopic suture, absent ear lobules)
• Leukodystrophy, neuronal migration defects especially perisylvian polymicrogyria
• Hepatomegaly (± hepatic insufficiency)
• Developmental delay with malabsorption features, retinopathy and sensorineural deafness
• Development delay with seizures and areflexia (peripheral neuropathy).
• School-age regression in a boy with previously normal development (+ Addison disease)
• Spastic paraplegia in older females.

Investigation results found in peroxisomal disorders

In neonatal and early manifesting peroxisomal disorders

• EEG trains of repetitive spikes shifting from side to side
• Slow nerve conduction (± EMG denervation)
• Low or absent ERG
• Brainstem auditory evoked potentials (BAEP) - gross abnormalities
  • High threshold
  • Delayed wave V latency
  • Lack of response
• MRI evidence of migration disorder
• MRI white matter altered signal
• Patellar or other chondral calcification
• Bone age retardation
• Echogenic renal cortex on ultrasound
• Increased CSF protein
• Liver biopsy: hepatic fibrosis
• Clotting defect of hepatic type
• Increased AST and ALT
• Low cholesterol
• Low vitamin E
• Increased phytanic acid
• Increased pipecolic acid
• Urinary excretion: amino acids, glycosaminoglycans, dicarboxylic acids.

In the regressing school child:

• Visual evoked potential (VEP) - latency increasem
• BAEP increased latency of wave V
• MRI increased signal in posterior central white matter on T2 images usually but not always

Special peroxisomal investigations

• Once sufficient clinical and investigational clues have been assembled, special tests for peroxisomal disorders may be sought at specialized laboratories. These primarily test

• The size of the peroxisomal compartment - simply, are peroxisomes missing and all functions depressed?
• The integrity of the peroxisomal beta-oxidation pathway, which metabolizes VLCFAs and also, in pan, bile acids

The tests of most direct help are as follows.

• VLCFAs in plasma

VLCFAs are increased in all disorders with absent or grossly diminished peroxisomes, and in most disorders of the VLCFA metabolic pathway (but not in all defects of isolated peroxisomal enzymes). This is the only test necessary for confirmation of the diagnosis of adrenoleukodystrophy in the regressing schoolchild.

• VLCFAs in fibroblasts

If a peroxisomopathy is suspected - as when there is characteristic dysmorphism, non-development and retinal and sensorineural hearing defects, then normal plasma studies (including normal plasma VLCFA) do not exclude D-bifunctional protein (DBP) deficiency. In this clinical situation, request full fibroblast studies including fibroblast C26:0 beta-oxidation rate and VLCFA ratios.

Bile acids

Abnormal bile acids are found in the urine, plasma and duodenal juice in infants with a lack of peroxisomes, and in those with peroxisomal DBP deficiency.

Dihydroxyacetone phosphate acyl transferase (DHAP-AT)

This is estimated in fibroblasts (and in Platelets). It is reduced in all cases of general lack of peroxisomes.

Liver biopsy with special studies.

It is possible to demonstrate lack of peroxisomes by special histochemistry and electronmicroscopy, and enlarged peroxisomes in biochemical lesions of the beta-oxidation pathway, but such studies are no longer essential for diagnosis, lmmunoblot methods allow determination of the individual beta-oxidation enzymes, but as these enzymes may be totally inactive despite demonstrable protein, it cannot at present be said that liver biopsy is a necessary investigation. 

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Mitochondrial disorders With the explosion of knowledge about disorders of the mitochondria, definitive investigations have become more complex and specialized. However, clinical clues can point towards a mitochondrial disorder and fairly simple tests support the diagnosis sufficiently to proceed to specific investigations of mitochondrial function.

To a certain extent there is a relationship between the type of disease and the site of metabolic defect along the pathway from the inner mitochondrial membrane to the termination of the respiratory chain, but there is considerable heterogeneity.

It has long been known that most of the genes responsible for mitochondrial function are nuclear rather than mitochondrial genes, and recently some of these nuclear genes have become much more accessible to analysis. The most important of these is POLG or POLG1, that is, mitochondrial DNA polymerase-gamma.

A major way by which mutations in POLG1 affect mitochondrial function is by inducing mitochondrial depletion. Such mitochondrial depletion may be either generalized or organ specific. Other genes of recent interest include Twinkle, DGUOK and the gene for thymidine kinase 2.

Clinical Clues TO mitochondrial disorders (most common genes in parentheses)

• Myopathy with fatigue (various mt tRNA mutations)
• 'Progressive neuronal degeneration of childhood' Alpers disease - with hemiclonic and focal myoclonic seizures often with epilepsia partialis continua, associated with developmental regression and cerebral atrophy ± terminal liver failure (POLG1).
• Leigh disease: regression with hypotonia, oculomotor and respiratory disturbances, with symmetrical lesions on imaging in basal ganglia, thalami, substantia nigra, red nuclei, cerebellum and commonly also in periaqueductal grey matter and spinal cord, and often COX (COX = cytochrome C oxidase = complex IV of the respiratory' chain) deficiency in muscle (genetically heterogeneous: many mitochondrial mutations, especially ATPase6 and genes for tRNA, nuclear genes, especially SURF1 - codes for assembly factor for COX - and genes for Complex 1, but only rarely POLG1).
• Leigh-like syndrome: similar to Leigh but oculomotor and respiratory impairment usually absent, more variable neurology (genes as in Leigh)
• Episodic, static or progressive ataxia ± other neurological deficits ± cerebellar lactate peak on H-MRS (POLG1, mtDNA depletion, ubiquinone deficiency).
• Chronic encephalomyopathy of childhood; fatigue, pigmentary retinopathy, oculomotor disturbance, sensorineural deafness, ataxia, pyramidal signs, regression, including NARP (mtDNA T8993G or C in complex V = ATPase6).
• Kearns-Sayre syndrome: ptosis and ophthalmoplegia, pigmentary retinopathy, heart block, short stature (large mitochondrial rearrangements). • Pearson syndrome: infantile-onset pancreatic disorder, sideroblastic anaemia, later Kearns-Sayre (large mitochondrial deletion).
• MELAS - mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes: severe migraines, partial epileptic seizures, hemipareses, and cerebral lesions on imaging which superficially look as if they were ischaemic but do not correspond to known vascular territories (mtDNA A3243C, or T3271C).
• MERRF - myoclonic epilepsy with ragged red fibres: myoclonic epileptic seizures with repetitive myoclonus and progressive ataxia and regression in late childhood (mtDNA A8344G).
• MNGIE-mitochondrial neurogastrointestinal encephalomyopathy (nDNA thymidine phosphorylase).
• LHON - Leber hereditary optic neuropathy (mtDNA C3460A, C11778A, T14484C in complex 1 subunits).
• Movement disoiders and various semiologies including paroxysmal dystonia (PDH deficiency, but also mt tRNA and LHON mutations).
• Guillain-Barre type acute polyneuropathy with atypical features including normal GSF protein but increased lactate, with or without recurrence (El a mutations in PDH complex).
• Infantile spinocerebellar ataxia with axonal neuropathy (perhaps Twinkle and twinky.
• Nonspecific combination of static or progressive neurodevelopmental disorder with paroxysmal events such as epileptic seizures and migraine, and evidence of more than one component of the nervous system (POLG1 especially).

INVESTIGATION RESULTS FOUND IN MITOCHONDRIAL DISORDERS

• Because these disorders of oxidative phosphorylation (OXPHOS) result in impaired aerobic energy metabolism, primary indicators are increased blood and CSF' lactates.
• However, normal lactates do not exclude a mitochondrial disorder, especially in POLG1
  

Combinations of the following investigations may give clues, depending on the systems involved:

• EEG polyspikes mini-bursts on rhythmic slow activity
• Slow nerve conduction velocity
• CT: calcification of the basal ganglia
• MRI: altered signal in basal ganglia structures (especially globus pallidus), substantia nigra, red nuclei, periaqueductal grey matter, alterations in white matter of centrum semiovale cerebellum
• Neuronal migration disorder in some, e.g. perisylvian polymicrogyria
• MR spectroscopy
  • Lacate peak on proton MRS in basal ganglia
  • Lactate peak on H-MRS in cerebellum - especially when ataxia
• CSF: increased protein (increased albumin).
• ECG: cardiac conduction defect including heart block and Wolff-Parkinson-White syndrome
• Haematology: marrow suppression
• Blood biochemistry
  • Low albumin
  • Increased alanine and proline
  • Raised transaminases
• Urine biochemistry
  • Generalized aminoaciduria
  • Impaired phosphate reabsorption
  • Krebs cycle organic acids (e.g. fumarate, succinate)

Special mitochondrial investigations

The combination of clinical and investigation results may point not only to a mitochondrial disorder but also to a specific phenotype.

Phenotypes such as MELAS, MERRFor Kearns-Sayre relate to a defect of the mitochondrial genome, whereas phenotypes such as Alpers-Huttenlocher would point towards a nuclear mitochondrial gene, in particular polymerase gamma (POLG1) or the mitochondrial DNA helicase Twinkle.

If the clinical picture points to a deficiency in pyruvate dehydrogenase (PDH) such as neonatal encephalopathy with lactic acidosis and absent, hypoplastic or dysplastic corups callosum or in the older child progressive dystonia with altered signal in globus pallidus on imaging, PDH activity may be measured in cultured skin fibroblasts.

While the clinical phenotype suggesting mitochondrial depletion will have pointed towards studies of POLG1 and Twinkle as indicated above, or when there is a less demined but persuasive suggestion of mitochondrial dysfunction, then the next stage is study of muscle and possibly liver.

Histological examination of muscle biopsy may show ragged red fibres on Gomori stain, lipid droplets and abnormal staining for cytochrome c oxidase. Mitochondria may be abnormal on electronmicroscopy. functional studies of all components of the mitochondrial respiratory chain will be undertaken on fresh specimens.

Liver biopsy with functional studies may be needed when the muscle biopsy studies are negative despite pointers to a mitochondrial disorder.

While until recently it has been recommended that CoQ10 be estimated in fresh muscle, while cell (or fibroblast) CoQ10 may be a better way of determining ubiquinone deficiency.

Mitochondrial 'confusion' Diagnostic confusion is not infrequent between mitochondrial disorders and other conditions. There may be 'pseudo-' or secondary mitochondrial deficiencies, but also true mitochondrial disorders may masquerade as for instance monoamine neurotransmitter conditions.