MECKEL (MECKEL-GRUBER) SYNDROME

Type of disorder: genic/chromosomal

Cause: genetically heterogeneous

Inheritance: autosomal recessive

Required testing: sequencing, large deletion/duplication testing (qPCR/MLPA), possibly FISH/CGH array.

Alterantive testing: exome sequencing

JOUBERT SYNDROME

The term Joubert syndrome (JS) has been coined to describe all disorders presenting with molar tooth sign on brain neuroimaging. Joubert syndrome is a clinically and genetically heterogeneous group of disorders also characterized by accompanying neurologic symptoms, including dysregulation of breathing pattern and developmental delay. Retinal dystrophy and renal anomalies are also included in the clinical spectrum. 

THREE IMPORTANT GENES IN LUNG CANCER

Molecular studies of lung cancer cells have been useful in the setting of advanced stage disease, whereas their usefulness in resectable disease is still under investigation. So far, molecular testing has been proved to be useful only for NSCLC (Non Small Cell Lung Cancer). Molecular testing can be done on samples from biopsies or on cytology specimens.

Amongst others, three genes have been frequently reèprted for showing mutations of diagnostic and predictive significance in lung cancer: EGFR, ALK and BRAF.

COMMERCIAL KITS FOR PHARMACOGENOMIC TESTING

Some commercial kits are already existing which can help any lab to do the screening of relevant pharmacogenetic variants known today. One of these is for instance the DMET™ Plus Solution kit by Affimetrix, which covers 1,936 genetic variants across 231 relevant genes.

Another commercial kit available for pharmacogenomic testing is the VeraCode ADME Core Panel by Illumina, which interrogates variants in 34 genes. This panel is based on the recommendation of PharmaADME working group, consisting of industry and academic experts who has developed a list of pharmacogenetic markers.

Another kit offering the screening of the ADME genes as proposed by the PharmaADME working group is the iPLEX® ADME PGx Pro Panel provided by Agena Bioscience.

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PHARMACOGENOMIC DATABASES

PHARMACOGENOMIC DATABASES

The best pharmacogenomic database today available is PharmGKB. At PharmGKB website it is also possible to download the current guidelines on selected gene variants as edited by the Clinical Pharmacogenetics Implementation Consortium (CPIC).

It’s not a database, but it’s worth to be highlighted for its activity of research and collaboration funding: it’s the PharmacoGenomics Reasearch Network (PGRN), supported by the National Institute of Health (NIH) since 2000.

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PHARMACOGENOMICS AND POPULATION DIVERSITY

Because of population genetic diversity, pharmacogenomics correlations are significantly varying from an ethnicity to another one. It happens so that a pharmacogenetic test shows to be of great epidemiological relevance within a certain geographical area, whereas it is of lower impact in some other ones. For instance, the variant 3673A of the VKORC1 gene, an allele to be considered in warfarin dosage, is rare in sub-Saharian Africans (less than 10%) but it is found at an extremely higher frequency in Southeast Asian populations (more than 90%). The Pharamcogenomics for Every Nation Initiative (PGENI) has exactly the aim of characterizing such pharmacogenomics differences among various populations. An online resource dedicated to multi-ethnic frequency data for pharmacogenetically relevant single nucleotide polymorphisms is FINDbase-PGx. Such data are also summarized at PharmaGKB website.

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GENETIC VARIANTS OF PHARMACOGENOMIC RELEVANCE

Many pharmacogenetically important genes belong to the family of the cytochrome P450, solute carrier (SLC), ATP-binding cassette (ABC), aldehyde dehydrogenase (ALDH) and UDP-glucoronyltransferase (UGT) families.

The first (and one of the more important) gene involved in pharmacogenomic studies is CYP2D6. It is believed that the enzyme encoded by this gene (cytochrome P450-2D6) is involved in the metabolism of about 25% of drugs available today. So far, more than 100 CYP2D6 alleles have been reported to be of pharmacogenomic relevance. In the last years CYP2D6 genotyping has been elaborated to classify patients into four metabolizer phenotypes: ultrarapid, extensive, intermediate and poor.

Many other cytochrome P450 genes like CYP2C9 and CYP2C19 have been pharmacogenetically characterized. CYP2C9 alleles, along with VKORC1 and CYP4F2 alleles, are important in warfarin efficacy and dosing. The CYP2C19*2 allele has been associated with a lower production of active metabolites of clopidrogel, higher platelet aggregation ratio and adverse clinical outcome. Other notable associations of these two genes are with phenytoin, acenocoumarol, glibenclamide, gliclazide, glimepiride, phenprocoumon, and tolbutamide (CYP2C9) and with carisoprodol, citalopram, clobazam, clopidogrel, dexlansoprazole, diazepam, esomeprazole, lansoprazole, nelfinavir, omeprazole, pantoprazole, prasugrel, rabeprazole, drospirenone ethinyl estradiol, atazanavir, axitinib, ticagrelor and voriconazole (CYP2C19).

Other important pharmacogenomic markers have been identified in the following gene/drug associations: ABCB1/aliskiren, CYP2B6/efavirenz, CYP34A/ticagrelor, aripiprazole, cabazitaxel, darunavir, dronedarone, fosamprenavir, gefitinib, indinavir, ivabradine, nelfinavir, posaconazole, ritonavir, ruxolitinib, sirolimus, sunitinib, telithromycin, tipranavir, voriconazole, zonisamide, CYP3A5/tacrolimus, DPYD/tegafur, capecitabine, fluorouracil, NAT2/isosorbide dinitrate and hydralazine hydrochloride, rifampin-isoniazid-pyrazinamid, SLC22A2/fampridine, SLCO1B1/simvastatin, TPMT/azathioprine, mercaptopurine, thioguanine, azathioprine, UGT1A1/irinotecan, IFNL3/PEG-interpheron-α (PEG-IFN alpha).

A working group of experts from the academy and the industry has actually collected the information on the most important genes so far recognized to be of relevance for their pharmacogenomic correlation. This is the PharmaADME working group, whose list have been used also to design commercial kits for genetic testing (the ADME gene list).

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CLINICAL VALIDITY OF PHARMACOGENOMIC TESTING

In genetic testing, clinical validity can be defined as the ability to predict a phenotype associated with a genotype. For typical genetic diseases (i.e. chromosomal aberrations and Mendelian disorders) clinical validity is approaching 100%, since there is a direct cause-effect relation between a certain genetic mutation and the presence of the disease.

Much different are things for what concerns pharmacogenomics since the scientific evidence of the association between certain genetic variant(s) and the response to a drug can be sometime controversial. It can happen so that a genetic variant found to be significantly associated with high sensitivity to a certain treatment in one study, is found to be of no pharmacogenomic relevance in another reseach. In extreme cases a certain genetic variant is found to exert a total opposite effect from another author.  This issue of controversial evidence is still a major obstacle to the diffusion of pharmacogenetic testing in daily routine since it mostly affects the trust of health professionals in pharmacogenomic solutions.

However, it must be said that in certain cases pharmacogenomic correlations have been well characterized and replicated by several independent studies. In such cases the pharmacogenomic evidence has been coded into clinical practice guidelines by known working groups of specialists (see for instance the guidelines from the Clinical Pharmacogenetics Implementation Consortium - CPIC, www.pharmagkb.org/page/cipc, the Royal Dutch Association for the Advancement of Pharmacy-Pharmacogenetics Working Group – KNMP-PWG, the EGAPP working group or the ACMG). By following these guidelines, health care operators can confidently take decisions in drug selection and dose adjustment.

An example of pharmacogenomic evidence successfully confirmed and applied in daily clinical practice is the one between the allele B*5701 in the HLA-B gene and the susceptibility to severe hypersensitivity reaction to abacavir treatment in HIV-positive patients. This pharmacogenetic test is now routinely performed to help infectivologists in selecting the most appropriate treatment combination for HIV-positive patients.

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PHARMACOGENOMICS

Pharmacogenomics (sometimes referred to also as pharmacogenetics) is a discipline aimed to identify human genetic traits which can be associated either to drug response (sensitivity/resistance) or to adverse drug reactions. Some individuals have certain genetic variants (also called pharmacogenetic or pharmacogenomic markers) which can affect the way a drug is metabolized and therefore influence the drug power and/or the appearance of side-effects. Therefore pharmacogenomics is essentially relevant for (1) dose adjustment and (2) drug selection.

Until yesterday, the costs and turn-around time of testing, a lack of skilled counselling staff and regulatory and reimbursement issues curbed the spreading of pharmacogenetic testing. Today, thanks to time-effective analyses and preventive genotyping programs, pharmacogenomics is about to become a reality in the context of the so-called personalized medicine.

Personalized medicine is not something new. Already in ancient Greece Hippocrates used to measure the balance of blood, phlegm, yellow bile, and black bile to select the best therapy for every patient. Today we prefer to look at genetics rather than to these four fanciful humors, but the point is the same: the “one-fit-for-all” model is, at least for some available drug, not even desirable, as considerable advantages in treatment outcome (and side-effects avoidance) can be achieved through a personalized approach.

Just to explain the concept with a few numbers: only 30-60 % of patients respond properly to beta-blockers, anti-depressants, statins and antipsycothic agents. Adverse drug reactions can cause significant prolongation of hospital visits and, only in the USA, they are estimated to cause approximately 100,000 deaths every year. To learn more about pharmacogenomics:

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GENETIC VARIANTS OF PHARMACOGENOMIC RELEVANCE
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PHARMACOGENOMIC DATABASES
COMMERCIAL KITS FOR PHARMACOGENOMIC TESTING

GENETIC CAUSES OF MALE INFERTILITY

Can male infertility be genetic? What are the causes?

Male infertility is diagnosed when a man has been unable to make a fertile woman pregnant after trying for at least a year. When caused by genetic factors, male infertility accounts for about 10 to 15 percent of cases of severe infertility. A man must produce healthy sperm in the right amounts to fertilize an ovum under normal circumstances.

DM1 AND DM2 ARE THE ONLY DEFINITE CAUSES OF MYOTONIC DYSTROPHY

Myotonic dystrophy is a genetic disease crucially characterized by myotonia (described as the difficulty in releasing musculature; typically these patients may find difficult to release the grip after shaking hands). The only known causes of myotonic dystrophy to date are either DMPK or ZNF9 repeat expansions (associated with DM1, also known as Steinert disease, and DM2 respectively).

DSMA and dHMN: WHAT'S THE DIFFERENCE?

Distal spinal muscular atrophy (DSMA) is also known as distal hereditary motor neuronopathy (dHMN or HMN). Properly, DSMA refers to the autosomal recessive forms of HMN: DSMA1 (IGHMBP2 mutations), DSMA2, DSMA3 (no identified genes) and DSMA4 (PLEKHG5 mutations).

GENETIC TESTING FOR TUMORS

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The scientific advances of recent years have allowed us to understand in detail many genetic aspects of cancer. A first distiction should be done between germline mutations, which cause a strong susceptibility to the onset of particular tumor types, commonly but erroneously called hereditary, and somatic mutations, most often the consequence and not the primary cause of the presence of tumor.

LYMPHEDEMA FOLLOWING BREAST CANCER TREATMENT: SURGICAL APPROACH

Breast cancer-related upper extremity lymphedema can be a complication with a reported incidence of about 1 in 5 (but almost 1 in 2 when complete axillary lymph node dissection was done).

Lymphedema may rise immediately after surgery but most often occurs after a latent period. The causes are still not full understood, but obesity and radiotherapy are risk factors. Increased volume and weight of the affected limb and  are the main signs. Although physically not much disabling, the condition may be an emetional and psychological burden, as the patient may develop sensations of funtional handicap as well as aesthetical concerns.

GENETIC SUBTYPES OF SPINOCEREBELLAR ATAXIA

Spinocerebellar ataxia (SCA) is one of the most heterogenous genetic disorders known to date. Mutations in different genes can cause the disease (through autosomal dominant, autosomal recessive or X-linked inheritance). Several type of mutations are described and different analytical approaches are therefore required for the test. Here below we present an updated list of all genetic subtypes known to date, divided by inheritance. For details about the method required for the analysis of each SCA subtype, please read here.

LABORATORY: DIAGNOSING SPINOCEREBELLAR ATAXIA

The genetic diagnosis of different spinocerebellar ataxia subtypes is based on different technical approaches. Three main methods can be used: fragment length analysis, sequencing, and deletion/duplication testing.

Note: for the latest update on all SCA subtypes known to date, please read here.

FAMILIAL NON-HODGKIN LYMPHOMA: GENETICS

Clinical presentation

Non-Hodgkin lymphoma (NHL) consists of a heterogeneous group of tumors, which are classified based on immunologic origin, clinical features, and treatment options. Most commonly non-Hodgkin lymphomas are manifesting as a painless swallowing of superficial lymph nodes stations (neck, axillary, inguinal), but sometimes can also rise in other areas (gastrointestinal, nervous, skin, bone marrow).

AFG3L2-RELATED SCA CAN ALSO BE INHERITED IN AN AUTOSOMAL RECESSIVE MANNER (SCAX5)

Spinocerebellar ataxia 28 (SCA28) is typically caused by autosomal dominant mutations in the AFG3L2 gene. However, Pierson TM et al (2011) have reported a family in which a homozygous hypomorphic allele (c.1847A>Gp.Y616C) is segregating as homozygous in affected patients. This form of AFG3L2-related SCA is called SCAX5 (see OMIM 614487). Hypomorphic alleles are consistent with mutations which can worsen the phenotype of some autosomal dominantly inherited genetic diseases. Examples of hypomorphic alleles have been reported in PKD1-related polycystic kidneys. However, homozygosity for hypomorphic alleles is not regularly reported as cause of disease. So this AFG3L2 mutation seems to be quite an exception.

References: see text.

THE DIFFERENTIAL DIAGNOSIS OF CMT4

The diagnosis of Charcot-Marie-Tooth neuropathy type 4 requires clinical, pathologic, and genetic confirmation. Mutations causing CMT4 can be detected in the following genes: GDAP1 (CMT4A), MTMR2 (CMT4B1), SBF2 (CMT4B2), SBF1 (CMT4B3), SH3TC2 (CMT4C), NDRG1 (CMT4D), EGR2 (CMT4E), PRX (CMT4F), FGD4 (CMT4H) and FIG4 (CMT4J). However, some additional loci for CMT4 have been mapped, without any identified gene yet: see for instance CTM4G linked to 10q22 and CMT linked to 8q21.3. Additional single reports have been published about cases of axonal neuropathy of late onset in a Costa Rican family linked to 19q13.3 (in which Rautenstrauss et al 2005 preliminarily reported a mutation in MED25), about an autosomal recessive neuropathy with mutations in LMNA, and about autosomal recessive motor and sensory axonal neuropathy with neuromyotonialoss caused by loss-of-function mutations in HINT1 causing (Zimoń et al 2012).

WHAT'S A SPLICE MUTATION?

WHAT'S A SPLICE MUTATION?

A splice mutation is a mutation impacting the splicing process. Genes are made up by coding region (exons) and non-coding regions (introns). The mRNA produced starting from the DNA sequence is containing exons and introns, but before the translation into protein the non coding parts a cut out to generate a filament including only coding sequence. This process is called the splicing. The splicing is operated by enzymes which recognize specific sequences, to which they bind to in order to cut out the non-coding parts of the messenger (mRNA). There are basically to type so sequences that can be targeted by the splicing enzymatic complex: the highly conserved donor and a acceptor sites and the so called enhancing or silencing sequences. The donor site is invariably constituted by a guanine and a timine (GT) at the first two nucleotide positions of the intron (identified with the cDNA position c.___+1 and c.___+2; the acceptor site is invariably constituted by an adenine-guanine sequence at the last two nucleotide positions of the intron c.___-1 and c.___-2. Any mutation affecting these positions is almost certainly disease-causing. In other words, any nucleotide substitution at cDNA positions +/-1 or +/-2 is very likely damaging. More difficult is to locate splice mutations not affecting the highly conserved donor or acceptor site, but they actually exists! These are difficult to individuate, because the splicing enhancing or silencing sequencing are not phylogenetically conserved and are varying from gene to gene. In general, it is good to evaluate Ba in silico analysis any mutation falling within the first 10 nucleotides from mthe exon/intron boundaries, but these mutations can fall also deeply in an intron or even within an exon. Deep intronic splice mutations are therefore difficult to be screened for because (1) the deep intronic regions, for cost-efficiency reasons, are currently not analyzed in standard genetic diagnosis. Secondly, their interpretation is always difficult because of the very high variability described above. As a very general rule, any deep intronic or exotic mutation creating an
GT or a AG di nucleotide sequence can be suspected to activate a cryptic donor or acceptor splice site, but this is not the rule in every case. In silico analysis with different software is often the best way to predict the effect of such mutations. The are several software available. The output of some of these software can be calculated in parallel with Alamut (Interactive Biosoftware).